Research Article Gastrodia elata Ameliorates High-Fructose ...
Transcript of Research Article Gastrodia elata Ameliorates High-Fructose ...
Research ArticleGastrodia elata Ameliorates High-Fructose Diet-Induced LipidMetabolism and Endothelial Dysfunction
Min Chul Kho12 Yun Jung Lee12 Jeong Dan Cha3 Kyung Min Choi3
Dae Gill Kang12 and Ho Sub Lee12
1 College of Oriental Medicine and Professional Graduate School of Oriental Medicine Wonkwang UniversityShinyong-dong Iksan Jeonbuk 570-749 Republic of Korea
2Hanbang Body-Fluid Research Center Wonkwang University Shinyong-dong Iksan Jeonbuk 570-749 Republic of Korea3 Department of Research Development Institute of Jinan Red Ginseng Jinan Jeonbuk 567-801 Republic of Korea
Correspondence should be addressed to Ho Sub Lee hostwkuackr
Received 13 November 2013 Accepted 27 December 2013 Published 26 February 2014
Academic Editor Waris Qidwai
Copyright copy 2014 Min Chul Kho et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Overconsumption of fructose results in dyslipidemia hypertension and impaired glucose tolerance which have documentedcorrelation with metabolic syndrome Gastrodia elata a widely used traditional herbal medicine was reported with anti-inflammatory and antidiabetes activities Thus this study examined whether ethanol extract of Gastrodia elata Blume (EGB)attenuate lipid metabolism and endothelial dysfunction in a high-fructose (HF) diet animal model Rats were fed the 65 HFdiet withwithout EGB 100mgkgday for 8 weeks Treatment with EGB significantly suppressed the increments of epididymalfat weight blood pressure plasma triglyceride total cholesterol levels and oral glucose tolerance respectively In addition EGBmarkedly prevented increase of adipocyte size and hepatic accumulation of triglycerides EGB ameliorated endothelial dysfunctionby downregulation of endothelin-1 (ET-1) and adhesion molecules in the aorta Moreover EGB significantly recovered theimpairment of vasorelaxation to acetylcholine and levels of endothelial nitric oxide synthase (eNOS) expression and inducedmarkedly upregulation of phosphorylation AMP-activated protein kinase (AMPK)120572 in the liver muscle and fat These resultsindicate that EGB ameliorates dyslipidemia hypertension and insulin resistance as well as impaired vascular endothelial functionin HF diet rats Taken together EGB may be a beneficial therapeutic approach for metabolic syndrome
1 Introduction
Metabolic syndrome a worldwide issue is characterized byinsulin resistance impaired glucose tolerance andor hyper-glycemia high blood serum triglycerides low concentrationof high-density lipoprotein (HDL) cholesterol high bloodpressure and central obesity The association of 3 (or more)of these factors leads to an increasedmorbidity andmortalityfrom several predominant diseases such as type 2 diabetescancer and cardiovascular diseases including atherosclerosismyocardial infarction and stroke [1 2]
Fructose is an isomer of glucose with a hydroxyl groupon carbon-4 reversed in position It is promptly absorbedand rapidly metabolized by liver Recent decades west-ernization of diets has resulted in significant increases inadded fructose enormous rised in fructose consumption
typical daily [3] The exposure of the liver to such enor-mous rising fructose consumption leads to rapid stimula-tion of lipogenesis and triglyceride accumulation which inturn leads to reduced insulin sensitivity and hepatic insulinresistanceglucose intolerance [4] Thus high-fructose dietinduces a well-characterised metabolic syndrome generallyresulting in hypertension dyslipidaemia and low level ofHDL-cholesterol [5] Recent studies suggest that high fruc-tose intake may be an important risk factor for the develop-ment of fatty liver [6] Rats are commonly used as a modelto mimic human disease including metabolic syndrome [7]Similarly emerging data suggest that experiment on fructose-diet rats tends to produce some of the changes associatedwithmetabolic syndrome such as altered lipid metabolism fattyliver hypertension obesity and dyslipidemia [8]
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 101624 10 pageshttpdxdoiorg1011552014101624
2 Evidence-Based Complementary and Alternative Medicine
Gastrodia elata Blume is a traditional herbal medicinein Korea China and Japan which has been used for thetreatment of headaches hypertension rheumatism and car-diovascular diseases [9] Several major physiological sub-stances have been identified from Gastrodia elata Blumesuch as gastrodin vanillyl alcohol vanillin glycoproteinp-endoxybenzyl alcohol and polysaccharides includingalpha-D-glucan [10ndash12] Our previous studies showed thatGastrodia elata Blume exhibits anti-inflammatory and anti-atherosclerotic properties by inhibiting the expression ofproinflammatory cytokines in vascular endothelial cells [1314] However the effect of ethanol extract of Gastrodia elataBlume on high-fructose (HF) diet animalmodel has not beenyet reported Thus the present study was designed to deter-mine whether an ethanol extract of Gastrodia elata Blume(EGB) improves high-fructose diet-induced lipidmetabolismand endothelial dysfunction
2 Materials and Methods
21 Preparation of Gastrodia elata Blume The Gastrodiaelata Blume was purchased from the Herbal Medicine Co-operative Association Iksan Jeonbuk Province Korea inMay 2012 A voucher specimen (no HBJ1041) was depositedin the herbarium of the Professional Graduate School ofOriental Medicine Wonkwang University Iksan JeonbukSouth Korea The dried Gastrodia elata Blume (400 g) wasextracted with 4 L of 95 ethanol at room temperaturefor 1 week The extract was filtered through Whatman no3 filter paper (Whatman International Ltd England) andconcentrated using rotary evaporator The resulting extract(12741 g) was lyophilized by using a freeze drier and retaineduntil required
22 Animal Experiments and Diet All experimental pro-cedures were carried out in accordance with the NationalInstitute of Health Guide for the Care and Use of LaboratoryAnimals and were approved by the Institutional Animal CareandUtilizationCommittee forMedical Science ofWonkwangUniversity Seven week-old male Sprague-Dawley (SD) ratswere obtained fromSamtako (Osan Korea) Rats were kept ina room automatically maintained at a temperature (23plusmn2∘C)humidity (50sim60) and 12-h lightdark cycle throughout theexperiments After 1 week of acclimatization animals wererandomly divided into three groups (119899 = 10 per group)Control group (Cont) was fed regular diet high-fructosegroup (HF) was fed 65 fructose diet (Research Diet USA)and the third group (HF + EGB) was fed with 65 fructosealong with a single dose of 100mgkgday of EGB orallyfor a period of 8 weeks The regular diet was composed of50 starch 21 protein 4 fat and standard vitamins andmineral mix The high-fructose diet was composed of 65fructose 20 protein 10 fat and standard vitamins andmineral mix
23 Blood and Tissue Sampling At the end of the experi-ments the aorta liver adipose tissue (epididymal fat pads)and muscle were separated and frozen until analysis after
being rinsed with cold saline The plasma was obtained fromthe coagulated blood by centrifugation at 3000 rpm 15minat 4∘C The separation of plasma was frozen at minus80∘C untilanalysis
24 Measurements of Blood Pressure Systolic blood pres-sure (SBP) was determined by using noninvasive tail-cuffplethysmography method and recorded with an automaticsphygmotonography (MK2000 Muromachi Kikai TokyoJapan) The systolic blood pressure (SBP) was measured atweek 1 week 3 and week 7 respectively At least sevendeterminations were made in every session Values werepresented as the mean plusmn SEM of five measurements
25 Analysis of Plasma Lipids The levels of triglyceride inplasma were measured by using commercial kits (ARKRAYInc MINAMI-KU KYOTO Japan) The levels of high-density lipoprotein (HDL)-cholesterol total cholesterol andLDL-cholesterol in plasma were measured by using HDL andLDL assay kit (E2HL-100 BioAssay Systems)
26 Estimation of Blood Glucose and Oral Glucose ToleranceTest The concentration of glucose in blood was measuredwhich was obtained from tail vein using glucometer (One-touch Ultra) and Test Strip (Life Scan Inc CA USA)respectively
The oral glucose tolerance test (OGTT) was performed2 days apart at 7 weeks For the OGTT briefly basalblood glucose concentrations were measured after 10sim12 h ofovernight food privation then the glucose solution (2 gkgbody weight) was immediately administered via oral gavageand fourth more tail vein blood samples were taken at 30 6090 and 120min after glucose administration
27 Preparation of Carotid Artery and Measurement of Vascu-lar Reactivity Thecarotid arteries of the rats were rapidly andcarefully isolated and placed into cold Krebrsquos solution of thefollowing composition (mM) NaCl 118 KCl 47 MgSO
411
KH2PO412 CaCl 15 NaHCO
325 glucose 10 and pH 74
The carotid arteries were removed to connective tissue andfat and cut into rings of approximately 3mm in length Alldissecting procedures were carried out for caring to protectthe endothelium from accidental damage The carotid arteryrings were suspended by means of two L-shaped stainless-steel wires inserted into the lumen in a tissue bath containingKrebrsquos solution at 37∘C and aerated with 95O
2and 5CO
2
The isometric forces of the rings were measured by using aGrass FT 03 force displacement transducer connected to aModel 7E polygraph recording system (Grass TechnologiesQuincy MA USA) In the carotid artery rings of rats apassive stretch of 1 g was determined to be optimal tensionfor maximal responsiveness to phenylephrine (10minus6M) Thepreparationswere allowed to equilibrate for approximately 1 hwith an exchange of Krebrsquos solution every 10minThe relaxanteffects of acetylcholine (ACh 10minus9sim 10minus6M) and sodiumnitroprusside (SNP 10minus10sim 10minus5M) were studied in carotidartery rings constricted submaximally with phenylephrine(10minus6M)
Evidence-Based Complementary and Alternative Medicine 3
Table 1 Effect of EGB on body weight epididymal fat pads and blood glucose
Groups Control HF HF + EGBInitial BW (g) 2458 plusmn 76 2444 plusmn 74 2444 plusmn 90
Terminal BW (g) 4494 plusmn 289 4398 plusmn 265 4025 plusmn 221
Epididymal fat pads weight (g) 25 plusmn 07 39 plusmn 12lowastlowast
25 plusmn 05
Blood glucose (mgdL) 9463 plusmn 648 9950 plusmn 730 9670 plusmn 854
Values were expressed as mean plusmn SD (119899 = 10) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF HF high fructose HF + EGB high fructose dietwith EGB BW body weight
28 Western Blot Analysis in the Rat Aorta Liver Muscleand Fat The aorta liver muscle and fat tissues homo-genate were prepared in ice-cold buffer containing 250mMsucrose 1mM EDTA 01mM phenylmethylsufonyl fluo-ride and 20mM potassium phosphate buffer (pH 76)The homogenates were then centrifuged at 8000 rpm for10min at 4∘C and the supernatant was centrifuged at13000 rpm for 5min at 4∘C and as a cytosolic fraction forthe analysis of protein The recovered proteins were sepa-rated by 10 SDS-polyacrylamide gel electrophoresis andelectrophoresis transferred to nitrocellulose membranesMembranes were blocked by 5 BSA powder in 005Tween 20-Tris-bufferd saline (TBS-T) for 1 h The anti-bodies against ICAM-1 VCAM-1 E-selectin eNOS ET-1 (in aorta) AMPK and p-AMPK (in liver muscle andfat) were purchased from Santa Cruz Biotechnology Inc(Santa Cruz CA USA) The nitrocellulose membranes wereincubated overnight at 4∘Cwith protein antibodiesThe blotswere washed several times with TBS-T and incubated withhorseradish peroxidase-conjugated secondary antibody for1 h and then the immunoreactive bands were visualized byusing enhanced chemiluminescence (Amersham Buching-hamshire UK) The bands were analyzed densitometricallyby using a Chemi-doc image analyzer (Bio-Rad HerculesCA USA)
29 Histopathological Staining of Aorta Epididymal Fat andLiver Aortic tissues were fixed in 10 (vv) formalin in001Mphosphate buffered saline (PBS) for 2 dayswith changeof formalin solution every day to remove traces of blood fromtissue The tissue samples were dehydrated and embeddedin paraffin and then thin sections (6 120583m) of the aortic archin each group were cut and stained with hematoxylin andeosin (HampE) Epididymal fat and liver tissues were fixedby immersion in 4 paraformaldehyde for 48 h at 4∘C andincubated with 30 sucrose for 2 days Each fat and liver wasembedded in OCT compound (Tissue-Tek Sakura FinetekTorrance CA USA) frozen in liquid nitrogen and stored atminus80∘C Frozen sections were cut with a Shandon CryotomeSME (Thermo Electron Corporation Pittsburg PA USA)and placed on poly-L-lysine-coated slide Epididymal fat sec-tions were stained with HampE Liver sections were assessed byusing Oil Red O staining For quantitative histopathologicalcomparisons each section was determined by Axiovision 4ImagingArchiving software
210 Immunihistochemical Staining of Aortic Tissues Paraf-fin sections for immunohistochemical staining were placed
on poly-L-lysine-coated slide (Fisher scientific PittsburghPA USA) Slides were immunostained by InvitrogenrsquosHISOTO-STAIN-SP kits using the Labeled-(strept) Avidin-Biotin (LAB-SA) method After antigen retrieval slideswere immersed in 3 hydrogen peroxide for 10min atroom temperature to block endogenous peroxidase activ-ity and rinsed with PBS After being rinsed slides wereincubated with 10 nonimmune goat serum for 10min atroom temperature and incubated with primary antibodies ofICAM-1 VCAM-1 and E-selectin (1200 Santa Cruz CAUSA) in humidified chambers overnight at 4∘C All slideswere then incubated with biotinylated secondary antibodyfor 20min at room temperature and then incubated withhorseradish peroxidase-conjugated streptavidin for 20min atroom temperature Peroxidase activity was visualized by 331015840-Diaminobenzidine (DAB Novex CA) substrate-chromogensystem counterstaining with hematoxylin (Zymed CAUSA) For quantitative analysis the average score of 10sim20randomly selected area was calculated by using NIH Imageanalysis software Image J (NIH Bethesda MD USA)
211 Statistical Analysis All the experiments were repeatedat least three timesThe results were expressed as amean plusmnSDormean plusmnSEThe data was analyzed using SIGMAPLOT 100program The Studentrsquos t-test was used to determine any sig-nificant differences 119875 lt 005 was considered as statisticallysignificant
3 Results
31 Characteristics of Experimental Animals During theentire experimental period all groups showed significantincrease in body weight There was no significant changein body weight after 8 weeks of fructose feeding in HFgroup However treatment of EGB group showed significantdecrease in body weight (4398 plusmn 265 versus 4025 plusmn 221119875 lt 005) (Table 1) Moreover HF diet results in a significantincrease in epididymal fat padsweightTheweight of epididy-mal fat pads was 608 plusmn 174 higher than that of the HFdiet group compared with control group However treatmentof EGB group significantly reduced the epididymal fat padsweight (575 plusmn 73) compared with HF diet group (Table 1)
32 Effect of EGB on Blood Pressure At the beginning ofthe experimental feeding period the levels of systolic bloodpressure in all groups were approximately 95sim100mmHg asinvestigated by the tail-cuff technique After 4 weeks systolicblood pressure of HF group was significantly increased than
4 Evidence-Based Complementary and Alternative Medicine
Week0 2 4 6 8 10
Systo
lic b
lood
pre
ssur
e (m
mH
g)
0
80
100
120
140
ContHF
HF + EGB
minus2
lowastlowastlowastlowast
(a)
(min)0 30 60 90 120 150
Glu
cose
(mg
dL)
80
100
120
140
160
180
ContHF
HF + EGB
lowast
lowast
lowast
lowastlowast
(b)
Figure 1 Effects of EGB on systolic blood pressure (a) and oral glucose tolerance test (b) Values were expressed as mean plusmn SE (119899 = 10)lowast
119875 lt 005 lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
that of control group (119875 lt 001) However EGB group wassignificantly decreased than that of HF group during all theexperimental period (13671 plusmn 124 versus 1164 plusmn 121 119875 lt001) (Figure 1(a))
33 Effect of EGB on Blood Glucose Level and Oral GlucoseTolerance Test Plasma blood glucose levels were not statisti-cally different in HF diet rats with chronic treatment of EGB(Table 1) Oral glucose tolerance test was carried out to checkinsulin resistance in high-fructose diet rats after 8 weeksTheresults showed that HF diet group maintained the significantincrease in blood glucose levels at 30 60 90 (119875 lt 001) and120min (119875 lt 005) respectivelyHowever the plasma glucoselevels in treatment of EGB were significantly decreased at30 and 90min as compared with HF diet group (119875 lt 005)(Figure 1(b))
34 Effect of EGB on Plasma Lipids Group fed a HF dietdisplayed was increased plasma triglyceride levels totalcholesterol levels and LDL-c levels however treatment ofEGB group significantly decreased plasma triglyceride levels(27267 plusmn 1070 versus 17733 plusmn 596 119875 lt 005) total cho-lesterol levels (10294 plusmn 197 versus 6779 plusmn 58 119875 lt 001)and LDL-c levels (4456 plusmn 81 versus 2428 plusmn 31 119875 lt 001)respectively Beside the plasma levels of HDL-c levels in EGBgroup increased compared with HF diet group (1602 plusmn 29versus 202 plusmn 22 119875 lt 005) (Table 2)
35 Effect of EGB on Vascular Tension Vascular responsesto ACh endothelium-dependent vasodilator (1 times 10minus9 to 1times 10minus6M) SNP and endothelium-independent vasodilator(1 times 10minus10 to 1 times 10minus7M) were measured in carotid arteryResponses to ACh-induced relaxation of carotid artery rings
were significantly decreased in the HF diet group comparedwith control group (1 times 10minus75 to 1 times 10minus6M 119875 lt 005)However the impairment of vasorelaxation was remarkablyattenuated by treatment with EGB (1 times 10minus85 to 1 times 10minus65M119875 lt 001 1 times 10minus6M 119875 lt 005) (Figure 2(a)) On theother hand response to SNP-induced relaxation of carotidartery rings had no significant difference in all the groups(Figure 2(b))
36 Effect of EGB on the Morphology of Aorta and EpididymalFat Pads EGB effectively decreased blood pressure andattenuated impairment of vasorelaxationThus we examinedhistological changes by staining with HampE in thoracic aortaFigure 3 showed that thoracic aorta of HF diet group revealedroughened endothelial layers and increased tunica intima-media of layers compared with control group (+2413119875 lt 001) However treatment of EGB group significantlymaintained the smooth character of the intima endotheliallayers and decreased tunica intima-media thickness in aorticsection (minus1610 119875 lt 001) (Figures 3(a) and 3(c))
Because EGB effectively reduced the epididymal fat padsweight we prepared frozen section of epididymal fat pads andstained with HampEThe adipocytes were hypertrophy inducedby HF diet compared with control group (+4097 119875 lt001) However treatment of EGB significantly decreased thehypertrophy of adipocytes (minus1304 119875 lt 005) (Figures 3(b)and 3(d))
37 Effect of EGB on the Hepatic Lipids To investigate theexistence of fat accumulation of liver in all experimentalgroups we prepared frozen section of liver and stained withOil Red O Lipid droplets were detected in HF diet groupsHowever treatment of EGB showed that the number of lipid
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Effect of EGB on plasma lipid levels
Groups Control HF HF + EGBT-Cho (mgdL) 6786 plusmn 76 10294 plusmn 197
lowastlowast
6779 plusmn 58
TG (mgdL) 8383 plusmn 164 27267 plusmn 1070lowastlowast
17733 plusmn 596
HDL-c (mgdL) 1375 plusmn 13 1602 plusmn 29 202 plusmn 22
LDL-c (mgdL) 2837 plusmn 39 4456 plusmn 81lowastlowast
2428 plusmn 31
Values were expressed as mean plusmn SD (119899 = 10) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF HF high fructose HF + EGB high-fructose dietwith EGB T-Cho total cholesterol TG triglyceride HDL-c high-density lipoprotein cholesterol LDL-c low-density lipoprotein cholesterol
60657075808590
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [ACh] (M)
lowastlowastlowast
lowast
(a)
707580859095100
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [SNP] (M)
(b)
Figure 2 Effect of EGB on relaxation of carotid arteries Cumulative concentration-response curves to acetylcholine (ACh) endothelium-dependent vasodilator (a) and sodiumnitroprusside (SNP) endothelium-independent vasodilator (b) in phenylephrine precontracted carotidarteries from experiment rats Values were expressed as mean plusmn SE (119899 = 5) lowast119875 lt 005 versus Cont 119875 lt 005 119875 lt 001 versus HF
droplets significantly decreased comparedwithHFdiet group(Figure 4)
38 Effect of EGB on the Expressions Levels of AdhesionMolecules eNOS andET-1 inAorta Protein expression levelsof VCAM-1 ICAM-1 E-selectin eNOS and ET-1 in aortawere determined by western blotting respectively Adhesionmolecules (VCAM-1 ICAM-1 and E-selectin) and ET-1 pro-tein levels were increased in theHF diet group comparedwithcontrol groupHowever treatment of EGBgroup significantlydecreased expression levels of protein compared with HFdiet group Moreover we examined the expression of eNOSlevels to evaluate vascular endothelial function The eNOSprotein levels decreased in the HF diet group compared withcontrol group However treatment of EGB group increasedexpression levels of protein compared with HF diet group(Figure 5)
Immunohistochemistry was performed to determine thedirect expression of adhesion molecules in the aortic wallAdhesion molecules expressions such as VCAM-1 ICAM-1
and E-selectinwere increased in theHFdiet group (119875 lt001)however treatment of EGB group significantly decreasedexpression levels of protein (VCAM-1 ICAM-1 119875 lt 001 E-selectin 119875 lt 005) (Figure 6)
39 Effect of EGB on the Expressions Levels of AMPK in LiverMuscle and Fat Tissues Because EGB effectively suppressedthe development of impaired glucose tolerance dyslipidemiafatty liver and endothelial dysfunction the expression ofAMPK was examined in liver muscle and fat tissues Theexpression of AMPK was significantly decreased in HFdiet group However treatment of EGB group increasedexpression levels of protein in liver muscle and fat tissues(Figure 7)
4 Discussion
Herb Acupuncture and Natural Medicine (HAN) one of themost ancient and revered forms of healing has been used todiagnose treat and prevent disease for over 3000 yearsHAN
6 Evidence-Based Complementary and Alternative Medicine
HF + EGBCont HF
(a)
(b)
(fold
of c
ontro
l)
00
05
10
15
Leng
th o
f tun
ica i
ntim
a-m
edia
(120583m
)
Cont HF HF + EGB
lowastlowast
(c)
Size
of a
dipo
se ce
lls (f
old
of co
ntro
l)
00
04
08
12
16
Cont HF HF + EGB
lowastlowast
(d)
Figure 3 Effects of EGB on aortic wall and adipocytes in HF diet rats Representative microscopic photographs of HampE stained section ofthe thoracic aorta (a) and epididymal fat pads (b) in HF diet rats Lower panel indicated the length of intima-media (c) and size of adiposecells (magnification times400) Values were expressed as mean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
HF + EGBCont HF
Figure 4 Effect of EGB on fatty liver in HF diet rats Representative microscopic photographs of Oil Red O stained section of the liver
Evidence-Based Complementary and Alternative Medicine 7
ICAM-1
VCAM-1
E-selection
eNOS
ET-1
120573-Actin
HF dietEGB (100mgkg)
minus
minus
+ +
minus +
Figure 5 Effects of EGB on the expression of adhesion moleculeseNOS andET-1 in the aorta ofHFdiet rats Each electrophoretogramis representative of the results from three individual experiments
is now used worldwide as an effective means of overcomingdisease Gastrodia elata is a well-known traditional Koreanmedicinal herb specifically for promoting blood circulationto remove blood stasis In the present study we provided theevidence for the beneficial effect of Gastrodia elata on lipidmetabolism and endothelial dysfunction in high fructose-induced metabolic syndrome rat model
Fructose is a lipogenic component its consumptionpromotes the development of atherogenic lipid profile andelevation of postprandial hypertriglycemia [15 16] In addi-tion HF diet animals develop hypertriglyceridemia obesityimpaired glucose tolerance fatty liver increased SBP andvascular remodeling [17 18] In the present study HF dietclearly increased visceral epididymal fat padsweight resultingfrom the increases in triglyceride and LDL cholesterolTreatment with EGB lowered epididymal fat pads weighttriglyceride and LDL cholesterol levels whereas it elevatedHDL cholesterol levels which assist lipid metabolism ThusEGB improves lipid metabolism by the decrease of triglyc-eride and LDL cholesterol Although increased epididymalfat pads body weight was not different from control diet andHF diet groupWe suppose that proper experimental periodsshould be longer than the present periods for 8 weeks toincrease body weight It is sure that EGB is effective in obesityin HF diet rats since EGB significantly decreased HF diet-induced increase in body weight
In addition disorder of lipid levels induced by HFdiet was associated with aortic lesion Histological analysisdemonstrated that the endothelial layers were rougher inaortic sections of HF diet rats associated with a trend towardsan increased development of atherosclerosis Intima-media
thickness of the thoracic aorta has been shown to corre-late with prognosis and extend of coronary artery disease[19] Treatment of EGB maintained smooth and soft intimaendothelial layers and decreased intima-media thickness inaortic sections of HF diet rats
Dyslipidemia impaired glucose tolerance and fatty liverare major features associated withmetabolic syndrome in HFdiet rats [19 20] Fructose induces impaired glucose tolerancevia the elevation of plasma triglyceride levels In additionprevious study demonstrated that an elevated fructose dietassociated with impaired glucose tolerance and endothelialdysfunction precedes the development of hypertension [21]Impaired glucose tolerance plays an important role in thedevelopment of such abnormalities as insulin resistance type2 diabetes and dyslipidemia [22] Similarly HF diet inducedimpaired glucose tolerance and dyslipidemia whereas treat-ment of EGB improved impaired glucose tolerance with theamelioration of dyslipidemia In addition EGB significantlysuppressed the increasing adipocyte size and fatty liverThusthese results suggest that EGB may be useful to suppress thedevelopment of atherosclerotic lesions obesity and amelio-rated lipid metabolism in metabolic syndrome model
Endothelial dysfunction plays an important role inhypertension and vascular inflammation other cardiovas-cular diseases and metabolic syndrome [23 24] In thisexperimental model the expression of ET-1 and inducibleadhesion molecules such as ICAM-1 VCAM-1 and E-selectin in the arterial wall represent a key event in thedevelopment of atherosclerosis EGB ameliorated vascularinflammation by downregulation of ET-1 as well as ICAM-1 VCAM-1 and E-selectin expressions in thoracic arotaSeveral studies have shown that lowering blood pressureand endothelial functions are related to an increase ofeNOS reactivity thereby increasing NO production rolesas a strong vasodilator [25 26] In the present study EGBupregulated eNOS levels in the aorta and recovered the HFdiet-induced impairment of endothelium-dependent vasore-laxation However endothelium-independent vasodilator-induced vasorelaxation was not affected by EGB Theseresults suggest that hypotensive effect of EGB is mediatedby endothelium-dependent NOcGMP pathway Histologicalstudy revealed that EGB suppressed vascular inflammationcompatible with the processes of atherosclerosis In factendothelial dysfunction was initially identified as impairedvasodilation to specific stimuli such as ACh or bradykinintherefore improvement of endothelial function is predictedto regulate lipid homeostasis [27] Thus antihypertensionand antivascular inflammatory effects of EGB contributeto the beneficial effects on endothelial function and lipidmetabolism in metabolic syndrome
To clarify the mechanism for EGB suppressing thedevelopment of visceral obesity impaired glucose tolerancedyslipidemia and fatty liver the study was focused on theexpression of AMP-activated protein kinase (AMPK) Thereis a strong correlation between low activation state of AMPKwith metabolic disorder associated with insulin resistancefat deposition and dyslipidemia [28ndash30] AMPK is a keyregulator of glucose and lipid metabolism In the liver andmuscle activation of AMPK results in enhanced fatty acid
8 Evidence-Based Complementary and Alternative Medicine
Dig
itize
an im
age-
VCA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
HF + EGBCont HF
lowastlowast
(a)
Dig
itize
an im
age-
ICA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
6
HF + EGBCont HF
lowastlowast
(b)
Dig
itize
an im
age-
E-se
lect
in(fo
ld o
f con
trol)
0
1
2
3
4
HF + EGBCont HF HF + EGBCont HF
lowastlowast
(c)
Figure 6 Effects of EGB on VCAM-1 (a) ICAM-1 (b) and E-selectin (c) immunoreactivity in aortic tissues of HF diet rats Representativeimmunohistochemistry (left) and quantifications (right) are shown Values were expressed as mean plusmn SE lowastlowast119875 lt 001 versus Cont 119875 lt 005119875 lt 001 versus HF
AMPK12057212
120573-Actin
HF dietEGB (100mgkg)
minusminus minus
minusminus minus
minusminus minus
++
+ + ++
+ ++
p-AMPK12057212
(a) (b) (c)
Figure 7 Effects of EGB on the expression of AMPK and p-AMPK in the liver (a) muscle (b) and fat (c) of HF diet rats Eachelectrophoretogram is representative of the results from three individual experiments
oxidation and decreased production of glucose cholesteroland triglycerides [31] Recently Misra reported that thesuspected role of AMPK appeared as a promising tool toprevent andor to treat metabolic disorders [32] Also theactivation of AMPK signaling pathway is associated with
eNOS regulation and alteration of systemic endothelin path-way in fructose diet animal models [25] AMPK is requiredfor adiponectin- thrombin- and histamine-induced eNOSphosphorylation and subsequent NO production in endothe-lium [33] However our study showed that EGB induced
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
Gastrodia elata Blume is a traditional herbal medicinein Korea China and Japan which has been used for thetreatment of headaches hypertension rheumatism and car-diovascular diseases [9] Several major physiological sub-stances have been identified from Gastrodia elata Blumesuch as gastrodin vanillyl alcohol vanillin glycoproteinp-endoxybenzyl alcohol and polysaccharides includingalpha-D-glucan [10ndash12] Our previous studies showed thatGastrodia elata Blume exhibits anti-inflammatory and anti-atherosclerotic properties by inhibiting the expression ofproinflammatory cytokines in vascular endothelial cells [1314] However the effect of ethanol extract of Gastrodia elataBlume on high-fructose (HF) diet animalmodel has not beenyet reported Thus the present study was designed to deter-mine whether an ethanol extract of Gastrodia elata Blume(EGB) improves high-fructose diet-induced lipidmetabolismand endothelial dysfunction
2 Materials and Methods
21 Preparation of Gastrodia elata Blume The Gastrodiaelata Blume was purchased from the Herbal Medicine Co-operative Association Iksan Jeonbuk Province Korea inMay 2012 A voucher specimen (no HBJ1041) was depositedin the herbarium of the Professional Graduate School ofOriental Medicine Wonkwang University Iksan JeonbukSouth Korea The dried Gastrodia elata Blume (400 g) wasextracted with 4 L of 95 ethanol at room temperaturefor 1 week The extract was filtered through Whatman no3 filter paper (Whatman International Ltd England) andconcentrated using rotary evaporator The resulting extract(12741 g) was lyophilized by using a freeze drier and retaineduntil required
22 Animal Experiments and Diet All experimental pro-cedures were carried out in accordance with the NationalInstitute of Health Guide for the Care and Use of LaboratoryAnimals and were approved by the Institutional Animal CareandUtilizationCommittee forMedical Science ofWonkwangUniversity Seven week-old male Sprague-Dawley (SD) ratswere obtained fromSamtako (Osan Korea) Rats were kept ina room automatically maintained at a temperature (23plusmn2∘C)humidity (50sim60) and 12-h lightdark cycle throughout theexperiments After 1 week of acclimatization animals wererandomly divided into three groups (119899 = 10 per group)Control group (Cont) was fed regular diet high-fructosegroup (HF) was fed 65 fructose diet (Research Diet USA)and the third group (HF + EGB) was fed with 65 fructosealong with a single dose of 100mgkgday of EGB orallyfor a period of 8 weeks The regular diet was composed of50 starch 21 protein 4 fat and standard vitamins andmineral mix The high-fructose diet was composed of 65fructose 20 protein 10 fat and standard vitamins andmineral mix
23 Blood and Tissue Sampling At the end of the experi-ments the aorta liver adipose tissue (epididymal fat pads)and muscle were separated and frozen until analysis after
being rinsed with cold saline The plasma was obtained fromthe coagulated blood by centrifugation at 3000 rpm 15minat 4∘C The separation of plasma was frozen at minus80∘C untilanalysis
24 Measurements of Blood Pressure Systolic blood pres-sure (SBP) was determined by using noninvasive tail-cuffplethysmography method and recorded with an automaticsphygmotonography (MK2000 Muromachi Kikai TokyoJapan) The systolic blood pressure (SBP) was measured atweek 1 week 3 and week 7 respectively At least sevendeterminations were made in every session Values werepresented as the mean plusmn SEM of five measurements
25 Analysis of Plasma Lipids The levels of triglyceride inplasma were measured by using commercial kits (ARKRAYInc MINAMI-KU KYOTO Japan) The levels of high-density lipoprotein (HDL)-cholesterol total cholesterol andLDL-cholesterol in plasma were measured by using HDL andLDL assay kit (E2HL-100 BioAssay Systems)
26 Estimation of Blood Glucose and Oral Glucose ToleranceTest The concentration of glucose in blood was measuredwhich was obtained from tail vein using glucometer (One-touch Ultra) and Test Strip (Life Scan Inc CA USA)respectively
The oral glucose tolerance test (OGTT) was performed2 days apart at 7 weeks For the OGTT briefly basalblood glucose concentrations were measured after 10sim12 h ofovernight food privation then the glucose solution (2 gkgbody weight) was immediately administered via oral gavageand fourth more tail vein blood samples were taken at 30 6090 and 120min after glucose administration
27 Preparation of Carotid Artery and Measurement of Vascu-lar Reactivity Thecarotid arteries of the rats were rapidly andcarefully isolated and placed into cold Krebrsquos solution of thefollowing composition (mM) NaCl 118 KCl 47 MgSO
411
KH2PO412 CaCl 15 NaHCO
325 glucose 10 and pH 74
The carotid arteries were removed to connective tissue andfat and cut into rings of approximately 3mm in length Alldissecting procedures were carried out for caring to protectthe endothelium from accidental damage The carotid arteryrings were suspended by means of two L-shaped stainless-steel wires inserted into the lumen in a tissue bath containingKrebrsquos solution at 37∘C and aerated with 95O
2and 5CO
2
The isometric forces of the rings were measured by using aGrass FT 03 force displacement transducer connected to aModel 7E polygraph recording system (Grass TechnologiesQuincy MA USA) In the carotid artery rings of rats apassive stretch of 1 g was determined to be optimal tensionfor maximal responsiveness to phenylephrine (10minus6M) Thepreparationswere allowed to equilibrate for approximately 1 hwith an exchange of Krebrsquos solution every 10minThe relaxanteffects of acetylcholine (ACh 10minus9sim 10minus6M) and sodiumnitroprusside (SNP 10minus10sim 10minus5M) were studied in carotidartery rings constricted submaximally with phenylephrine(10minus6M)
Evidence-Based Complementary and Alternative Medicine 3
Table 1 Effect of EGB on body weight epididymal fat pads and blood glucose
Groups Control HF HF + EGBInitial BW (g) 2458 plusmn 76 2444 plusmn 74 2444 plusmn 90
Terminal BW (g) 4494 plusmn 289 4398 plusmn 265 4025 plusmn 221
Epididymal fat pads weight (g) 25 plusmn 07 39 plusmn 12lowastlowast
25 plusmn 05
Blood glucose (mgdL) 9463 plusmn 648 9950 plusmn 730 9670 plusmn 854
Values were expressed as mean plusmn SD (119899 = 10) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF HF high fructose HF + EGB high fructose dietwith EGB BW body weight
28 Western Blot Analysis in the Rat Aorta Liver Muscleand Fat The aorta liver muscle and fat tissues homo-genate were prepared in ice-cold buffer containing 250mMsucrose 1mM EDTA 01mM phenylmethylsufonyl fluo-ride and 20mM potassium phosphate buffer (pH 76)The homogenates were then centrifuged at 8000 rpm for10min at 4∘C and the supernatant was centrifuged at13000 rpm for 5min at 4∘C and as a cytosolic fraction forthe analysis of protein The recovered proteins were sepa-rated by 10 SDS-polyacrylamide gel electrophoresis andelectrophoresis transferred to nitrocellulose membranesMembranes were blocked by 5 BSA powder in 005Tween 20-Tris-bufferd saline (TBS-T) for 1 h The anti-bodies against ICAM-1 VCAM-1 E-selectin eNOS ET-1 (in aorta) AMPK and p-AMPK (in liver muscle andfat) were purchased from Santa Cruz Biotechnology Inc(Santa Cruz CA USA) The nitrocellulose membranes wereincubated overnight at 4∘Cwith protein antibodiesThe blotswere washed several times with TBS-T and incubated withhorseradish peroxidase-conjugated secondary antibody for1 h and then the immunoreactive bands were visualized byusing enhanced chemiluminescence (Amersham Buching-hamshire UK) The bands were analyzed densitometricallyby using a Chemi-doc image analyzer (Bio-Rad HerculesCA USA)
29 Histopathological Staining of Aorta Epididymal Fat andLiver Aortic tissues were fixed in 10 (vv) formalin in001Mphosphate buffered saline (PBS) for 2 dayswith changeof formalin solution every day to remove traces of blood fromtissue The tissue samples were dehydrated and embeddedin paraffin and then thin sections (6 120583m) of the aortic archin each group were cut and stained with hematoxylin andeosin (HampE) Epididymal fat and liver tissues were fixedby immersion in 4 paraformaldehyde for 48 h at 4∘C andincubated with 30 sucrose for 2 days Each fat and liver wasembedded in OCT compound (Tissue-Tek Sakura FinetekTorrance CA USA) frozen in liquid nitrogen and stored atminus80∘C Frozen sections were cut with a Shandon CryotomeSME (Thermo Electron Corporation Pittsburg PA USA)and placed on poly-L-lysine-coated slide Epididymal fat sec-tions were stained with HampE Liver sections were assessed byusing Oil Red O staining For quantitative histopathologicalcomparisons each section was determined by Axiovision 4ImagingArchiving software
210 Immunihistochemical Staining of Aortic Tissues Paraf-fin sections for immunohistochemical staining were placed
on poly-L-lysine-coated slide (Fisher scientific PittsburghPA USA) Slides were immunostained by InvitrogenrsquosHISOTO-STAIN-SP kits using the Labeled-(strept) Avidin-Biotin (LAB-SA) method After antigen retrieval slideswere immersed in 3 hydrogen peroxide for 10min atroom temperature to block endogenous peroxidase activ-ity and rinsed with PBS After being rinsed slides wereincubated with 10 nonimmune goat serum for 10min atroom temperature and incubated with primary antibodies ofICAM-1 VCAM-1 and E-selectin (1200 Santa Cruz CAUSA) in humidified chambers overnight at 4∘C All slideswere then incubated with biotinylated secondary antibodyfor 20min at room temperature and then incubated withhorseradish peroxidase-conjugated streptavidin for 20min atroom temperature Peroxidase activity was visualized by 331015840-Diaminobenzidine (DAB Novex CA) substrate-chromogensystem counterstaining with hematoxylin (Zymed CAUSA) For quantitative analysis the average score of 10sim20randomly selected area was calculated by using NIH Imageanalysis software Image J (NIH Bethesda MD USA)
211 Statistical Analysis All the experiments were repeatedat least three timesThe results were expressed as amean plusmnSDormean plusmnSEThe data was analyzed using SIGMAPLOT 100program The Studentrsquos t-test was used to determine any sig-nificant differences 119875 lt 005 was considered as statisticallysignificant
3 Results
31 Characteristics of Experimental Animals During theentire experimental period all groups showed significantincrease in body weight There was no significant changein body weight after 8 weeks of fructose feeding in HFgroup However treatment of EGB group showed significantdecrease in body weight (4398 plusmn 265 versus 4025 plusmn 221119875 lt 005) (Table 1) Moreover HF diet results in a significantincrease in epididymal fat padsweightTheweight of epididy-mal fat pads was 608 plusmn 174 higher than that of the HFdiet group compared with control group However treatmentof EGB group significantly reduced the epididymal fat padsweight (575 plusmn 73) compared with HF diet group (Table 1)
32 Effect of EGB on Blood Pressure At the beginning ofthe experimental feeding period the levels of systolic bloodpressure in all groups were approximately 95sim100mmHg asinvestigated by the tail-cuff technique After 4 weeks systolicblood pressure of HF group was significantly increased than
4 Evidence-Based Complementary and Alternative Medicine
Week0 2 4 6 8 10
Systo
lic b
lood
pre
ssur
e (m
mH
g)
0
80
100
120
140
ContHF
HF + EGB
minus2
lowastlowastlowastlowast
(a)
(min)0 30 60 90 120 150
Glu
cose
(mg
dL)
80
100
120
140
160
180
ContHF
HF + EGB
lowast
lowast
lowast
lowastlowast
(b)
Figure 1 Effects of EGB on systolic blood pressure (a) and oral glucose tolerance test (b) Values were expressed as mean plusmn SE (119899 = 10)lowast
119875 lt 005 lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
that of control group (119875 lt 001) However EGB group wassignificantly decreased than that of HF group during all theexperimental period (13671 plusmn 124 versus 1164 plusmn 121 119875 lt001) (Figure 1(a))
33 Effect of EGB on Blood Glucose Level and Oral GlucoseTolerance Test Plasma blood glucose levels were not statisti-cally different in HF diet rats with chronic treatment of EGB(Table 1) Oral glucose tolerance test was carried out to checkinsulin resistance in high-fructose diet rats after 8 weeksTheresults showed that HF diet group maintained the significantincrease in blood glucose levels at 30 60 90 (119875 lt 001) and120min (119875 lt 005) respectivelyHowever the plasma glucoselevels in treatment of EGB were significantly decreased at30 and 90min as compared with HF diet group (119875 lt 005)(Figure 1(b))
34 Effect of EGB on Plasma Lipids Group fed a HF dietdisplayed was increased plasma triglyceride levels totalcholesterol levels and LDL-c levels however treatment ofEGB group significantly decreased plasma triglyceride levels(27267 plusmn 1070 versus 17733 plusmn 596 119875 lt 005) total cho-lesterol levels (10294 plusmn 197 versus 6779 plusmn 58 119875 lt 001)and LDL-c levels (4456 plusmn 81 versus 2428 plusmn 31 119875 lt 001)respectively Beside the plasma levels of HDL-c levels in EGBgroup increased compared with HF diet group (1602 plusmn 29versus 202 plusmn 22 119875 lt 005) (Table 2)
35 Effect of EGB on Vascular Tension Vascular responsesto ACh endothelium-dependent vasodilator (1 times 10minus9 to 1times 10minus6M) SNP and endothelium-independent vasodilator(1 times 10minus10 to 1 times 10minus7M) were measured in carotid arteryResponses to ACh-induced relaxation of carotid artery rings
were significantly decreased in the HF diet group comparedwith control group (1 times 10minus75 to 1 times 10minus6M 119875 lt 005)However the impairment of vasorelaxation was remarkablyattenuated by treatment with EGB (1 times 10minus85 to 1 times 10minus65M119875 lt 001 1 times 10minus6M 119875 lt 005) (Figure 2(a)) On theother hand response to SNP-induced relaxation of carotidartery rings had no significant difference in all the groups(Figure 2(b))
36 Effect of EGB on the Morphology of Aorta and EpididymalFat Pads EGB effectively decreased blood pressure andattenuated impairment of vasorelaxationThus we examinedhistological changes by staining with HampE in thoracic aortaFigure 3 showed that thoracic aorta of HF diet group revealedroughened endothelial layers and increased tunica intima-media of layers compared with control group (+2413119875 lt 001) However treatment of EGB group significantlymaintained the smooth character of the intima endotheliallayers and decreased tunica intima-media thickness in aorticsection (minus1610 119875 lt 001) (Figures 3(a) and 3(c))
Because EGB effectively reduced the epididymal fat padsweight we prepared frozen section of epididymal fat pads andstained with HampEThe adipocytes were hypertrophy inducedby HF diet compared with control group (+4097 119875 lt001) However treatment of EGB significantly decreased thehypertrophy of adipocytes (minus1304 119875 lt 005) (Figures 3(b)and 3(d))
37 Effect of EGB on the Hepatic Lipids To investigate theexistence of fat accumulation of liver in all experimentalgroups we prepared frozen section of liver and stained withOil Red O Lipid droplets were detected in HF diet groupsHowever treatment of EGB showed that the number of lipid
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Effect of EGB on plasma lipid levels
Groups Control HF HF + EGBT-Cho (mgdL) 6786 plusmn 76 10294 plusmn 197
lowastlowast
6779 plusmn 58
TG (mgdL) 8383 plusmn 164 27267 plusmn 1070lowastlowast
17733 plusmn 596
HDL-c (mgdL) 1375 plusmn 13 1602 plusmn 29 202 plusmn 22
LDL-c (mgdL) 2837 plusmn 39 4456 plusmn 81lowastlowast
2428 plusmn 31
Values were expressed as mean plusmn SD (119899 = 10) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF HF high fructose HF + EGB high-fructose dietwith EGB T-Cho total cholesterol TG triglyceride HDL-c high-density lipoprotein cholesterol LDL-c low-density lipoprotein cholesterol
60657075808590
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [ACh] (M)
lowastlowastlowast
lowast
(a)
707580859095100
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [SNP] (M)
(b)
Figure 2 Effect of EGB on relaxation of carotid arteries Cumulative concentration-response curves to acetylcholine (ACh) endothelium-dependent vasodilator (a) and sodiumnitroprusside (SNP) endothelium-independent vasodilator (b) in phenylephrine precontracted carotidarteries from experiment rats Values were expressed as mean plusmn SE (119899 = 5) lowast119875 lt 005 versus Cont 119875 lt 005 119875 lt 001 versus HF
droplets significantly decreased comparedwithHFdiet group(Figure 4)
38 Effect of EGB on the Expressions Levels of AdhesionMolecules eNOS andET-1 inAorta Protein expression levelsof VCAM-1 ICAM-1 E-selectin eNOS and ET-1 in aortawere determined by western blotting respectively Adhesionmolecules (VCAM-1 ICAM-1 and E-selectin) and ET-1 pro-tein levels were increased in theHF diet group comparedwithcontrol groupHowever treatment of EGBgroup significantlydecreased expression levels of protein compared with HFdiet group Moreover we examined the expression of eNOSlevels to evaluate vascular endothelial function The eNOSprotein levels decreased in the HF diet group compared withcontrol group However treatment of EGB group increasedexpression levels of protein compared with HF diet group(Figure 5)
Immunohistochemistry was performed to determine thedirect expression of adhesion molecules in the aortic wallAdhesion molecules expressions such as VCAM-1 ICAM-1
and E-selectinwere increased in theHFdiet group (119875 lt001)however treatment of EGB group significantly decreasedexpression levels of protein (VCAM-1 ICAM-1 119875 lt 001 E-selectin 119875 lt 005) (Figure 6)
39 Effect of EGB on the Expressions Levels of AMPK in LiverMuscle and Fat Tissues Because EGB effectively suppressedthe development of impaired glucose tolerance dyslipidemiafatty liver and endothelial dysfunction the expression ofAMPK was examined in liver muscle and fat tissues Theexpression of AMPK was significantly decreased in HFdiet group However treatment of EGB group increasedexpression levels of protein in liver muscle and fat tissues(Figure 7)
4 Discussion
Herb Acupuncture and Natural Medicine (HAN) one of themost ancient and revered forms of healing has been used todiagnose treat and prevent disease for over 3000 yearsHAN
6 Evidence-Based Complementary and Alternative Medicine
HF + EGBCont HF
(a)
(b)
(fold
of c
ontro
l)
00
05
10
15
Leng
th o
f tun
ica i
ntim
a-m
edia
(120583m
)
Cont HF HF + EGB
lowastlowast
(c)
Size
of a
dipo
se ce
lls (f
old
of co
ntro
l)
00
04
08
12
16
Cont HF HF + EGB
lowastlowast
(d)
Figure 3 Effects of EGB on aortic wall and adipocytes in HF diet rats Representative microscopic photographs of HampE stained section ofthe thoracic aorta (a) and epididymal fat pads (b) in HF diet rats Lower panel indicated the length of intima-media (c) and size of adiposecells (magnification times400) Values were expressed as mean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
HF + EGBCont HF
Figure 4 Effect of EGB on fatty liver in HF diet rats Representative microscopic photographs of Oil Red O stained section of the liver
Evidence-Based Complementary and Alternative Medicine 7
ICAM-1
VCAM-1
E-selection
eNOS
ET-1
120573-Actin
HF dietEGB (100mgkg)
minus
minus
+ +
minus +
Figure 5 Effects of EGB on the expression of adhesion moleculeseNOS andET-1 in the aorta ofHFdiet rats Each electrophoretogramis representative of the results from three individual experiments
is now used worldwide as an effective means of overcomingdisease Gastrodia elata is a well-known traditional Koreanmedicinal herb specifically for promoting blood circulationto remove blood stasis In the present study we provided theevidence for the beneficial effect of Gastrodia elata on lipidmetabolism and endothelial dysfunction in high fructose-induced metabolic syndrome rat model
Fructose is a lipogenic component its consumptionpromotes the development of atherogenic lipid profile andelevation of postprandial hypertriglycemia [15 16] In addi-tion HF diet animals develop hypertriglyceridemia obesityimpaired glucose tolerance fatty liver increased SBP andvascular remodeling [17 18] In the present study HF dietclearly increased visceral epididymal fat padsweight resultingfrom the increases in triglyceride and LDL cholesterolTreatment with EGB lowered epididymal fat pads weighttriglyceride and LDL cholesterol levels whereas it elevatedHDL cholesterol levels which assist lipid metabolism ThusEGB improves lipid metabolism by the decrease of triglyc-eride and LDL cholesterol Although increased epididymalfat pads body weight was not different from control diet andHF diet groupWe suppose that proper experimental periodsshould be longer than the present periods for 8 weeks toincrease body weight It is sure that EGB is effective in obesityin HF diet rats since EGB significantly decreased HF diet-induced increase in body weight
In addition disorder of lipid levels induced by HFdiet was associated with aortic lesion Histological analysisdemonstrated that the endothelial layers were rougher inaortic sections of HF diet rats associated with a trend towardsan increased development of atherosclerosis Intima-media
thickness of the thoracic aorta has been shown to corre-late with prognosis and extend of coronary artery disease[19] Treatment of EGB maintained smooth and soft intimaendothelial layers and decreased intima-media thickness inaortic sections of HF diet rats
Dyslipidemia impaired glucose tolerance and fatty liverare major features associated withmetabolic syndrome in HFdiet rats [19 20] Fructose induces impaired glucose tolerancevia the elevation of plasma triglyceride levels In additionprevious study demonstrated that an elevated fructose dietassociated with impaired glucose tolerance and endothelialdysfunction precedes the development of hypertension [21]Impaired glucose tolerance plays an important role in thedevelopment of such abnormalities as insulin resistance type2 diabetes and dyslipidemia [22] Similarly HF diet inducedimpaired glucose tolerance and dyslipidemia whereas treat-ment of EGB improved impaired glucose tolerance with theamelioration of dyslipidemia In addition EGB significantlysuppressed the increasing adipocyte size and fatty liverThusthese results suggest that EGB may be useful to suppress thedevelopment of atherosclerotic lesions obesity and amelio-rated lipid metabolism in metabolic syndrome model
Endothelial dysfunction plays an important role inhypertension and vascular inflammation other cardiovas-cular diseases and metabolic syndrome [23 24] In thisexperimental model the expression of ET-1 and inducibleadhesion molecules such as ICAM-1 VCAM-1 and E-selectin in the arterial wall represent a key event in thedevelopment of atherosclerosis EGB ameliorated vascularinflammation by downregulation of ET-1 as well as ICAM-1 VCAM-1 and E-selectin expressions in thoracic arotaSeveral studies have shown that lowering blood pressureand endothelial functions are related to an increase ofeNOS reactivity thereby increasing NO production rolesas a strong vasodilator [25 26] In the present study EGBupregulated eNOS levels in the aorta and recovered the HFdiet-induced impairment of endothelium-dependent vasore-laxation However endothelium-independent vasodilator-induced vasorelaxation was not affected by EGB Theseresults suggest that hypotensive effect of EGB is mediatedby endothelium-dependent NOcGMP pathway Histologicalstudy revealed that EGB suppressed vascular inflammationcompatible with the processes of atherosclerosis In factendothelial dysfunction was initially identified as impairedvasodilation to specific stimuli such as ACh or bradykinintherefore improvement of endothelial function is predictedto regulate lipid homeostasis [27] Thus antihypertensionand antivascular inflammatory effects of EGB contributeto the beneficial effects on endothelial function and lipidmetabolism in metabolic syndrome
To clarify the mechanism for EGB suppressing thedevelopment of visceral obesity impaired glucose tolerancedyslipidemia and fatty liver the study was focused on theexpression of AMP-activated protein kinase (AMPK) Thereis a strong correlation between low activation state of AMPKwith metabolic disorder associated with insulin resistancefat deposition and dyslipidemia [28ndash30] AMPK is a keyregulator of glucose and lipid metabolism In the liver andmuscle activation of AMPK results in enhanced fatty acid
8 Evidence-Based Complementary and Alternative Medicine
Dig
itize
an im
age-
VCA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
HF + EGBCont HF
lowastlowast
(a)
Dig
itize
an im
age-
ICA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
6
HF + EGBCont HF
lowastlowast
(b)
Dig
itize
an im
age-
E-se
lect
in(fo
ld o
f con
trol)
0
1
2
3
4
HF + EGBCont HF HF + EGBCont HF
lowastlowast
(c)
Figure 6 Effects of EGB on VCAM-1 (a) ICAM-1 (b) and E-selectin (c) immunoreactivity in aortic tissues of HF diet rats Representativeimmunohistochemistry (left) and quantifications (right) are shown Values were expressed as mean plusmn SE lowastlowast119875 lt 001 versus Cont 119875 lt 005119875 lt 001 versus HF
AMPK12057212
120573-Actin
HF dietEGB (100mgkg)
minusminus minus
minusminus minus
minusminus minus
++
+ + ++
+ ++
p-AMPK12057212
(a) (b) (c)
Figure 7 Effects of EGB on the expression of AMPK and p-AMPK in the liver (a) muscle (b) and fat (c) of HF diet rats Eachelectrophoretogram is representative of the results from three individual experiments
oxidation and decreased production of glucose cholesteroland triglycerides [31] Recently Misra reported that thesuspected role of AMPK appeared as a promising tool toprevent andor to treat metabolic disorders [32] Also theactivation of AMPK signaling pathway is associated with
eNOS regulation and alteration of systemic endothelin path-way in fructose diet animal models [25] AMPK is requiredfor adiponectin- thrombin- and histamine-induced eNOSphosphorylation and subsequent NO production in endothe-lium [33] However our study showed that EGB induced
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
Table 1 Effect of EGB on body weight epididymal fat pads and blood glucose
Groups Control HF HF + EGBInitial BW (g) 2458 plusmn 76 2444 plusmn 74 2444 plusmn 90
Terminal BW (g) 4494 plusmn 289 4398 plusmn 265 4025 plusmn 221
Epididymal fat pads weight (g) 25 plusmn 07 39 plusmn 12lowastlowast
25 plusmn 05
Blood glucose (mgdL) 9463 plusmn 648 9950 plusmn 730 9670 plusmn 854
Values were expressed as mean plusmn SD (119899 = 10) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF HF high fructose HF + EGB high fructose dietwith EGB BW body weight
28 Western Blot Analysis in the Rat Aorta Liver Muscleand Fat The aorta liver muscle and fat tissues homo-genate were prepared in ice-cold buffer containing 250mMsucrose 1mM EDTA 01mM phenylmethylsufonyl fluo-ride and 20mM potassium phosphate buffer (pH 76)The homogenates were then centrifuged at 8000 rpm for10min at 4∘C and the supernatant was centrifuged at13000 rpm for 5min at 4∘C and as a cytosolic fraction forthe analysis of protein The recovered proteins were sepa-rated by 10 SDS-polyacrylamide gel electrophoresis andelectrophoresis transferred to nitrocellulose membranesMembranes were blocked by 5 BSA powder in 005Tween 20-Tris-bufferd saline (TBS-T) for 1 h The anti-bodies against ICAM-1 VCAM-1 E-selectin eNOS ET-1 (in aorta) AMPK and p-AMPK (in liver muscle andfat) were purchased from Santa Cruz Biotechnology Inc(Santa Cruz CA USA) The nitrocellulose membranes wereincubated overnight at 4∘Cwith protein antibodiesThe blotswere washed several times with TBS-T and incubated withhorseradish peroxidase-conjugated secondary antibody for1 h and then the immunoreactive bands were visualized byusing enhanced chemiluminescence (Amersham Buching-hamshire UK) The bands were analyzed densitometricallyby using a Chemi-doc image analyzer (Bio-Rad HerculesCA USA)
29 Histopathological Staining of Aorta Epididymal Fat andLiver Aortic tissues were fixed in 10 (vv) formalin in001Mphosphate buffered saline (PBS) for 2 dayswith changeof formalin solution every day to remove traces of blood fromtissue The tissue samples were dehydrated and embeddedin paraffin and then thin sections (6 120583m) of the aortic archin each group were cut and stained with hematoxylin andeosin (HampE) Epididymal fat and liver tissues were fixedby immersion in 4 paraformaldehyde for 48 h at 4∘C andincubated with 30 sucrose for 2 days Each fat and liver wasembedded in OCT compound (Tissue-Tek Sakura FinetekTorrance CA USA) frozen in liquid nitrogen and stored atminus80∘C Frozen sections were cut with a Shandon CryotomeSME (Thermo Electron Corporation Pittsburg PA USA)and placed on poly-L-lysine-coated slide Epididymal fat sec-tions were stained with HampE Liver sections were assessed byusing Oil Red O staining For quantitative histopathologicalcomparisons each section was determined by Axiovision 4ImagingArchiving software
210 Immunihistochemical Staining of Aortic Tissues Paraf-fin sections for immunohistochemical staining were placed
on poly-L-lysine-coated slide (Fisher scientific PittsburghPA USA) Slides were immunostained by InvitrogenrsquosHISOTO-STAIN-SP kits using the Labeled-(strept) Avidin-Biotin (LAB-SA) method After antigen retrieval slideswere immersed in 3 hydrogen peroxide for 10min atroom temperature to block endogenous peroxidase activ-ity and rinsed with PBS After being rinsed slides wereincubated with 10 nonimmune goat serum for 10min atroom temperature and incubated with primary antibodies ofICAM-1 VCAM-1 and E-selectin (1200 Santa Cruz CAUSA) in humidified chambers overnight at 4∘C All slideswere then incubated with biotinylated secondary antibodyfor 20min at room temperature and then incubated withhorseradish peroxidase-conjugated streptavidin for 20min atroom temperature Peroxidase activity was visualized by 331015840-Diaminobenzidine (DAB Novex CA) substrate-chromogensystem counterstaining with hematoxylin (Zymed CAUSA) For quantitative analysis the average score of 10sim20randomly selected area was calculated by using NIH Imageanalysis software Image J (NIH Bethesda MD USA)
211 Statistical Analysis All the experiments were repeatedat least three timesThe results were expressed as amean plusmnSDormean plusmnSEThe data was analyzed using SIGMAPLOT 100program The Studentrsquos t-test was used to determine any sig-nificant differences 119875 lt 005 was considered as statisticallysignificant
3 Results
31 Characteristics of Experimental Animals During theentire experimental period all groups showed significantincrease in body weight There was no significant changein body weight after 8 weeks of fructose feeding in HFgroup However treatment of EGB group showed significantdecrease in body weight (4398 plusmn 265 versus 4025 plusmn 221119875 lt 005) (Table 1) Moreover HF diet results in a significantincrease in epididymal fat padsweightTheweight of epididy-mal fat pads was 608 plusmn 174 higher than that of the HFdiet group compared with control group However treatmentof EGB group significantly reduced the epididymal fat padsweight (575 plusmn 73) compared with HF diet group (Table 1)
32 Effect of EGB on Blood Pressure At the beginning ofthe experimental feeding period the levels of systolic bloodpressure in all groups were approximately 95sim100mmHg asinvestigated by the tail-cuff technique After 4 weeks systolicblood pressure of HF group was significantly increased than
4 Evidence-Based Complementary and Alternative Medicine
Week0 2 4 6 8 10
Systo
lic b
lood
pre
ssur
e (m
mH
g)
0
80
100
120
140
ContHF
HF + EGB
minus2
lowastlowastlowastlowast
(a)
(min)0 30 60 90 120 150
Glu
cose
(mg
dL)
80
100
120
140
160
180
ContHF
HF + EGB
lowast
lowast
lowast
lowastlowast
(b)
Figure 1 Effects of EGB on systolic blood pressure (a) and oral glucose tolerance test (b) Values were expressed as mean plusmn SE (119899 = 10)lowast
119875 lt 005 lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
that of control group (119875 lt 001) However EGB group wassignificantly decreased than that of HF group during all theexperimental period (13671 plusmn 124 versus 1164 plusmn 121 119875 lt001) (Figure 1(a))
33 Effect of EGB on Blood Glucose Level and Oral GlucoseTolerance Test Plasma blood glucose levels were not statisti-cally different in HF diet rats with chronic treatment of EGB(Table 1) Oral glucose tolerance test was carried out to checkinsulin resistance in high-fructose diet rats after 8 weeksTheresults showed that HF diet group maintained the significantincrease in blood glucose levels at 30 60 90 (119875 lt 001) and120min (119875 lt 005) respectivelyHowever the plasma glucoselevels in treatment of EGB were significantly decreased at30 and 90min as compared with HF diet group (119875 lt 005)(Figure 1(b))
34 Effect of EGB on Plasma Lipids Group fed a HF dietdisplayed was increased plasma triglyceride levels totalcholesterol levels and LDL-c levels however treatment ofEGB group significantly decreased plasma triglyceride levels(27267 plusmn 1070 versus 17733 plusmn 596 119875 lt 005) total cho-lesterol levels (10294 plusmn 197 versus 6779 plusmn 58 119875 lt 001)and LDL-c levels (4456 plusmn 81 versus 2428 plusmn 31 119875 lt 001)respectively Beside the plasma levels of HDL-c levels in EGBgroup increased compared with HF diet group (1602 plusmn 29versus 202 plusmn 22 119875 lt 005) (Table 2)
35 Effect of EGB on Vascular Tension Vascular responsesto ACh endothelium-dependent vasodilator (1 times 10minus9 to 1times 10minus6M) SNP and endothelium-independent vasodilator(1 times 10minus10 to 1 times 10minus7M) were measured in carotid arteryResponses to ACh-induced relaxation of carotid artery rings
were significantly decreased in the HF diet group comparedwith control group (1 times 10minus75 to 1 times 10minus6M 119875 lt 005)However the impairment of vasorelaxation was remarkablyattenuated by treatment with EGB (1 times 10minus85 to 1 times 10minus65M119875 lt 001 1 times 10minus6M 119875 lt 005) (Figure 2(a)) On theother hand response to SNP-induced relaxation of carotidartery rings had no significant difference in all the groups(Figure 2(b))
36 Effect of EGB on the Morphology of Aorta and EpididymalFat Pads EGB effectively decreased blood pressure andattenuated impairment of vasorelaxationThus we examinedhistological changes by staining with HampE in thoracic aortaFigure 3 showed that thoracic aorta of HF diet group revealedroughened endothelial layers and increased tunica intima-media of layers compared with control group (+2413119875 lt 001) However treatment of EGB group significantlymaintained the smooth character of the intima endotheliallayers and decreased tunica intima-media thickness in aorticsection (minus1610 119875 lt 001) (Figures 3(a) and 3(c))
Because EGB effectively reduced the epididymal fat padsweight we prepared frozen section of epididymal fat pads andstained with HampEThe adipocytes were hypertrophy inducedby HF diet compared with control group (+4097 119875 lt001) However treatment of EGB significantly decreased thehypertrophy of adipocytes (minus1304 119875 lt 005) (Figures 3(b)and 3(d))
37 Effect of EGB on the Hepatic Lipids To investigate theexistence of fat accumulation of liver in all experimentalgroups we prepared frozen section of liver and stained withOil Red O Lipid droplets were detected in HF diet groupsHowever treatment of EGB showed that the number of lipid
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Effect of EGB on plasma lipid levels
Groups Control HF HF + EGBT-Cho (mgdL) 6786 plusmn 76 10294 plusmn 197
lowastlowast
6779 plusmn 58
TG (mgdL) 8383 plusmn 164 27267 plusmn 1070lowastlowast
17733 plusmn 596
HDL-c (mgdL) 1375 plusmn 13 1602 plusmn 29 202 plusmn 22
LDL-c (mgdL) 2837 plusmn 39 4456 plusmn 81lowastlowast
2428 plusmn 31
Values were expressed as mean plusmn SD (119899 = 10) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF HF high fructose HF + EGB high-fructose dietwith EGB T-Cho total cholesterol TG triglyceride HDL-c high-density lipoprotein cholesterol LDL-c low-density lipoprotein cholesterol
60657075808590
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [ACh] (M)
lowastlowastlowast
lowast
(a)
707580859095100
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [SNP] (M)
(b)
Figure 2 Effect of EGB on relaxation of carotid arteries Cumulative concentration-response curves to acetylcholine (ACh) endothelium-dependent vasodilator (a) and sodiumnitroprusside (SNP) endothelium-independent vasodilator (b) in phenylephrine precontracted carotidarteries from experiment rats Values were expressed as mean plusmn SE (119899 = 5) lowast119875 lt 005 versus Cont 119875 lt 005 119875 lt 001 versus HF
droplets significantly decreased comparedwithHFdiet group(Figure 4)
38 Effect of EGB on the Expressions Levels of AdhesionMolecules eNOS andET-1 inAorta Protein expression levelsof VCAM-1 ICAM-1 E-selectin eNOS and ET-1 in aortawere determined by western blotting respectively Adhesionmolecules (VCAM-1 ICAM-1 and E-selectin) and ET-1 pro-tein levels were increased in theHF diet group comparedwithcontrol groupHowever treatment of EGBgroup significantlydecreased expression levels of protein compared with HFdiet group Moreover we examined the expression of eNOSlevels to evaluate vascular endothelial function The eNOSprotein levels decreased in the HF diet group compared withcontrol group However treatment of EGB group increasedexpression levels of protein compared with HF diet group(Figure 5)
Immunohistochemistry was performed to determine thedirect expression of adhesion molecules in the aortic wallAdhesion molecules expressions such as VCAM-1 ICAM-1
and E-selectinwere increased in theHFdiet group (119875 lt001)however treatment of EGB group significantly decreasedexpression levels of protein (VCAM-1 ICAM-1 119875 lt 001 E-selectin 119875 lt 005) (Figure 6)
39 Effect of EGB on the Expressions Levels of AMPK in LiverMuscle and Fat Tissues Because EGB effectively suppressedthe development of impaired glucose tolerance dyslipidemiafatty liver and endothelial dysfunction the expression ofAMPK was examined in liver muscle and fat tissues Theexpression of AMPK was significantly decreased in HFdiet group However treatment of EGB group increasedexpression levels of protein in liver muscle and fat tissues(Figure 7)
4 Discussion
Herb Acupuncture and Natural Medicine (HAN) one of themost ancient and revered forms of healing has been used todiagnose treat and prevent disease for over 3000 yearsHAN
6 Evidence-Based Complementary and Alternative Medicine
HF + EGBCont HF
(a)
(b)
(fold
of c
ontro
l)
00
05
10
15
Leng
th o
f tun
ica i
ntim
a-m
edia
(120583m
)
Cont HF HF + EGB
lowastlowast
(c)
Size
of a
dipo
se ce
lls (f
old
of co
ntro
l)
00
04
08
12
16
Cont HF HF + EGB
lowastlowast
(d)
Figure 3 Effects of EGB on aortic wall and adipocytes in HF diet rats Representative microscopic photographs of HampE stained section ofthe thoracic aorta (a) and epididymal fat pads (b) in HF diet rats Lower panel indicated the length of intima-media (c) and size of adiposecells (magnification times400) Values were expressed as mean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
HF + EGBCont HF
Figure 4 Effect of EGB on fatty liver in HF diet rats Representative microscopic photographs of Oil Red O stained section of the liver
Evidence-Based Complementary and Alternative Medicine 7
ICAM-1
VCAM-1
E-selection
eNOS
ET-1
120573-Actin
HF dietEGB (100mgkg)
minus
minus
+ +
minus +
Figure 5 Effects of EGB on the expression of adhesion moleculeseNOS andET-1 in the aorta ofHFdiet rats Each electrophoretogramis representative of the results from three individual experiments
is now used worldwide as an effective means of overcomingdisease Gastrodia elata is a well-known traditional Koreanmedicinal herb specifically for promoting blood circulationto remove blood stasis In the present study we provided theevidence for the beneficial effect of Gastrodia elata on lipidmetabolism and endothelial dysfunction in high fructose-induced metabolic syndrome rat model
Fructose is a lipogenic component its consumptionpromotes the development of atherogenic lipid profile andelevation of postprandial hypertriglycemia [15 16] In addi-tion HF diet animals develop hypertriglyceridemia obesityimpaired glucose tolerance fatty liver increased SBP andvascular remodeling [17 18] In the present study HF dietclearly increased visceral epididymal fat padsweight resultingfrom the increases in triglyceride and LDL cholesterolTreatment with EGB lowered epididymal fat pads weighttriglyceride and LDL cholesterol levels whereas it elevatedHDL cholesterol levels which assist lipid metabolism ThusEGB improves lipid metabolism by the decrease of triglyc-eride and LDL cholesterol Although increased epididymalfat pads body weight was not different from control diet andHF diet groupWe suppose that proper experimental periodsshould be longer than the present periods for 8 weeks toincrease body weight It is sure that EGB is effective in obesityin HF diet rats since EGB significantly decreased HF diet-induced increase in body weight
In addition disorder of lipid levels induced by HFdiet was associated with aortic lesion Histological analysisdemonstrated that the endothelial layers were rougher inaortic sections of HF diet rats associated with a trend towardsan increased development of atherosclerosis Intima-media
thickness of the thoracic aorta has been shown to corre-late with prognosis and extend of coronary artery disease[19] Treatment of EGB maintained smooth and soft intimaendothelial layers and decreased intima-media thickness inaortic sections of HF diet rats
Dyslipidemia impaired glucose tolerance and fatty liverare major features associated withmetabolic syndrome in HFdiet rats [19 20] Fructose induces impaired glucose tolerancevia the elevation of plasma triglyceride levels In additionprevious study demonstrated that an elevated fructose dietassociated with impaired glucose tolerance and endothelialdysfunction precedes the development of hypertension [21]Impaired glucose tolerance plays an important role in thedevelopment of such abnormalities as insulin resistance type2 diabetes and dyslipidemia [22] Similarly HF diet inducedimpaired glucose tolerance and dyslipidemia whereas treat-ment of EGB improved impaired glucose tolerance with theamelioration of dyslipidemia In addition EGB significantlysuppressed the increasing adipocyte size and fatty liverThusthese results suggest that EGB may be useful to suppress thedevelopment of atherosclerotic lesions obesity and amelio-rated lipid metabolism in metabolic syndrome model
Endothelial dysfunction plays an important role inhypertension and vascular inflammation other cardiovas-cular diseases and metabolic syndrome [23 24] In thisexperimental model the expression of ET-1 and inducibleadhesion molecules such as ICAM-1 VCAM-1 and E-selectin in the arterial wall represent a key event in thedevelopment of atherosclerosis EGB ameliorated vascularinflammation by downregulation of ET-1 as well as ICAM-1 VCAM-1 and E-selectin expressions in thoracic arotaSeveral studies have shown that lowering blood pressureand endothelial functions are related to an increase ofeNOS reactivity thereby increasing NO production rolesas a strong vasodilator [25 26] In the present study EGBupregulated eNOS levels in the aorta and recovered the HFdiet-induced impairment of endothelium-dependent vasore-laxation However endothelium-independent vasodilator-induced vasorelaxation was not affected by EGB Theseresults suggest that hypotensive effect of EGB is mediatedby endothelium-dependent NOcGMP pathway Histologicalstudy revealed that EGB suppressed vascular inflammationcompatible with the processes of atherosclerosis In factendothelial dysfunction was initially identified as impairedvasodilation to specific stimuli such as ACh or bradykinintherefore improvement of endothelial function is predictedto regulate lipid homeostasis [27] Thus antihypertensionand antivascular inflammatory effects of EGB contributeto the beneficial effects on endothelial function and lipidmetabolism in metabolic syndrome
To clarify the mechanism for EGB suppressing thedevelopment of visceral obesity impaired glucose tolerancedyslipidemia and fatty liver the study was focused on theexpression of AMP-activated protein kinase (AMPK) Thereis a strong correlation between low activation state of AMPKwith metabolic disorder associated with insulin resistancefat deposition and dyslipidemia [28ndash30] AMPK is a keyregulator of glucose and lipid metabolism In the liver andmuscle activation of AMPK results in enhanced fatty acid
8 Evidence-Based Complementary and Alternative Medicine
Dig
itize
an im
age-
VCA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
HF + EGBCont HF
lowastlowast
(a)
Dig
itize
an im
age-
ICA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
6
HF + EGBCont HF
lowastlowast
(b)
Dig
itize
an im
age-
E-se
lect
in(fo
ld o
f con
trol)
0
1
2
3
4
HF + EGBCont HF HF + EGBCont HF
lowastlowast
(c)
Figure 6 Effects of EGB on VCAM-1 (a) ICAM-1 (b) and E-selectin (c) immunoreactivity in aortic tissues of HF diet rats Representativeimmunohistochemistry (left) and quantifications (right) are shown Values were expressed as mean plusmn SE lowastlowast119875 lt 001 versus Cont 119875 lt 005119875 lt 001 versus HF
AMPK12057212
120573-Actin
HF dietEGB (100mgkg)
minusminus minus
minusminus minus
minusminus minus
++
+ + ++
+ ++
p-AMPK12057212
(a) (b) (c)
Figure 7 Effects of EGB on the expression of AMPK and p-AMPK in the liver (a) muscle (b) and fat (c) of HF diet rats Eachelectrophoretogram is representative of the results from three individual experiments
oxidation and decreased production of glucose cholesteroland triglycerides [31] Recently Misra reported that thesuspected role of AMPK appeared as a promising tool toprevent andor to treat metabolic disorders [32] Also theactivation of AMPK signaling pathway is associated with
eNOS regulation and alteration of systemic endothelin path-way in fructose diet animal models [25] AMPK is requiredfor adiponectin- thrombin- and histamine-induced eNOSphosphorylation and subsequent NO production in endothe-lium [33] However our study showed that EGB induced
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Evidence-Based Complementary and Alternative Medicine
Week0 2 4 6 8 10
Systo
lic b
lood
pre
ssur
e (m
mH
g)
0
80
100
120
140
ContHF
HF + EGB
minus2
lowastlowastlowastlowast
(a)
(min)0 30 60 90 120 150
Glu
cose
(mg
dL)
80
100
120
140
160
180
ContHF
HF + EGB
lowast
lowast
lowast
lowastlowast
(b)
Figure 1 Effects of EGB on systolic blood pressure (a) and oral glucose tolerance test (b) Values were expressed as mean plusmn SE (119899 = 10)lowast
119875 lt 005 lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
that of control group (119875 lt 001) However EGB group wassignificantly decreased than that of HF group during all theexperimental period (13671 plusmn 124 versus 1164 plusmn 121 119875 lt001) (Figure 1(a))
33 Effect of EGB on Blood Glucose Level and Oral GlucoseTolerance Test Plasma blood glucose levels were not statisti-cally different in HF diet rats with chronic treatment of EGB(Table 1) Oral glucose tolerance test was carried out to checkinsulin resistance in high-fructose diet rats after 8 weeksTheresults showed that HF diet group maintained the significantincrease in blood glucose levels at 30 60 90 (119875 lt 001) and120min (119875 lt 005) respectivelyHowever the plasma glucoselevels in treatment of EGB were significantly decreased at30 and 90min as compared with HF diet group (119875 lt 005)(Figure 1(b))
34 Effect of EGB on Plasma Lipids Group fed a HF dietdisplayed was increased plasma triglyceride levels totalcholesterol levels and LDL-c levels however treatment ofEGB group significantly decreased plasma triglyceride levels(27267 plusmn 1070 versus 17733 plusmn 596 119875 lt 005) total cho-lesterol levels (10294 plusmn 197 versus 6779 plusmn 58 119875 lt 001)and LDL-c levels (4456 plusmn 81 versus 2428 plusmn 31 119875 lt 001)respectively Beside the plasma levels of HDL-c levels in EGBgroup increased compared with HF diet group (1602 plusmn 29versus 202 plusmn 22 119875 lt 005) (Table 2)
35 Effect of EGB on Vascular Tension Vascular responsesto ACh endothelium-dependent vasodilator (1 times 10minus9 to 1times 10minus6M) SNP and endothelium-independent vasodilator(1 times 10minus10 to 1 times 10minus7M) were measured in carotid arteryResponses to ACh-induced relaxation of carotid artery rings
were significantly decreased in the HF diet group comparedwith control group (1 times 10minus75 to 1 times 10minus6M 119875 lt 005)However the impairment of vasorelaxation was remarkablyattenuated by treatment with EGB (1 times 10minus85 to 1 times 10minus65M119875 lt 001 1 times 10minus6M 119875 lt 005) (Figure 2(a)) On theother hand response to SNP-induced relaxation of carotidartery rings had no significant difference in all the groups(Figure 2(b))
36 Effect of EGB on the Morphology of Aorta and EpididymalFat Pads EGB effectively decreased blood pressure andattenuated impairment of vasorelaxationThus we examinedhistological changes by staining with HampE in thoracic aortaFigure 3 showed that thoracic aorta of HF diet group revealedroughened endothelial layers and increased tunica intima-media of layers compared with control group (+2413119875 lt 001) However treatment of EGB group significantlymaintained the smooth character of the intima endotheliallayers and decreased tunica intima-media thickness in aorticsection (minus1610 119875 lt 001) (Figures 3(a) and 3(c))
Because EGB effectively reduced the epididymal fat padsweight we prepared frozen section of epididymal fat pads andstained with HampEThe adipocytes were hypertrophy inducedby HF diet compared with control group (+4097 119875 lt001) However treatment of EGB significantly decreased thehypertrophy of adipocytes (minus1304 119875 lt 005) (Figures 3(b)and 3(d))
37 Effect of EGB on the Hepatic Lipids To investigate theexistence of fat accumulation of liver in all experimentalgroups we prepared frozen section of liver and stained withOil Red O Lipid droplets were detected in HF diet groupsHowever treatment of EGB showed that the number of lipid
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Effect of EGB on plasma lipid levels
Groups Control HF HF + EGBT-Cho (mgdL) 6786 plusmn 76 10294 plusmn 197
lowastlowast
6779 plusmn 58
TG (mgdL) 8383 plusmn 164 27267 plusmn 1070lowastlowast
17733 plusmn 596
HDL-c (mgdL) 1375 plusmn 13 1602 plusmn 29 202 plusmn 22
LDL-c (mgdL) 2837 plusmn 39 4456 plusmn 81lowastlowast
2428 plusmn 31
Values were expressed as mean plusmn SD (119899 = 10) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF HF high fructose HF + EGB high-fructose dietwith EGB T-Cho total cholesterol TG triglyceride HDL-c high-density lipoprotein cholesterol LDL-c low-density lipoprotein cholesterol
60657075808590
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [ACh] (M)
lowastlowastlowast
lowast
(a)
707580859095100
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [SNP] (M)
(b)
Figure 2 Effect of EGB on relaxation of carotid arteries Cumulative concentration-response curves to acetylcholine (ACh) endothelium-dependent vasodilator (a) and sodiumnitroprusside (SNP) endothelium-independent vasodilator (b) in phenylephrine precontracted carotidarteries from experiment rats Values were expressed as mean plusmn SE (119899 = 5) lowast119875 lt 005 versus Cont 119875 lt 005 119875 lt 001 versus HF
droplets significantly decreased comparedwithHFdiet group(Figure 4)
38 Effect of EGB on the Expressions Levels of AdhesionMolecules eNOS andET-1 inAorta Protein expression levelsof VCAM-1 ICAM-1 E-selectin eNOS and ET-1 in aortawere determined by western blotting respectively Adhesionmolecules (VCAM-1 ICAM-1 and E-selectin) and ET-1 pro-tein levels were increased in theHF diet group comparedwithcontrol groupHowever treatment of EGBgroup significantlydecreased expression levels of protein compared with HFdiet group Moreover we examined the expression of eNOSlevels to evaluate vascular endothelial function The eNOSprotein levels decreased in the HF diet group compared withcontrol group However treatment of EGB group increasedexpression levels of protein compared with HF diet group(Figure 5)
Immunohistochemistry was performed to determine thedirect expression of adhesion molecules in the aortic wallAdhesion molecules expressions such as VCAM-1 ICAM-1
and E-selectinwere increased in theHFdiet group (119875 lt001)however treatment of EGB group significantly decreasedexpression levels of protein (VCAM-1 ICAM-1 119875 lt 001 E-selectin 119875 lt 005) (Figure 6)
39 Effect of EGB on the Expressions Levels of AMPK in LiverMuscle and Fat Tissues Because EGB effectively suppressedthe development of impaired glucose tolerance dyslipidemiafatty liver and endothelial dysfunction the expression ofAMPK was examined in liver muscle and fat tissues Theexpression of AMPK was significantly decreased in HFdiet group However treatment of EGB group increasedexpression levels of protein in liver muscle and fat tissues(Figure 7)
4 Discussion
Herb Acupuncture and Natural Medicine (HAN) one of themost ancient and revered forms of healing has been used todiagnose treat and prevent disease for over 3000 yearsHAN
6 Evidence-Based Complementary and Alternative Medicine
HF + EGBCont HF
(a)
(b)
(fold
of c
ontro
l)
00
05
10
15
Leng
th o
f tun
ica i
ntim
a-m
edia
(120583m
)
Cont HF HF + EGB
lowastlowast
(c)
Size
of a
dipo
se ce
lls (f
old
of co
ntro
l)
00
04
08
12
16
Cont HF HF + EGB
lowastlowast
(d)
Figure 3 Effects of EGB on aortic wall and adipocytes in HF diet rats Representative microscopic photographs of HampE stained section ofthe thoracic aorta (a) and epididymal fat pads (b) in HF diet rats Lower panel indicated the length of intima-media (c) and size of adiposecells (magnification times400) Values were expressed as mean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
HF + EGBCont HF
Figure 4 Effect of EGB on fatty liver in HF diet rats Representative microscopic photographs of Oil Red O stained section of the liver
Evidence-Based Complementary and Alternative Medicine 7
ICAM-1
VCAM-1
E-selection
eNOS
ET-1
120573-Actin
HF dietEGB (100mgkg)
minus
minus
+ +
minus +
Figure 5 Effects of EGB on the expression of adhesion moleculeseNOS andET-1 in the aorta ofHFdiet rats Each electrophoretogramis representative of the results from three individual experiments
is now used worldwide as an effective means of overcomingdisease Gastrodia elata is a well-known traditional Koreanmedicinal herb specifically for promoting blood circulationto remove blood stasis In the present study we provided theevidence for the beneficial effect of Gastrodia elata on lipidmetabolism and endothelial dysfunction in high fructose-induced metabolic syndrome rat model
Fructose is a lipogenic component its consumptionpromotes the development of atherogenic lipid profile andelevation of postprandial hypertriglycemia [15 16] In addi-tion HF diet animals develop hypertriglyceridemia obesityimpaired glucose tolerance fatty liver increased SBP andvascular remodeling [17 18] In the present study HF dietclearly increased visceral epididymal fat padsweight resultingfrom the increases in triglyceride and LDL cholesterolTreatment with EGB lowered epididymal fat pads weighttriglyceride and LDL cholesterol levels whereas it elevatedHDL cholesterol levels which assist lipid metabolism ThusEGB improves lipid metabolism by the decrease of triglyc-eride and LDL cholesterol Although increased epididymalfat pads body weight was not different from control diet andHF diet groupWe suppose that proper experimental periodsshould be longer than the present periods for 8 weeks toincrease body weight It is sure that EGB is effective in obesityin HF diet rats since EGB significantly decreased HF diet-induced increase in body weight
In addition disorder of lipid levels induced by HFdiet was associated with aortic lesion Histological analysisdemonstrated that the endothelial layers were rougher inaortic sections of HF diet rats associated with a trend towardsan increased development of atherosclerosis Intima-media
thickness of the thoracic aorta has been shown to corre-late with prognosis and extend of coronary artery disease[19] Treatment of EGB maintained smooth and soft intimaendothelial layers and decreased intima-media thickness inaortic sections of HF diet rats
Dyslipidemia impaired glucose tolerance and fatty liverare major features associated withmetabolic syndrome in HFdiet rats [19 20] Fructose induces impaired glucose tolerancevia the elevation of plasma triglyceride levels In additionprevious study demonstrated that an elevated fructose dietassociated with impaired glucose tolerance and endothelialdysfunction precedes the development of hypertension [21]Impaired glucose tolerance plays an important role in thedevelopment of such abnormalities as insulin resistance type2 diabetes and dyslipidemia [22] Similarly HF diet inducedimpaired glucose tolerance and dyslipidemia whereas treat-ment of EGB improved impaired glucose tolerance with theamelioration of dyslipidemia In addition EGB significantlysuppressed the increasing adipocyte size and fatty liverThusthese results suggest that EGB may be useful to suppress thedevelopment of atherosclerotic lesions obesity and amelio-rated lipid metabolism in metabolic syndrome model
Endothelial dysfunction plays an important role inhypertension and vascular inflammation other cardiovas-cular diseases and metabolic syndrome [23 24] In thisexperimental model the expression of ET-1 and inducibleadhesion molecules such as ICAM-1 VCAM-1 and E-selectin in the arterial wall represent a key event in thedevelopment of atherosclerosis EGB ameliorated vascularinflammation by downregulation of ET-1 as well as ICAM-1 VCAM-1 and E-selectin expressions in thoracic arotaSeveral studies have shown that lowering blood pressureand endothelial functions are related to an increase ofeNOS reactivity thereby increasing NO production rolesas a strong vasodilator [25 26] In the present study EGBupregulated eNOS levels in the aorta and recovered the HFdiet-induced impairment of endothelium-dependent vasore-laxation However endothelium-independent vasodilator-induced vasorelaxation was not affected by EGB Theseresults suggest that hypotensive effect of EGB is mediatedby endothelium-dependent NOcGMP pathway Histologicalstudy revealed that EGB suppressed vascular inflammationcompatible with the processes of atherosclerosis In factendothelial dysfunction was initially identified as impairedvasodilation to specific stimuli such as ACh or bradykinintherefore improvement of endothelial function is predictedto regulate lipid homeostasis [27] Thus antihypertensionand antivascular inflammatory effects of EGB contributeto the beneficial effects on endothelial function and lipidmetabolism in metabolic syndrome
To clarify the mechanism for EGB suppressing thedevelopment of visceral obesity impaired glucose tolerancedyslipidemia and fatty liver the study was focused on theexpression of AMP-activated protein kinase (AMPK) Thereis a strong correlation between low activation state of AMPKwith metabolic disorder associated with insulin resistancefat deposition and dyslipidemia [28ndash30] AMPK is a keyregulator of glucose and lipid metabolism In the liver andmuscle activation of AMPK results in enhanced fatty acid
8 Evidence-Based Complementary and Alternative Medicine
Dig
itize
an im
age-
VCA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
HF + EGBCont HF
lowastlowast
(a)
Dig
itize
an im
age-
ICA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
6
HF + EGBCont HF
lowastlowast
(b)
Dig
itize
an im
age-
E-se
lect
in(fo
ld o
f con
trol)
0
1
2
3
4
HF + EGBCont HF HF + EGBCont HF
lowastlowast
(c)
Figure 6 Effects of EGB on VCAM-1 (a) ICAM-1 (b) and E-selectin (c) immunoreactivity in aortic tissues of HF diet rats Representativeimmunohistochemistry (left) and quantifications (right) are shown Values were expressed as mean plusmn SE lowastlowast119875 lt 001 versus Cont 119875 lt 005119875 lt 001 versus HF
AMPK12057212
120573-Actin
HF dietEGB (100mgkg)
minusminus minus
minusminus minus
minusminus minus
++
+ + ++
+ ++
p-AMPK12057212
(a) (b) (c)
Figure 7 Effects of EGB on the expression of AMPK and p-AMPK in the liver (a) muscle (b) and fat (c) of HF diet rats Eachelectrophoretogram is representative of the results from three individual experiments
oxidation and decreased production of glucose cholesteroland triglycerides [31] Recently Misra reported that thesuspected role of AMPK appeared as a promising tool toprevent andor to treat metabolic disorders [32] Also theactivation of AMPK signaling pathway is associated with
eNOS regulation and alteration of systemic endothelin path-way in fructose diet animal models [25] AMPK is requiredfor adiponectin- thrombin- and histamine-induced eNOSphosphorylation and subsequent NO production in endothe-lium [33] However our study showed that EGB induced
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 5
Table 2 Effect of EGB on plasma lipid levels
Groups Control HF HF + EGBT-Cho (mgdL) 6786 plusmn 76 10294 plusmn 197
lowastlowast
6779 plusmn 58
TG (mgdL) 8383 plusmn 164 27267 plusmn 1070lowastlowast
17733 plusmn 596
HDL-c (mgdL) 1375 plusmn 13 1602 plusmn 29 202 plusmn 22
LDL-c (mgdL) 2837 plusmn 39 4456 plusmn 81lowastlowast
2428 plusmn 31
Values were expressed as mean plusmn SD (119899 = 10) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF HF high fructose HF + EGB high-fructose dietwith EGB T-Cho total cholesterol TG triglyceride HDL-c high-density lipoprotein cholesterol LDL-c low-density lipoprotein cholesterol
60657075808590
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [ACh] (M)
lowastlowastlowast
lowast
(a)
707580859095100
Rela
xatio
n (
)
0
20
40
60
80
100
120
ContHF
HF + EGB
minus20
minuslog [SNP] (M)
(b)
Figure 2 Effect of EGB on relaxation of carotid arteries Cumulative concentration-response curves to acetylcholine (ACh) endothelium-dependent vasodilator (a) and sodiumnitroprusside (SNP) endothelium-independent vasodilator (b) in phenylephrine precontracted carotidarteries from experiment rats Values were expressed as mean plusmn SE (119899 = 5) lowast119875 lt 005 versus Cont 119875 lt 005 119875 lt 001 versus HF
droplets significantly decreased comparedwithHFdiet group(Figure 4)
38 Effect of EGB on the Expressions Levels of AdhesionMolecules eNOS andET-1 inAorta Protein expression levelsof VCAM-1 ICAM-1 E-selectin eNOS and ET-1 in aortawere determined by western blotting respectively Adhesionmolecules (VCAM-1 ICAM-1 and E-selectin) and ET-1 pro-tein levels were increased in theHF diet group comparedwithcontrol groupHowever treatment of EGBgroup significantlydecreased expression levels of protein compared with HFdiet group Moreover we examined the expression of eNOSlevels to evaluate vascular endothelial function The eNOSprotein levels decreased in the HF diet group compared withcontrol group However treatment of EGB group increasedexpression levels of protein compared with HF diet group(Figure 5)
Immunohistochemistry was performed to determine thedirect expression of adhesion molecules in the aortic wallAdhesion molecules expressions such as VCAM-1 ICAM-1
and E-selectinwere increased in theHFdiet group (119875 lt001)however treatment of EGB group significantly decreasedexpression levels of protein (VCAM-1 ICAM-1 119875 lt 001 E-selectin 119875 lt 005) (Figure 6)
39 Effect of EGB on the Expressions Levels of AMPK in LiverMuscle and Fat Tissues Because EGB effectively suppressedthe development of impaired glucose tolerance dyslipidemiafatty liver and endothelial dysfunction the expression ofAMPK was examined in liver muscle and fat tissues Theexpression of AMPK was significantly decreased in HFdiet group However treatment of EGB group increasedexpression levels of protein in liver muscle and fat tissues(Figure 7)
4 Discussion
Herb Acupuncture and Natural Medicine (HAN) one of themost ancient and revered forms of healing has been used todiagnose treat and prevent disease for over 3000 yearsHAN
6 Evidence-Based Complementary and Alternative Medicine
HF + EGBCont HF
(a)
(b)
(fold
of c
ontro
l)
00
05
10
15
Leng
th o
f tun
ica i
ntim
a-m
edia
(120583m
)
Cont HF HF + EGB
lowastlowast
(c)
Size
of a
dipo
se ce
lls (f
old
of co
ntro
l)
00
04
08
12
16
Cont HF HF + EGB
lowastlowast
(d)
Figure 3 Effects of EGB on aortic wall and adipocytes in HF diet rats Representative microscopic photographs of HampE stained section ofthe thoracic aorta (a) and epididymal fat pads (b) in HF diet rats Lower panel indicated the length of intima-media (c) and size of adiposecells (magnification times400) Values were expressed as mean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
HF + EGBCont HF
Figure 4 Effect of EGB on fatty liver in HF diet rats Representative microscopic photographs of Oil Red O stained section of the liver
Evidence-Based Complementary and Alternative Medicine 7
ICAM-1
VCAM-1
E-selection
eNOS
ET-1
120573-Actin
HF dietEGB (100mgkg)
minus
minus
+ +
minus +
Figure 5 Effects of EGB on the expression of adhesion moleculeseNOS andET-1 in the aorta ofHFdiet rats Each electrophoretogramis representative of the results from three individual experiments
is now used worldwide as an effective means of overcomingdisease Gastrodia elata is a well-known traditional Koreanmedicinal herb specifically for promoting blood circulationto remove blood stasis In the present study we provided theevidence for the beneficial effect of Gastrodia elata on lipidmetabolism and endothelial dysfunction in high fructose-induced metabolic syndrome rat model
Fructose is a lipogenic component its consumptionpromotes the development of atherogenic lipid profile andelevation of postprandial hypertriglycemia [15 16] In addi-tion HF diet animals develop hypertriglyceridemia obesityimpaired glucose tolerance fatty liver increased SBP andvascular remodeling [17 18] In the present study HF dietclearly increased visceral epididymal fat padsweight resultingfrom the increases in triglyceride and LDL cholesterolTreatment with EGB lowered epididymal fat pads weighttriglyceride and LDL cholesterol levels whereas it elevatedHDL cholesterol levels which assist lipid metabolism ThusEGB improves lipid metabolism by the decrease of triglyc-eride and LDL cholesterol Although increased epididymalfat pads body weight was not different from control diet andHF diet groupWe suppose that proper experimental periodsshould be longer than the present periods for 8 weeks toincrease body weight It is sure that EGB is effective in obesityin HF diet rats since EGB significantly decreased HF diet-induced increase in body weight
In addition disorder of lipid levels induced by HFdiet was associated with aortic lesion Histological analysisdemonstrated that the endothelial layers were rougher inaortic sections of HF diet rats associated with a trend towardsan increased development of atherosclerosis Intima-media
thickness of the thoracic aorta has been shown to corre-late with prognosis and extend of coronary artery disease[19] Treatment of EGB maintained smooth and soft intimaendothelial layers and decreased intima-media thickness inaortic sections of HF diet rats
Dyslipidemia impaired glucose tolerance and fatty liverare major features associated withmetabolic syndrome in HFdiet rats [19 20] Fructose induces impaired glucose tolerancevia the elevation of plasma triglyceride levels In additionprevious study demonstrated that an elevated fructose dietassociated with impaired glucose tolerance and endothelialdysfunction precedes the development of hypertension [21]Impaired glucose tolerance plays an important role in thedevelopment of such abnormalities as insulin resistance type2 diabetes and dyslipidemia [22] Similarly HF diet inducedimpaired glucose tolerance and dyslipidemia whereas treat-ment of EGB improved impaired glucose tolerance with theamelioration of dyslipidemia In addition EGB significantlysuppressed the increasing adipocyte size and fatty liverThusthese results suggest that EGB may be useful to suppress thedevelopment of atherosclerotic lesions obesity and amelio-rated lipid metabolism in metabolic syndrome model
Endothelial dysfunction plays an important role inhypertension and vascular inflammation other cardiovas-cular diseases and metabolic syndrome [23 24] In thisexperimental model the expression of ET-1 and inducibleadhesion molecules such as ICAM-1 VCAM-1 and E-selectin in the arterial wall represent a key event in thedevelopment of atherosclerosis EGB ameliorated vascularinflammation by downregulation of ET-1 as well as ICAM-1 VCAM-1 and E-selectin expressions in thoracic arotaSeveral studies have shown that lowering blood pressureand endothelial functions are related to an increase ofeNOS reactivity thereby increasing NO production rolesas a strong vasodilator [25 26] In the present study EGBupregulated eNOS levels in the aorta and recovered the HFdiet-induced impairment of endothelium-dependent vasore-laxation However endothelium-independent vasodilator-induced vasorelaxation was not affected by EGB Theseresults suggest that hypotensive effect of EGB is mediatedby endothelium-dependent NOcGMP pathway Histologicalstudy revealed that EGB suppressed vascular inflammationcompatible with the processes of atherosclerosis In factendothelial dysfunction was initially identified as impairedvasodilation to specific stimuli such as ACh or bradykinintherefore improvement of endothelial function is predictedto regulate lipid homeostasis [27] Thus antihypertensionand antivascular inflammatory effects of EGB contributeto the beneficial effects on endothelial function and lipidmetabolism in metabolic syndrome
To clarify the mechanism for EGB suppressing thedevelopment of visceral obesity impaired glucose tolerancedyslipidemia and fatty liver the study was focused on theexpression of AMP-activated protein kinase (AMPK) Thereis a strong correlation between low activation state of AMPKwith metabolic disorder associated with insulin resistancefat deposition and dyslipidemia [28ndash30] AMPK is a keyregulator of glucose and lipid metabolism In the liver andmuscle activation of AMPK results in enhanced fatty acid
8 Evidence-Based Complementary and Alternative Medicine
Dig
itize
an im
age-
VCA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
HF + EGBCont HF
lowastlowast
(a)
Dig
itize
an im
age-
ICA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
6
HF + EGBCont HF
lowastlowast
(b)
Dig
itize
an im
age-
E-se
lect
in(fo
ld o
f con
trol)
0
1
2
3
4
HF + EGBCont HF HF + EGBCont HF
lowastlowast
(c)
Figure 6 Effects of EGB on VCAM-1 (a) ICAM-1 (b) and E-selectin (c) immunoreactivity in aortic tissues of HF diet rats Representativeimmunohistochemistry (left) and quantifications (right) are shown Values were expressed as mean plusmn SE lowastlowast119875 lt 001 versus Cont 119875 lt 005119875 lt 001 versus HF
AMPK12057212
120573-Actin
HF dietEGB (100mgkg)
minusminus minus
minusminus minus
minusminus minus
++
+ + ++
+ ++
p-AMPK12057212
(a) (b) (c)
Figure 7 Effects of EGB on the expression of AMPK and p-AMPK in the liver (a) muscle (b) and fat (c) of HF diet rats Eachelectrophoretogram is representative of the results from three individual experiments
oxidation and decreased production of glucose cholesteroland triglycerides [31] Recently Misra reported that thesuspected role of AMPK appeared as a promising tool toprevent andor to treat metabolic disorders [32] Also theactivation of AMPK signaling pathway is associated with
eNOS regulation and alteration of systemic endothelin path-way in fructose diet animal models [25] AMPK is requiredfor adiponectin- thrombin- and histamine-induced eNOSphosphorylation and subsequent NO production in endothe-lium [33] However our study showed that EGB induced
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Evidence-Based Complementary and Alternative Medicine
HF + EGBCont HF
(a)
(b)
(fold
of c
ontro
l)
00
05
10
15
Leng
th o
f tun
ica i
ntim
a-m
edia
(120583m
)
Cont HF HF + EGB
lowastlowast
(c)
Size
of a
dipo
se ce
lls (f
old
of co
ntro
l)
00
04
08
12
16
Cont HF HF + EGB
lowastlowast
(d)
Figure 3 Effects of EGB on aortic wall and adipocytes in HF diet rats Representative microscopic photographs of HampE stained section ofthe thoracic aorta (a) and epididymal fat pads (b) in HF diet rats Lower panel indicated the length of intima-media (c) and size of adiposecells (magnification times400) Values were expressed as mean plusmn SE (119899 = 5) lowastlowast119875 lt 001 versus Cont 119875 lt 005 119875 lt 001 versus HF
HF + EGBCont HF
Figure 4 Effect of EGB on fatty liver in HF diet rats Representative microscopic photographs of Oil Red O stained section of the liver
Evidence-Based Complementary and Alternative Medicine 7
ICAM-1
VCAM-1
E-selection
eNOS
ET-1
120573-Actin
HF dietEGB (100mgkg)
minus
minus
+ +
minus +
Figure 5 Effects of EGB on the expression of adhesion moleculeseNOS andET-1 in the aorta ofHFdiet rats Each electrophoretogramis representative of the results from three individual experiments
is now used worldwide as an effective means of overcomingdisease Gastrodia elata is a well-known traditional Koreanmedicinal herb specifically for promoting blood circulationto remove blood stasis In the present study we provided theevidence for the beneficial effect of Gastrodia elata on lipidmetabolism and endothelial dysfunction in high fructose-induced metabolic syndrome rat model
Fructose is a lipogenic component its consumptionpromotes the development of atherogenic lipid profile andelevation of postprandial hypertriglycemia [15 16] In addi-tion HF diet animals develop hypertriglyceridemia obesityimpaired glucose tolerance fatty liver increased SBP andvascular remodeling [17 18] In the present study HF dietclearly increased visceral epididymal fat padsweight resultingfrom the increases in triglyceride and LDL cholesterolTreatment with EGB lowered epididymal fat pads weighttriglyceride and LDL cholesterol levels whereas it elevatedHDL cholesterol levels which assist lipid metabolism ThusEGB improves lipid metabolism by the decrease of triglyc-eride and LDL cholesterol Although increased epididymalfat pads body weight was not different from control diet andHF diet groupWe suppose that proper experimental periodsshould be longer than the present periods for 8 weeks toincrease body weight It is sure that EGB is effective in obesityin HF diet rats since EGB significantly decreased HF diet-induced increase in body weight
In addition disorder of lipid levels induced by HFdiet was associated with aortic lesion Histological analysisdemonstrated that the endothelial layers were rougher inaortic sections of HF diet rats associated with a trend towardsan increased development of atherosclerosis Intima-media
thickness of the thoracic aorta has been shown to corre-late with prognosis and extend of coronary artery disease[19] Treatment of EGB maintained smooth and soft intimaendothelial layers and decreased intima-media thickness inaortic sections of HF diet rats
Dyslipidemia impaired glucose tolerance and fatty liverare major features associated withmetabolic syndrome in HFdiet rats [19 20] Fructose induces impaired glucose tolerancevia the elevation of plasma triglyceride levels In additionprevious study demonstrated that an elevated fructose dietassociated with impaired glucose tolerance and endothelialdysfunction precedes the development of hypertension [21]Impaired glucose tolerance plays an important role in thedevelopment of such abnormalities as insulin resistance type2 diabetes and dyslipidemia [22] Similarly HF diet inducedimpaired glucose tolerance and dyslipidemia whereas treat-ment of EGB improved impaired glucose tolerance with theamelioration of dyslipidemia In addition EGB significantlysuppressed the increasing adipocyte size and fatty liverThusthese results suggest that EGB may be useful to suppress thedevelopment of atherosclerotic lesions obesity and amelio-rated lipid metabolism in metabolic syndrome model
Endothelial dysfunction plays an important role inhypertension and vascular inflammation other cardiovas-cular diseases and metabolic syndrome [23 24] In thisexperimental model the expression of ET-1 and inducibleadhesion molecules such as ICAM-1 VCAM-1 and E-selectin in the arterial wall represent a key event in thedevelopment of atherosclerosis EGB ameliorated vascularinflammation by downregulation of ET-1 as well as ICAM-1 VCAM-1 and E-selectin expressions in thoracic arotaSeveral studies have shown that lowering blood pressureand endothelial functions are related to an increase ofeNOS reactivity thereby increasing NO production rolesas a strong vasodilator [25 26] In the present study EGBupregulated eNOS levels in the aorta and recovered the HFdiet-induced impairment of endothelium-dependent vasore-laxation However endothelium-independent vasodilator-induced vasorelaxation was not affected by EGB Theseresults suggest that hypotensive effect of EGB is mediatedby endothelium-dependent NOcGMP pathway Histologicalstudy revealed that EGB suppressed vascular inflammationcompatible with the processes of atherosclerosis In factendothelial dysfunction was initially identified as impairedvasodilation to specific stimuli such as ACh or bradykinintherefore improvement of endothelial function is predictedto regulate lipid homeostasis [27] Thus antihypertensionand antivascular inflammatory effects of EGB contributeto the beneficial effects on endothelial function and lipidmetabolism in metabolic syndrome
To clarify the mechanism for EGB suppressing thedevelopment of visceral obesity impaired glucose tolerancedyslipidemia and fatty liver the study was focused on theexpression of AMP-activated protein kinase (AMPK) Thereis a strong correlation between low activation state of AMPKwith metabolic disorder associated with insulin resistancefat deposition and dyslipidemia [28ndash30] AMPK is a keyregulator of glucose and lipid metabolism In the liver andmuscle activation of AMPK results in enhanced fatty acid
8 Evidence-Based Complementary and Alternative Medicine
Dig
itize
an im
age-
VCA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
HF + EGBCont HF
lowastlowast
(a)
Dig
itize
an im
age-
ICA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
6
HF + EGBCont HF
lowastlowast
(b)
Dig
itize
an im
age-
E-se
lect
in(fo
ld o
f con
trol)
0
1
2
3
4
HF + EGBCont HF HF + EGBCont HF
lowastlowast
(c)
Figure 6 Effects of EGB on VCAM-1 (a) ICAM-1 (b) and E-selectin (c) immunoreactivity in aortic tissues of HF diet rats Representativeimmunohistochemistry (left) and quantifications (right) are shown Values were expressed as mean plusmn SE lowastlowast119875 lt 001 versus Cont 119875 lt 005119875 lt 001 versus HF
AMPK12057212
120573-Actin
HF dietEGB (100mgkg)
minusminus minus
minusminus minus
minusminus minus
++
+ + ++
+ ++
p-AMPK12057212
(a) (b) (c)
Figure 7 Effects of EGB on the expression of AMPK and p-AMPK in the liver (a) muscle (b) and fat (c) of HF diet rats Eachelectrophoretogram is representative of the results from three individual experiments
oxidation and decreased production of glucose cholesteroland triglycerides [31] Recently Misra reported that thesuspected role of AMPK appeared as a promising tool toprevent andor to treat metabolic disorders [32] Also theactivation of AMPK signaling pathway is associated with
eNOS regulation and alteration of systemic endothelin path-way in fructose diet animal models [25] AMPK is requiredfor adiponectin- thrombin- and histamine-induced eNOSphosphorylation and subsequent NO production in endothe-lium [33] However our study showed that EGB induced
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
ICAM-1
VCAM-1
E-selection
eNOS
ET-1
120573-Actin
HF dietEGB (100mgkg)
minus
minus
+ +
minus +
Figure 5 Effects of EGB on the expression of adhesion moleculeseNOS andET-1 in the aorta ofHFdiet rats Each electrophoretogramis representative of the results from three individual experiments
is now used worldwide as an effective means of overcomingdisease Gastrodia elata is a well-known traditional Koreanmedicinal herb specifically for promoting blood circulationto remove blood stasis In the present study we provided theevidence for the beneficial effect of Gastrodia elata on lipidmetabolism and endothelial dysfunction in high fructose-induced metabolic syndrome rat model
Fructose is a lipogenic component its consumptionpromotes the development of atherogenic lipid profile andelevation of postprandial hypertriglycemia [15 16] In addi-tion HF diet animals develop hypertriglyceridemia obesityimpaired glucose tolerance fatty liver increased SBP andvascular remodeling [17 18] In the present study HF dietclearly increased visceral epididymal fat padsweight resultingfrom the increases in triglyceride and LDL cholesterolTreatment with EGB lowered epididymal fat pads weighttriglyceride and LDL cholesterol levels whereas it elevatedHDL cholesterol levels which assist lipid metabolism ThusEGB improves lipid metabolism by the decrease of triglyc-eride and LDL cholesterol Although increased epididymalfat pads body weight was not different from control diet andHF diet groupWe suppose that proper experimental periodsshould be longer than the present periods for 8 weeks toincrease body weight It is sure that EGB is effective in obesityin HF diet rats since EGB significantly decreased HF diet-induced increase in body weight
In addition disorder of lipid levels induced by HFdiet was associated with aortic lesion Histological analysisdemonstrated that the endothelial layers were rougher inaortic sections of HF diet rats associated with a trend towardsan increased development of atherosclerosis Intima-media
thickness of the thoracic aorta has been shown to corre-late with prognosis and extend of coronary artery disease[19] Treatment of EGB maintained smooth and soft intimaendothelial layers and decreased intima-media thickness inaortic sections of HF diet rats
Dyslipidemia impaired glucose tolerance and fatty liverare major features associated withmetabolic syndrome in HFdiet rats [19 20] Fructose induces impaired glucose tolerancevia the elevation of plasma triglyceride levels In additionprevious study demonstrated that an elevated fructose dietassociated with impaired glucose tolerance and endothelialdysfunction precedes the development of hypertension [21]Impaired glucose tolerance plays an important role in thedevelopment of such abnormalities as insulin resistance type2 diabetes and dyslipidemia [22] Similarly HF diet inducedimpaired glucose tolerance and dyslipidemia whereas treat-ment of EGB improved impaired glucose tolerance with theamelioration of dyslipidemia In addition EGB significantlysuppressed the increasing adipocyte size and fatty liverThusthese results suggest that EGB may be useful to suppress thedevelopment of atherosclerotic lesions obesity and amelio-rated lipid metabolism in metabolic syndrome model
Endothelial dysfunction plays an important role inhypertension and vascular inflammation other cardiovas-cular diseases and metabolic syndrome [23 24] In thisexperimental model the expression of ET-1 and inducibleadhesion molecules such as ICAM-1 VCAM-1 and E-selectin in the arterial wall represent a key event in thedevelopment of atherosclerosis EGB ameliorated vascularinflammation by downregulation of ET-1 as well as ICAM-1 VCAM-1 and E-selectin expressions in thoracic arotaSeveral studies have shown that lowering blood pressureand endothelial functions are related to an increase ofeNOS reactivity thereby increasing NO production rolesas a strong vasodilator [25 26] In the present study EGBupregulated eNOS levels in the aorta and recovered the HFdiet-induced impairment of endothelium-dependent vasore-laxation However endothelium-independent vasodilator-induced vasorelaxation was not affected by EGB Theseresults suggest that hypotensive effect of EGB is mediatedby endothelium-dependent NOcGMP pathway Histologicalstudy revealed that EGB suppressed vascular inflammationcompatible with the processes of atherosclerosis In factendothelial dysfunction was initially identified as impairedvasodilation to specific stimuli such as ACh or bradykinintherefore improvement of endothelial function is predictedto regulate lipid homeostasis [27] Thus antihypertensionand antivascular inflammatory effects of EGB contributeto the beneficial effects on endothelial function and lipidmetabolism in metabolic syndrome
To clarify the mechanism for EGB suppressing thedevelopment of visceral obesity impaired glucose tolerancedyslipidemia and fatty liver the study was focused on theexpression of AMP-activated protein kinase (AMPK) Thereis a strong correlation between low activation state of AMPKwith metabolic disorder associated with insulin resistancefat deposition and dyslipidemia [28ndash30] AMPK is a keyregulator of glucose and lipid metabolism In the liver andmuscle activation of AMPK results in enhanced fatty acid
8 Evidence-Based Complementary and Alternative Medicine
Dig
itize
an im
age-
VCA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
HF + EGBCont HF
lowastlowast
(a)
Dig
itize
an im
age-
ICA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
6
HF + EGBCont HF
lowastlowast
(b)
Dig
itize
an im
age-
E-se
lect
in(fo
ld o
f con
trol)
0
1
2
3
4
HF + EGBCont HF HF + EGBCont HF
lowastlowast
(c)
Figure 6 Effects of EGB on VCAM-1 (a) ICAM-1 (b) and E-selectin (c) immunoreactivity in aortic tissues of HF diet rats Representativeimmunohistochemistry (left) and quantifications (right) are shown Values were expressed as mean plusmn SE lowastlowast119875 lt 001 versus Cont 119875 lt 005119875 lt 001 versus HF
AMPK12057212
120573-Actin
HF dietEGB (100mgkg)
minusminus minus
minusminus minus
minusminus minus
++
+ + ++
+ ++
p-AMPK12057212
(a) (b) (c)
Figure 7 Effects of EGB on the expression of AMPK and p-AMPK in the liver (a) muscle (b) and fat (c) of HF diet rats Eachelectrophoretogram is representative of the results from three individual experiments
oxidation and decreased production of glucose cholesteroland triglycerides [31] Recently Misra reported that thesuspected role of AMPK appeared as a promising tool toprevent andor to treat metabolic disorders [32] Also theactivation of AMPK signaling pathway is associated with
eNOS regulation and alteration of systemic endothelin path-way in fructose diet animal models [25] AMPK is requiredfor adiponectin- thrombin- and histamine-induced eNOSphosphorylation and subsequent NO production in endothe-lium [33] However our study showed that EGB induced
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Evidence-Based Complementary and Alternative Medicine
Dig
itize
an im
age-
VCA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
HF + EGBCont HF
lowastlowast
(a)
Dig
itize
an im
age-
ICA
M-1
(fold
of c
ontro
l)
0
1
2
3
4
5
6
HF + EGBCont HF
lowastlowast
(b)
Dig
itize
an im
age-
E-se
lect
in(fo
ld o
f con
trol)
0
1
2
3
4
HF + EGBCont HF HF + EGBCont HF
lowastlowast
(c)
Figure 6 Effects of EGB on VCAM-1 (a) ICAM-1 (b) and E-selectin (c) immunoreactivity in aortic tissues of HF diet rats Representativeimmunohistochemistry (left) and quantifications (right) are shown Values were expressed as mean plusmn SE lowastlowast119875 lt 001 versus Cont 119875 lt 005119875 lt 001 versus HF
AMPK12057212
120573-Actin
HF dietEGB (100mgkg)
minusminus minus
minusminus minus
minusminus minus
++
+ + ++
+ ++
p-AMPK12057212
(a) (b) (c)
Figure 7 Effects of EGB on the expression of AMPK and p-AMPK in the liver (a) muscle (b) and fat (c) of HF diet rats Eachelectrophoretogram is representative of the results from three individual experiments
oxidation and decreased production of glucose cholesteroland triglycerides [31] Recently Misra reported that thesuspected role of AMPK appeared as a promising tool toprevent andor to treat metabolic disorders [32] Also theactivation of AMPK signaling pathway is associated with
eNOS regulation and alteration of systemic endothelin path-way in fructose diet animal models [25] AMPK is requiredfor adiponectin- thrombin- and histamine-induced eNOSphosphorylation and subsequent NO production in endothe-lium [33] However our study showed that EGB induced
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 9
markedly not only activation of phosphorylation AMPK120572 inthe liver muscle and fat but also activation of eNOS levelsin aorta It could be hypothesized that EGB could lead tonovel AMPK-mediated eNOS pathways which could in turnrecover HF diet-induced metabolic disorders
5 Conclusion
These results suppose that EGB ameliorates lipidmetabolismimpaired glucose tolerance hypertension and endothelialdysfunction in HF diet-induced metabolic syndrome at leastin part via activation of AMPK and eNOSNO pathwayTherefore Gastrodia elata Blume might be a beneficial thera-peutic approach for metabolic syndrome
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was supported by the National Research Founda-tion of Korea (NRF) Grant funded by the Korea government(MSIP) (2008-0062484) and the Ministry of KnowledgeEconomy (MKE-R0002038)
References
[1] B M Egan E L Greene and T L Goodfriend ldquoInsulinresistance and cardiovascular diseaserdquoThe American Journal ofHypertension vol 14 no 6 pp S116ndashS125 2001
[2] S M Grundy ldquoObesity metabolic syndrome and coronaryatherosclerosisrdquo Circulation vol 105 no 23 pp 2696ndash26982002
[3] K L Stanhope and P J Havel ldquoFructose consumption recentresults and their potential implicationsrdquoAnnals of the New YorkAcademy of Sciences vol 1190 pp 15ndash24 2010
[4] H Basciano L Federico and K Adeli ldquoFructose insulin resis-tance and metabolic dyslipidemiardquo Nutrition and Metabolismvol 2 article 5 2005
[5] H Y Kim T Okubo L R Juneja and T Yokozawa ldquoTheprotective role of amla (Emblica officinalis Gaertn) againstfructose-induced metabolic syndrome in a rat modelrdquo BritishJournal of Nutrition vol 103 no 4 pp 502ndash512 2010
[6] X Ouyang P Cirillo Y Sautin et al ldquoFructose consumptionas a risk factor for non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 48 no 6 pp 993ndash999 2008
[7] S K Panchal and L Brown ldquoRodent models for metabolicsyndrome researchrdquo Journal of Biomedicine and Biotechnologyvol 2011 Article ID 351982 2011
[8] L FerderMD Ferder and F Inserra ldquoThe role of high-fructosecorn syrup in metabolic syndrome and hypertensionrdquo CurrentHypertension Reports vol 12 no 2 pp 105ndash112 2010
[9] W Tang and G Eisenbrand Chinese Drugs of Plant OriginChemistry Pharmacology and Use in Traditional and ModernMedicine Springer Berlin Germany 1992
[10] C-S Ding Y-S Shen G Li Z Wei and F Wei ldquoStudy of aglycoprotein from Gastrodia elata its effects of anticoagulation
and antithrombosisrdquo Zhongguo Zhongyao Zazhi vol 32 no 11pp 1060ndash1064 2007
[11] M Musharof Hossain ldquoTherapeutic orchids traditional usesand recent advances an overviewrdquo Fitoterapia vol 82 no 2 pp102ndash140 2011
[12] J-H Ha S-M Shin S-K Lee et al ldquoIn vitro effects ofhydroxybenzaldehydes from Gastrodia elata and their ana-logues on GABAergic neurotransmission and a structure-activity correlationrdquo Planta Medica vol 67 no 9 pp 877ndash8802001
[13] Y J Lee S M Hwang D G Kang J S Kim and H S LeeldquoEffect of Gastrodia elata on tumor necrosis factor-alpha-induced matrix metalloproteinase activity in endothelial cellsrdquoJournal of Natural Medicines vol 63 no 4 pp 463ndash467 2009
[14] S M Hwang Y J Lee D G Kang and H S Lee ldquoAnti-inflammatory effect of Gastrodia elata rhizome in humanumbilical vein endothelial cellsrdquo The American Journal ofChinese Medicine vol 37 no 2 pp 395ndash406 2009
[15] H Tilg and G S Hotamisligil ldquoNonalcoholic fatty liver diseasecytokine-adipokine interplay and regulation of insulin resis-tancerdquo Gastroenterology vol 131 no 3 pp 934ndash945 2006
[16] G L Kelley G Allan and S Azhar ldquoHigh dietary fructoseinduces a hepatic stress response resulting in cholesterol andlipid dysregulationrdquo Endocrinology vol 145 no 2 pp 548ndash5552004
[17] R Miatello M Vazquez N Renna M Cruzado A P Zuminoand N Risler ldquoChronic administration of resveratrol preventsbiochemical cardiovascular changes in fructose-fed ratsrdquo TheAmerican Journal of Hypertension vol 18 no 6 pp 864ndash8702005
[18] N F Renna M A Vazquez M C Lama E S Gonzalez andR M Miatello ldquoEffect of chronic aspirin administration onan experimental model of metabolic syndromerdquo Clinical andExperimental Pharmacology and Physiology vol 36 no 2 pp162ndash168 2009
[19] A Miller and K Adeli ldquoDietary fructose and the metabolicsyndromerdquo Current Opinion in Gastroenterology vol 24 no 2pp 204ndash209 2008
[20] L T Tran V G Yuen and J H McNeill ldquoThe fructose-fed rat a review on the mechanisms of fructose-inducedinsulin resistance and hypertensionrdquo Molecular and CellularBiochemistry vol 332 no 1-2 pp 145ndash159 2009
[21] T Suzuki and H Hara ldquoIngestion of guar gum hydrolysatea soluble and fermentable nondigestible saccharide improvesglucose intolerance and prevents hypertriglyceridemia in ratsfed fructoserdquo Journal of Nutrition vol 134 no 8 pp 1942ndash19472004
[22] K M Rexrode J E Manson and C H Hennekens ldquoObesityand cardiovascular diseaserdquoCurrent Opinion in Cardiology vol11 no 5 pp 490ndash495 1996
[23] C J Pepine ldquoClinical implications of endothelial dysfunctionrdquoClinical Cardiology vol 21 no 11 pp 795ndash799 1998
[24] K Tziomalos V G Athyros A Karagiannis and D P Mikhai-lidis ldquoEndothelial dysfunction in metabolic syndrome preva-lence pathogenesis and managementrdquo Nutrition Metabolismand Cardiovascular Diseases vol 20 no 2 pp 140ndash146 2010
[25] X Z Chun X Xu Y Cui et al ldquoIncreased endothelial nitric-oxide synthase expression reduces hypertension and hyperin-sulinemia in fructose-treated ratsrdquo Journal of Pharmacology andExperimental Therapeutics vol 328 no 2 pp 610ndash620 2009
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Evidence-Based Complementary and Alternative Medicine
[26] M F Mahmoud M El-Nagar and H M El-Bassossy ldquoAnti-inflammatory effect of atorvastatin on vascular reactivity andinsulin resistance in fructose fed ratsrdquo Archives of PharmacalResearch vol 35 no 1 pp 155ndash162 2012
[27] D H Endemann and E L Schiffrin ldquoNitric oxide oxidativeexcess and vascular complications of diabetesmellitusrdquoCurrentHypertension Reports vol 6 no 2 pp 85ndash89 2004
[28] W J Lee K L In S K Hyoun et al ldquo120572-lipoic acid preventsendothelial dysfunction in obese rats via activation of AMP-activated protein kinaserdquo Arteriosclerosis Thrombosis and Vas-cular Biology vol 25 no 12 pp 2488ndash2494 2005
[29] Z Luo A K Saha X Xiang and N B Ruderman ldquoAMPKthemetabolic syndrome and cancerrdquo Trends in PharmacologicalSciences vol 26 no 2 pp 69ndash76 2005
[30] J B Majithiya and R Balaraman ldquoMetformin reducesblood pressure and restores endothelial function in aorta ofstreptozotocin-induced diabetic ratsrdquo Life Sciences vol 78 no22 pp 2615ndash2624 2006
[31] P Misra and R Chakrabarti ldquoThe role of AMP kinase indiabetesrdquo Indian Journal of Medical Research vol 125 no 3 pp389ndash398 2007
[32] PMisra ldquoAMPactivated protein kinase a next generation targetfor total metabolic controlrdquo Expert Opinion on TherapeuticTargets vol 12 no 1 pp 91ndash100 2008
[33] Y Zhang T-S Lee E M Kolb et al ldquoAMP-activated proteinkinase is involved in endothelial NO synthase activation inresponse to shear stressrdquo Arteriosclerosis Thrombosis andVascular Biology vol 26 no 6 pp 1281ndash1287 2006
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom