Schisandrae Fructus Supplementation Ameliorates Sciatic ......2.8.4. GSH Content Measurement....

Research ArticleSchisandrae Fructus Supplementation Ameliorates SciaticNeurectomy-Induced Muscle Atrophy in Mice

Joo Wan Kim1 Sae-Kwang Ku2 Ki Young Kim1 Sung Goo Kim1

Min Ho Han3 Gi-Young Kim4 Hye Jin Hwang56 Byung Woo Kim57

Cheol Min Kim8 and Yung Hyun Choi35

1Research Institute Bio-Port Korea INC Marine Bio-Industry Development Center Busan 619-912 Republic of Korea2Department of Anatomy and Histology College of Korean Medicine Daegu Haany University Gyeongsan 712-715 Republic of Korea3Department of Biochemistry Dong-eui University College of Korean Medicine Busan 614-052 Republic of Korea4Laboratory of Immunobiology Department of Marine Life Sciences Jeju National University Jeju 690-756 Republic of Korea5Anti-Aging Research Center and Blue-Bio Industry RIC College of Natural Sciences amp Human EcologyDong-eui University Busan 614-714 Republic of Korea6Department of Food and Nutrition College of Natural Sciences amp Human Ecology Dong-eui UniversityBusan 614-714 Republic of Korea7Department of Life Science and Biotechnology College of Natural Sciences amp Human Ecology Dong-eui UniversityBusan 614-714 Republic of Korea8Department of Biochemistry Busan National University College of Medicine Yangsan 626-870 Republic of Korea

Correspondence should be addressed to Yung Hyun Choi choiyhdeuackr

Received 4 October 2014 Accepted 20 April 2015

Academic Editor Angel Catala

Copyright copy 2015 Joo Wan Kim 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

The objective of this study was to assess the possible beneficial skeletal muscle preserving effects of ethanol extract of SchisandraeFructus (EESF) on sciatic neurectomy- (NTX-) induced hindlimb muscle atrophy in mice Here calf muscle atrophy was inducedby unilateral right sciatic NTX In order to investigate whether administration of EESF prevents or improves sciatic NTX-inducedmuscle atrophy EESF was administered orally Our results indicated that EESF dose-dependently diminished the decreases inmarkers of muscle mass and activity levels and the increases in markers of muscle damage and fibrosis inflammatory cellinfiltration cytokines and apoptotic events in the gastrocnemius muscle bundles are induced by NTX Additionally destructionof gastrocnemius antioxidant defense systems after NTX was dose-dependently protected by treatment with EESF EESF alsoupregulatedmuscle-specificmRNAs involved inmuscle protein synthesis but downregulated those involved in protein degradationThe overall effects of 500mgkg EESF were similar to those of 50mgkg oxymetholone but it showed more favorable antioxidanteffects The present results suggested that EESF exerts a favorable ameliorating effect on muscle atrophy induced by NTX throughanti-inflammatory and antioxidant effects related to muscle fiber protective effects and via an increase in protein synthesis and adecrease in protein degradation

1 Introduction

Muscle atrophy known as a sarcopenia is defined as aloss of muscle mass resulting from a reduction in musclefiber area or density due to a decrease in muscle proteinsynthesis and an increase in protein breakdown [1 2]Many conditions are associated with muscle atrophy such

as aging denervation disuse starvation severe injury andinflammation prolonged bed rest glucocorticoid treatmentsepsis cancer and other cachectic diseases [3ndash5] Moreoverthe loss of skeletal muscle mass has a profound effect ona patientrsquos daily life especially physical activity The result-ing reduction in physical activity induces further skeletalmuscle atrophy leading to a vicious circle of the atrophic

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 872428 17 pageshttpdxdoiorg1011552015872428

2 Oxidative Medicine and Cellular Longevity

process The disuse of the muscles will also lead to disuseatrophy while neurogenic atrophy is muscle atrophy thatresults from damage to the nerve that stimulates the muscle[4 6 7]

There is a common genetic program involved in muscleproteolysis regardless of its etiology however the signalingpathways that modulate this system are distinct [6 8] It iswell established that the serum levels of proinflammatorycytokines particularly interleukin- (IL-) 1 and tumor necrosisfactor- (TNF-) 120572 are elevated in cachectic patients contribut-ing to muscle atrophy [8 9] Moreover the oxidative stressis an important inducer of muscle atrophy in both disuseand muscle cachexia [10] In addition apoptosis of musclefibers is also involved in the early phase of muscle atrophyregardless of its etiology [11 12] Up till now various animalmodels of skeletal muscle atrophy have been used in researchfields including those of denervation [13] unloading [1415] immobilization [16] starvation [17] and administrationof dexamethasone [18] Among them denervation (sciaticneurectomy NTX) causes numerous changes in contractileelectrical metabolic and molecular properties of the musclefiber membrane and sarcoplasm in hindlimb skeletal muscles[19 20] Previous studies have shown that denervation altersinterstitial spaces between muscle fibers mononucleatedcells are infiltrated into interstitial spaces of muscle fibersand fibrosis occasionally occurs in the muscle [21ndash24]These findings suggest that NTX-induced hindlimb muscleatrophy is a useful and rapid model in vivo for screeningthe agents that can reverse the abnormalities in muscleatrophy

Schisandrae Fructus the dried fruits of Schisandra chi-nensis (Turcz) Baill (Magnoliaceae) one of the most famousherbal medicines has been extensively used in Asia includingKorea China and Japanwith Russia [25 26] It was originallyused as a tonic and has been traditionally used for thetreatment of many uncomfortable symptoms such as coughdyspnea dysentery insomnia and amnesia for a long time[25 26] Previous reports have shown that SchisandraeFructusand its related compounds possess various biologicalactivities such as antioxidant anti-inflammatory antimicro-bial antiseptic antiaging hepatoprotective immunostim-ulating and anticancer effects [27ndash31] In addition recentreports have suggested that Schisandrae Fructus has favorableeffects on diabetes and it is related [32ndash34] with smoothmus-cle relaxant effects [35 36] However the effect of SchisandraeFructus on muscle atrophy has not yet been evaluated Inthe present study we assessed the possible beneficial skeletalmuscle preserving effects of ethanol extract of SchisandraeFructus (EESF) on sciatic NTX-induced hindlimb muscleatrophy in mice In this experiment calf muscle atrophy wasinduced by unilateral right sciatic NTX according to previousstudies [23 37] In order to investigate whether adminis-tration of EESF prevents or improves sciatic NTX-inducedmuscle atrophy with possible action mechanisms 125 250and 500mgkg of EESF were administered orally once a dayfor 28 days from2weeks afterNTX inmice for the therapeuticstudy

2 Materials and Methods

21 Test Substance Schisandrae Fructuswere collected aroundMungyeong- (Gyeongsangbuk-do Republic of Korea) andwashed three times with tap water before storage at minus20∘CThe frozen samples were lyophilized and homogenized usinga grinder before extractionThematerials were extractedwith20 ethanol (EESF) at room temperature for 24 h filteredand concentrated using a rotary vacuum evaporator (BuchiRotavapor R-144 BUCHI Labortechnik Flawil Switzerland)The extract was dissolved in dimethyl sulfoxide (DMSOSigma-Aldrich) as a 50mgmL stock solution and stored at4∘C until use In addition oxymetholone 17120573-hydroxy-2-hydroxymethylidene-17120572-methyl-3-androstanone (CelltrionPharm Inc Jincheon Republic of Korea) which is anorally active 17120572-alkylated anabolic-androgenic steroid firstdescribed by Ringold et al [38] was used as the referencedrug it was also stored in a refrigerator at 4∘C to protect fromlight and degeneration until use

22 Animals and Experimental Design Total 60 adultSPFVAF Outbred CrljOriCD1 (ICR) mice (Orient Bio IncSeongnam Republic of Korea) weighing 29sim32 g were usedin this experiment after allowing for 7 days of acclimati-zation The animals were allocated five per polycarbonatecage in a temperature (20ndash25∘C) and humidity (40ndash45)controlled room The light dark cycle was 12 hr 12 hr andnormal rodent pellet diet (Samyang Co Seoul Republicof Korea) and water were supplied during acclimatizationad libitum After acclimatization unilateral sciatic NTX wasperformed to induce hindlimb muscle atrophy according tothe previously established method [23 37] Briefly all of themice were anesthetized with 2 to 3 isoflurane (Hana PharmCo Hwasung Republic of Korea) in the mixture of 70N2O and 285 O

2 and anesthesia was maintained with 1

to 15 isoflurane in the mixture of 70 N2O and 285

O2 Body temperature was maintained at 370 plusmn 1∘C and

mice were subjected to operation Right sciatic neurectomywas performed by removing a 05 cm section of the nerve inthe mid thigh and a sham operation (nerve identification)was performed in the case of the sham groups as followsanimals were randomly divided into 6 groups of 10 mice eachat 2 weeks after NTX treatment sham control NTX controloxymetholone-treated group and EESF-treated groups (125250 and 500mgkg) based on the body weights (3761 plusmn163 g range 3570sim4040 g in the vehicle sham control group3724 plusmn 220 g range 3400sim4390 g in the NTX group) andcalf thicknesses (286 plusmn 011mm range 271sim300mm in thevehicle sham control group 230 plusmn 006mm range 218sim241mm in the NTX group) The dosage of oxymetholonewas selected as 50mgkg based on the previous efficacy testin mice [39 40] Prepared and stored EESF stock solution(500mgmL in DMSO) was 10- 20- or 40-fold diluted as 5025 and 125mgmL by distilled water and then orally admin-istered in a volume of 10mLkg of body weight as matchedas 500 250 and 125mgkg of body weight by oral gavagesonce a day for 28 days from2weeks afterNTX respectively Inaddition oxymetholone was also dissolve in distilled water as5mgmL concentrations and orally administered in a volume

Oxidative Medicine and Cellular Longevity 3

of 10mLkg of body weight as matched as 50mgkg of bodyweight as same period as EESF An equal volume of distilledwater was administered orally in sham and NTX controlmice instead of EESF or oxymetholoneThis experiment wasconducted according to the international regulations for theuse and welfare of laboratory animals and was approved bythe Institutional Animal Care and Use Committee of DaeguHaany University (Gyeongsan Republic of Korea) (Approvalnumber DHU2013-077)

23 BodyWeightMeasurements Bodyweights of eachmousewere measured from the day of NTX to sacrifice using anautomatic electronic balance (Precisa Instrument DietikonSwitzerland) In addition the body weight gains during thefour-week period of test substance administration were alsocalculated to reduce individual differences as follows

Body weight (g) gains during the total 4 week periodof administration = body weight at sacrifice ndash bodyweight at the first administration (2weeks afterNTX)

24 Calf and Gastrocnemius Muscle Thickness MeasurementsThe thickness of NTX-operated hind calf muscles (right side)was measured at the time of NTX 1 day before the start ofadministration first administration 1 7 14 21 and 27 daysand at sacrifice using an electronic digital caliper (MitutoyoTokyo Japan) as mmmouse levels In addition the gastroc-nemius muscle thicknesses of both sides of the hindlimbwere also measured after muscle exposure at sacrifice toreduce the differences due to surrounding tissues using thesame methods and the changes in calf thickness during thefour week period of test substance administration were alsocalculated to reduce individual differences as follows

Calf thickness changes (mm) after 4 weeks of con-tinuous administration = calf thickness at sacrifice ndashcalf thickness at the first administration (2 weeks afterNTX)

25 Gastrocnemius Muscle Weight Measurements After per-forming gastrocnemius muscle thickness measurement atsacrifice the gastrocnemius muscle masses were carefullyseparated from the tibia and fibula bones Then weightsof individual induced gastrocnemius muscle masses weremeasured at g levels (absolute wet-weights) using an auto-matic electronic balance and to reduce the differences dueto individual body weights relative weights ( of bodyweights) were also calculated using body weight at sacrificeand absolute weight as follows

Relative muscle mass weights ( of body weights) =((absolute organ weightsbody weight at sacrifice) times100)

26 Calf Muscle Strength Measurements At 1 hr after the last28th administration of vehicle oxymetholone or SF calfmuscle strengths of individual mice were measured as tensilestrengths using a computerized testing machine (SV-H1000Japan Instrumentation System Co Tokyo Japan) in Newton

(N) Briefly animals were restrained in machines using twoseparate 1-0 silk suture ties on the NTX-operated ankle andchest and the peak tensile loads were recorded as calf musclestrengths when knee angles reached 0∘ (10sim20mmdistances)in this experiment

27 Serum Biochemistry To obtain the sera for blood bio-chemistry blood samples in a separation tube were cen-trifuged at 3000 rpm for 10min on the day of necropsySerum creatine and creatine kinase (CK) levels were mea-sured using an autoanalyzer (Hemagen Analyst HemagenDiagnostic Inc Columbia MD USA) and lactate dehydro-genase (LDH) levels weremeasured using another automatedserum biochemistry analyzer (Spotchem SP-4410 ArkrayInc Tokyo Japan) respectively

28 AntioxidantDefense Systems After performingmeasure-ments of muscle mass weights gastrocnemius muscles wereseparated and the malondialdehyde (MDA) reactive oxygenspecies (ROS) and glutathione (GSH) contents catalase(CAT) and superoxide dismutase (SOD) enzyme activities inindividual muscles were assessed

281 Muscle Homogenate Preparation Separated gastrocne-mius muscles were weighed and homogenized in ice-cold001M Tris-HCl (pH 74) and then centrifuged at 12000 g for15min as described by Del Rio et al [41] Contents of totalprotein were measured by the previously described method[42] using bovine serum albumin (BSA Invitrogen CarlsbadCA USA) as the internal standard

282 Determination of Lipid Peroxidation The levels ofmuscle lipid peroxidation were determined by estimatingMDA with the thiobarbituric acid test at 525 nm absorbanceusing UVVis spectrometer (OPTIZEN POP Mecasys CoDaejeon Republic of Korea) as nM of MDAg tissue [43]

283 Determination of Reactive Oxygen Species Skeletalmuscles were homogenized and ROS level analysis was per-formed using 2101584071015840-dichlorofluorescein diacetate fluorescentdye as a probe and fluorescence density was measured at490520 nm according to manufactory guidance (ROS assaykit Abcam Cambridge MN USA) and the measured valuesof optical density (a relative fluorescence unit (RFU)) werecorrected by the protein concentrations of samples and wereexpressed as RFU120583g protein [44]

284 GSH Content Measurement Prepared homogenateswere mixed with 01mL of 25 trichloroacetic acid (MerckSan Francisco CA USA) and then centrifuged at 4200 rpmfor 40min at 4∘C GSH content was measured at 412 nmabsorbance using 2-nitrobenzoic acid (Sigma-Aldrich) asmgg tissue [45]

285 Tissue CAT Activity Decomposition of H2O2in the

presence of CAT was followed at 240 nm [46] CAT activitywas defined as the amount of enzyme required to decompose1 nM of H

2O2per minute at 25∘C and pH 78 Results were

expressed as Umg protein


Table 1 Oligonucleotides for quantitative RT-PCR used in this study

Target 51015840-31015840 Sequence Size (bp) Gene ID

Atrogin-1 Forward CAGCTTCGTGAGCGACCTC 244 67731Reverse GGCAGTCGAGAAGTCCAGTC

MuRF 1 Forward GACAGTCGCATTTCAAAGCA 194 433766Reverse GCCTAGCACTGACCTGGAAG

PI3K p85120572 Forward GCCAGTGGTCATTTGTGTTG 236 18708Reverse ACACAACCAGGGAAGTCCAG

Akt 1 Forward ATGAACGACGTAGCCATTGTG 116 11651Reverse TTGTAGCCAATAAAGGTGCCAT

Adenosine A1R Forward TGTTCCCAGGGCCTTTCAC 155 11539Reverse TAATGGACTGAGACTAGCTTGACTGGTA

TRPV4 Forward CAGGACCTCTGGAAGAGTGC 165 63873Reverse AAGAGCTAGCCTGGACACCA

Myostatin Forward CCTCCACTCCGGGAACTGA 185 17700Reverse AAGAGCCATCACTGCTGTCATC

SIRT1 Forward TTCACATTGCATGTGTGTGG 175 93759Reverse TGAGGCCCAGTGCTCTAACT

18s ribosomal RNA Forward AGCCTGAGAAACGGCTACC 252 19791Reverse TCCCAAGATCCAACTACGAG

286 Tissue SOD Activity Measurements of SOD activitieswere made according to Sun et al [47] SOD estimationwas based on the generation of superoxide radicals pro-duced by xanthine and xanthine oxidase which react withnitrotetrazolium blue to form formazan dye SOD activitywas then measured at 560 nm by the degree of inhibition ofthis reaction and was expressed as Umg protein One unit ofSOD enzymatic activity is equal to the amount of enzyme thatdiminishes the initial absorbance of nitroblue tetrazolium by50 during 1min

29 Cytokine Content Measurements Levels of IL-1120573 andTNF-120572weremeasured by sandwich enzyme-linked immuno-assay (ELISA) according to the manufacturerrsquos instructions(RampD Systems IncMinneapolisMNUSA) Approximately10ndash15mg of gastrocnemius muscle samples were homoge-nized in a tissue grinder containing 1mL of lysis buffer (PBScontaining 2mMPMSF and 1mgmL of aprotinin leupeptinand pepstatin A) as described by Clark et al [48] A 96-well plate was coated with 2 120583gmL of monoclonal anti-mouse IL-1120573 or TNF-120572 antibody at 4∘C overnight and thenblocked with 1 BSA in PBS for 1 hr The plates were washedthree times with PBS containing 02 Tween 20 Aliquots oftissue lysates were diluted to 100 120583L with Hanksrsquo balancedsalt solution with calcium and magnesium 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 1 fetalbovine serum added to the plates and then incubated for2 hrs at room temperature The plates were washed threetimes with PBS and 100 120583L aliquots of 01 120583gmL biotinylatedmouse IL-1120573 or TNF-120572 affinity-purified polyclonal antibodywas added and incubated for 2 hrs After further three washeswith PBS containing 02 Tween 20 the immune complexeswere colorimetrically detected using horseradish peroxidase-(HRP-) streptavidin conjugate The reaction was halted by

the addition of 1M H2SO4 and the absorbance at 450 nm

was measured using a microplate reader (Tecan MannedorfSwitzerland)

210 Quantitative RT-PCR RNA was extracted using Tri-zol reagent (Invitrogen Carlsbad CA USA) according tothe method recommended The RNA concentrations andquality were determined by CFX96 Real-Time System (Bio-Rad Hercules CA USA) To remove contaminating DNAsamples were treated with recombinant DNase I (DNA-free Ambion Austin TX USA) RNA was reverse tran-scribed using the reagent High-Capacity cDNA ReverseTranscriptionKit (Applied Biosystems Foster City CAUSA)according to the manufacturerrsquos instructions The internalcontrol was 18s ribosomal RNA The sequences of the PCRoligonucleotide primers are listed in Table 1

211 Histopathology Samples from gastrocnemius muscleswere separated and fixed in 10 neutral buffered forma-lin then embedded in paraffin sectioned (3sim4 120583m) andstained with Hematoxylin and eosin (HampE) for generalhistopathology [23] or Sirius red for collagen fiber [49]and after that the histopathological profiles of each samplewere observed under light microscope (Nikon Japan) Toobserve more detailed changes mean muscle fiber diameter(120583mfiber) number of inflammatory cells infiltrated into themuscle bundles (cellsmm2 of muscle bundles) and regionsoccupied by collagen fibers (mm2 of muscle bundles)were calculated for general histomorphometrical analysis ofgastrocnemius muscle samples using an automated imageanalyzer (iSolution FL ver 91 IMT iSolution Inc QuebecCanada) according to the previously described methods [1523] with some modifications


Table 2 Primary antisera and detection kits used in this study

Antisera or detection kits Code Source DilutionPrimary antisera

Anticleaved caspase-3 (Asp175) polyclonal antibody 9661 Cell Signaling Technology Inc Danvers MA USA 1 400Anticleaved PARP (Asp214) specific antibody 9545 Cell Signaling Technology Inc Danvers MA USA 1 100Anti-4-hydroxynonenal polyclonal antibody Ab46545 Abcam Cambridge UK 1 100Antinitrotyrosine polyclonal antibody 06-284 Millipore Corporation Billerica CA USA 1 200Anti-TNF-120572 (4E1) antibody sc-130349 Santa Cruz Biotechnology Burlingame CA USA 1 200Anti-COX-2 (murine) polyclonal antibody 160126 Cayman Chemical Ann Arbor MI USA 1 200Anti-iNOS (N-20) polyclonal antibody sc-651 Santa Cruz Biotechnology Burlingame CA USA 1 100Anti-GDF8myostatin antibody Ab71808 Abcam Cambridge UK 1 50

Detection kitsVectastain Elite ABC Kit PK-6200 Vector Lab Inc Burlingame CA USA 1 50Peroxidase substrate kit SK-4100 Vector Lab Inc Burlingame CA USA 1 50

212 Immunohistochemistry After deparaffinization of pre-pared gastrocnemius muscle histological paraffin sectionscitrate buffer antigen (epitope) retrieval pretreatment wasconducted as described previously [50] Briefly water bathwas preheated with staining dish containing 10mM citratebuffer (pH 60) until the temperature reached 95ndash100∘CSlides were immersed in the staining dish and the lid wasplaced loosely on the staining dish Slides were incubatedfor 20 minutes and the water bath was turned off Thestaining dish was kept at room temperature and the slideswere allowed to cool for 20 minutes After epitope retrievalsections were immunostained using avidin-biotin complex(ABC) methods for caspase-3 poly (ADP-ribose) poly-merase (PARP) nitrotyrosine 4-hydroxynonenal (4-HNE)inducible nitric oxide synthase (iNOS) cyclooxygenase-2(COX-2) TNF-120572 and myostatin (Table 2) according to theprevious study [51] Briefly endogenous peroxidase activitywas blocked by incubation in methanol and 03 H

2O2

for 30 minutes and nonspecific binding of immunoglobulinwas blocked with normal horse serum blocking solution(Vector Lab Burlingame CA USA Dilution 1 100) for1 hr in the humidity chamber Sections were treated withprimary antiserum (Table 2) overnight at 4∘C in the humiditychamber and then incubated with biotinylated universalsecondary antibody (Vector Lab Dilution 1 50) and ABCreagents (Vectastain Elite ABC Kit Vector Lab Dilution1 50) for 1 hr at room temperature in the humidity chamberand finally reacted with peroxidase substrate kit (VectorLab) for 3min at room temperature All sections were rinsedPBS for 3 times between steps The cells or muscle fibersoccupyingmore than 20 of immunoreactivities the densityof each antiserum for caspase-3 PARP nitrotyrosine 4-HNE iNOS COX-2 TNF-120572 and myostatin as comparedwith intact muscles were regarded as positive and the meannumber of caspase-3 PARP nitrotyrosine 4-HNE iNOSand myostatin immunoreactive fibers or COX-2 and TNF-120572 immunoreactive cells dispersed in the mm2 of musclebundles was counted using an automated image analysisprocess as per the established methods [52 53] with someof our modifications respectively The histopathologist was

blinded to the group distribution while performing theanalysis

213 Statistical Analyses Multiple comparison tests wereconducted for different dose groups Variance homogeneitywas examined using Levenersquos test [54] When Levenersquos testshowed no significant deviations from variance homogene-ity the obtained data were analyzed by one-way ANOVAtest followed by least-significant difference multicomparison(LSD) test to determine which pairs of group comparisonwere significantly differentWhen significant deviations fromvariance homogeneity were observed in Levenersquos test anonparametric comparison test Kruskal-Wallis 119867 test wasconducted When a significant difference was observed inthe Kruskal-Wallis 119867 test the Mann-Whitney 119880 (MW) testwas conducted to determine the specific pairs of groupcomparison which were significantly different Statisticalanalyses were conducted using SPSS for Windows (Release14 K SPSS Inc USA) [55] Differences were consideredsignificant at 119901 lt 005

3 Results

31 Effects of EESF Supplementation on BodyWeight ChangesExcept for the significant (119901 lt 005) increases in bodyweightsdetected in 250mgkg EESF-treated mice limited to the dayof sacrifice as compared with NTX vehicle control mice nosciatic NTX or test substance treatment-related meaningfulor significant changes in the bodyweights were demonstratedduring 6 weeks of the experimental period in this experiment(Figure 1) In addition there were no critical changes in bodyweight gains during the 4-week test substance administrationperiod in all oxymetholone-treated mice or EESF-treatedmice as compared with sham or NTX control mice exceptfor the significant (119901 lt 001 or 119901 lt 005) increasesin body weight gains detected in 250mgkg EESF-treatedmice as compared to those in sham and NTX control micerespectively (Table 3)


Table 3 Changes in body weight gains in mice with NTX-induced muscle atrophy

GroupsBody weights (g) Body weight gains during

the test article treatmentperiod (g) [119861 minus 119860]At NTX At 14 days after NTX

first administration [119860] Sacrifice [119861]

ControlsSham 3448 plusmn 112 3280 plusmn 126 3484 plusmn 127 204 plusmn 110NTX 3433 plusmn 098 3200 plusmn 118 3369 plusmn 162 169 plusmn 132

ReferenceOxymetholone 3471 plusmn 142 3247 plusmn 231 3458 plusmn 322 211 plusmn 132

EESF treated500mgkg 3477 plusmn 159 3282 plusmn 272 3453 plusmn 275 171 plusmn 098250mgkg 3426 plusmn 141 3265 plusmn 189 3600 plusmn 331c 335 plusmn 189ab

125mgkg 3447 plusmn 168 3263 plusmn 203 3440 plusmn 248 177 plusmn 119Values are expressed as mean plusmn SD of 10 micea119901 lt 005 as compared with sham control mice by the LSD test

b119901 lt 001 and c

119901 lt 005 as compared with NTX control mice by the LSD test

Days of administrationNTX 0 1 7 14 21 27 28

Body

wei

ghts

(g)

28

30

32

34

36

38

40

42

44

46

Sham control miceNTX control mice

a

Oxymetholone 50 mgkg treated miceEESF 500mgkg treated miceEESF 250mgkg treated miceEESF 125mgkg treated mice

minus1

Figure 1 Effects of EESF supplementation on body weight changesin mice with NTX-induced muscle atrophy Body weights of eachmouse were measured from the day of NTX to sacrifice as describedin Section 2 Values are expressed as mean plusmn SD of 10 mice (EESFSchisandrae Fructus ethanol extract NTX Sciatic neurectomy)Day minus1 means 1 day before the start of administration at 13 daysafter NTX surgery Zero means at the start of administration at14 days after NTX Twenty-eight means 28 days after the start ofadministration at sacrifice All animals were fasted overnight beforeNTX first administration and sacrifice (arrows) a

119901 lt 005 ascompared with NTX control mice by the LSD test

32 EESF Supplementation Mitigated NTX-Induced Loss ofCalf Thickness Significant (119901 lt 001) decreases in calf thick-nesses were demonstrated in NTX control mice as comparedwith sham control mice from 1 day before first administra-tion of the test substance to throughout the experimental


Calf

thic

knes

ses (

mm

)

18

20

22

24

26

28

30

32

34

36

ab

abab

a

minus1

Sham control miceNTX control miceOxymetholone 50 mgkg treated miceEESF 500mgkg treated miceEESF 250mgkg treated miceEESF 125mgkg treated mice

Figure 2 Effects of EESF supplementation on calf thickness changesin mice with NTX-induced muscle atrophy The thickness of NTXoperated hind calf (right side)wasmeasured atNTX 1 day before thestart of administration first administration 1 7 14 21 and 27 daysand sacrifice Values are expressed as mean plusmn SD of 10 mice Day minus1means 1 day before the start of administration at 13 days after NTXsurgery Zeromeans at the start of administration and at 14 days afterNTX Twenty-eight means 28 days after the start of administrationat sacrifice a119901 lt 001 as compared with sham control mice by theLSD test b119901 lt 001 as compared with NTX control mice by the LSDtest

period (Figure 2) Accordingly the calf thickness after 4weeks was significantly (119901 lt 001) decreased in NTXcontrol mice as compared with sham vehicle control mice(Table 4) However these decreases in calf thicknesseswere significantly (119901 lt 001) diminished by treatmentwith oxymetholone and EESF from 7 days after first


Table 4 Changes in the calf thickness in mice with NTX-induced muscle atrophy

GroupsCalf thicknesses (mm) Changes after the test

article treatment period(mm) [119861 minus 119860]At NTX At 14 days after NTX


ControlsSham 270 plusmn 007 285 plusmn 010 325 plusmn 012 040 plusmn 012NTX 269 plusmn 009 222 plusmn 006c 203 plusmn 011a minus019 plusmn 011a

ReferenceOxymetholone 270 plusmn 008 222 plusmn 007c 245 plusmn 010ab 023 plusmn 011ab

EESF treated500mgkg 269 plusmn 005 223 plusmn 004c 244 plusmn 012ab 021 plusmn 010ab

250mgkg 269 plusmn 007 222 plusmn 004c 233 plusmn 007ab 011 plusmn 006ab


Values are expressed as mean plusmn SD of 10 micea119901 lt 001 as compared with sham control mice by the LSD test

b119901 lt 001 as compared with NTX control mice by the LSD test

c119901 lt 001 as compared with sham control mice by the MW test

administration of the drug and the calf thickness wasrestored during the drug administration period (119901 lt 001)in these groups as compared with NTX control grouprespectively (Table 4)

33 EESF Supplementation Ameliorated NTX-Induced Lossof Gastrocnemius Muscle Thickness Significant (119901 lt 001)decreases in gastrocnemius muscle thicknesses after mus-cle exposure (through skin removal) were observed inNTX control mice as compared with sham vehicle con-trol mice (Figure 3) However significant (119901 lt 001)increases in gastrocnemius muscle thicknesses were detectedin oxymetholone-treated mice and EESF-treated mice ascompared with NTX control mice respectively

34 EESF Supplementation Ameliorated NTX-Induced Lossof Gastrocnemius Muscle Mass Weights Significant (119901 lt001) decreases in absolute wet-weight and relative weightsof gastrocnemius muscle mass were demonstrated in NTXcontrol mice as compared with sham vehicle control mice(Figure 4) However significant (119901 lt 001) increases ingastrocnemiusmusclemass were observed in oxymetholone-treated mice and EESF-treated mice as compared with NTXcontrol mice respectively

35 EESF Supplementation Attenuated NTX-Induced Loss ofCalf Muscle Strengths Significant (119901 lt 001) decreasesin tensile strengths of calf muscles were demonstrated inNTX control mice as compared with sham vehicle controlmice (Figure 5) However significant (119901 lt 001) increasesin calf muscle strengths were observed in oxymetholone-treated mice and EESF-treated mice as compared with NTXcontrol mice respectively In particular EESF-treated miceshowed dose-dependent increases in calf muscle strengths ascompared with NTX control mice

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

Gas

trocn

emiu

s mus

cle th

ickn

ess (

mm

)

00

05

10

15

20

25

30

35

a

ab abab ab

Figure 3 Effects of EESF supplementation on changes in gas-trocnemius muscle thicknesses after muscle exposure in mice withNTX-induced muscle atrophy mice The gastrocnemius musclethicknesses of both sides of the hindlimb were measured aftermuscle exposure at sacrifice Values are expressed as mean plusmn SDof 10 mice a119901 lt 001 as compared with sham control mice by theLSD test b119901 lt 001 as compared with NTX control mice by the LSDtest

36 Effects of EESF Supplementation on Serum BiochemistrySignificant (119901 lt 001) decreases in serum creatine levels andincreases in serum CK and LDH levels were demonstratedin NTX control mice as compared with sham vehicle controlmice However significant (119901 lt 001) increases in serumcreatine levels were observed in oxymetholone-treated miceand EESF-treated mice as compared with NTX control micealong with significant (119901 lt 001 or 119901 lt 005) decreases inserum CK and LDH levels respectively (Table 5)

8 Oxidative Medicine and Cellular LongevityG

astro

cnem

ius m

uscle

wei

ghts

00

01

02

03

04

05

06

07

Absolute wet-weights (g)Relative weights ( of body weights)

ab ab

ab aba

ccd cd cd cd

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

Figure 4 Effects of EESF supplementation on changes in gastroc-nemius muscle weights in mice with NTX-induced muscle atrophyAfter performing gastrocnemius muscle thickness measurement atsacrifice the gastrocnemius muscle masses were carefully separatedfrom the tibia and fibula bonesThen weights of individual inducedgastrocnemius muscle masses were measured at g levels (absolutewet-weights) and relative weights ( of body weights) were alsocalculated Values are expressed as mean plusmn SD of 10 mice a119901 lt 001as compared with sham control mice by the LSD test b

119901 lt 001

as compared with NTX control mice by the LSD test c119901 lt 001 ascompared with sham control mice by the MW test d

119901 lt 001 ascompared with NTX control mice by the MW test

37 Effects of EESF Supplementation on the GastrocnemiusMuscle Antioxidant Defense Systems Significant (119901 lt 001)increases in muscle lipid peroxidation elevation of MDAlevels and ROS content were observed in NTX control miceas compared with sham control mice (Table 6) However theelevated levels were significantly (119901 lt 001) reduced by treat-ment with EESF in a dose-dependent manner In additionthe increases in lipid peroxidation and ROS content were alsosignificantly (119901 lt 001) decreased in oxymetholone-treatedmice as compared with NTX control mice In addition asignificant (119901 lt 001) decrease in the muscle endogenousantioxidant GSH content and antioxidative enzyme (SODand CAT) activity was detected in NTX control mice ascompared with sham control mice however these decreaseswere significantly (119901 lt 001) dose-dependently diminishedby continuous oral treatment with oxymetholone and EESFfor 28 days respectively

38 Effects of EESF Supplementation on the GastrocnemiusMuscle Cytokine Content Significant (119901 lt 001) increasesin the levels of gastrocnemius muscle proinflammatorycytokines such as IL-1120573 and TNF-120572 were observed in NTXcontrol mice as compared with sham control mice (Table 7)However these elevations were significantly (119901 lt 001)

Calf

mus

cle st

reng

th (N

)

0

2

4

6

8

10

ab abab

ab

a

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

Figure 5 Effects of EESF supplementation on changes in calfmusclestrengths in mice with NTX-induced muscle atrophy mice At 1 hrafter the last 28th administration of vehicle oxymetholone or EESFcalf muscle strengths of individual mice were measured as tensilestrengths using a computerized testing machine in Newton (N)Values are expressed as mean plusmn SD of 10 mice (N = Newton)a119901 lt 001 as compared with sham control mice by the LSD test


decreased by treatment with EESF in a dose-dependentmanner In addition the IL-1120573 and TNF-120572 levels inoxymetholone-treated mice were significantly (119901 lt 001)decreased as compared with those in NTX control mice inthis experiment

39 Effects of EESF Supplementation on the GastrocnemiusMuscle mRNA Expression Levels Significantly (119901 lt 001)increased gastrocnemius muscle mRNAs expression levelsof Atrogin-1 and RING-finger protein-1 (MuRF1) which areinvolved in protein degradation myostatin a potent negativeregulator of muscle growth and Sirtuin 1 (SIRT1) a rep-resentative inhibitor of muscle regeneration were observedin NTX control mice as compared with sham controlmice (Table 8) However these elevations were significantly(119901 lt 001) dose-dependently decreased by treatment withoxymetholone and EESF as compared with those in NTXcontrol mice (Table 8) On the other hand significant (119901 lt001) decreases in mRNA expression levels of phosphatidyli-nositol 3-kinase (PI3K) Akt1 adenosine A1 receptor (A1R)and transient receptor potential cation channel subfamilyV member 4 (TRPV4) which are involved in activatingprotein synthesis and muscle growth were observed in NTXcontrol mice as compared with intact control mice Howeversignificant (119901 lt 001) increases in their expression levels weredemonstrated in EESF-treated mice as compared with NTXcontrol mice in a concentration-dependent manner

310 Effects of EESF Supplementation on the GastrocnemiusMuscle Histopathology Marked and classic muscle atrophicchanges including decline in the number of muscle fibers


Table 5 Changes in serum biochemistry in mice with NTX-induced muscle atrophy

Groups Serum levelsCreatine (mgdL) CK (IUL) LDH (IUL)

ControlsSham 039 plusmn 008 7810 plusmn 1448 48420 plusmn 14642NTX 017 plusmn 005c 24110 plusmn 4291c 178190 plusmn 18435a

ReferenceOxymetholone 028 plusmn 003cd 16130 plusmn 3746cd 108660 plusmn 21718ab

EESF treated500mgkg 028 plusmn 004cd 16200 plusmn 2906cd 108190 plusmn 23712ab

250mgkg 025 plusmn 004cd 18130 plusmn 2623cd 121260 plusmn 22403ab

125mgkg 023 plusmn 004cd 19590 plusmn 2567ce 148380 plusmn 16062ab




d119901 lt 001 and e

119901 lt 005 as compared with NTX control mice by the MW test

Table 6 Changes in the gastrocnemius muscle antioxidant defense systems in mice with NTX-induced muscle atrophy

GroupsFundus antioxidant defense systems

MDA(nMmg protein)

ROS(RFU120583g protein)

GSH(nMmg protein)

SOD(nMmimmg protein)

CAT(Umg protein)

ControlsSham 151 plusmn 041 2286 plusmn 581 067 plusmn 014 2430 plusmn 615 513 plusmn 140NTX 866 plusmn 188c 6711 plusmn 1225a 024 plusmn 007a 961 plusmn 146c 145 plusmn 057c

ReferenceOxymetholone 411 plusmn 122cd 4308 plusmn 1028ab 045 plusmn 012ab 1813 plusmn 366cd 263 plusmn 056cd

EESF treated500mgkg 438 plusmn 090cd 4235 plusmn 948ab 049 plusmn 009ab 1811 plusmn 236cd 266 plusmn 069cd

250mgkg 521 plusmn 117cd 4973 plusmn 1192ab 042 plusmn 008ab 1570 plusmn 405cd 238 plusmn 062cd

125mgkg 639 plusmn 083cd 5289 plusmn 1130ab 037 plusmn 007ab 1421 plusmn 197cd 206 plusmn 040ce




d119901 lt 001 and e


inflammatory cell infiltration and focal fibrosis in musclebundles were induced by NTX and accordingly significant(119901 lt 001) decreases in the mean muscle fiber diameterincreases in the mean number of inflammatory cells infil-trating into and percentages of regions occupied by collagenfibers in muscle bundles were detected in NTX control miceas compared with sham control mice (Table 9 and Figure 6)However these NTX-treatment related gastrocnemius mus-cle atrophic changes were dramatically and significantly (119901 lt001) reduced by treatment with EESF in a dose-dependentmanner In addition the muscle atrophic changes were alsosignificantly (119901 lt 001) diminished in oxymetholone-treated mice as compared with NTX control mice in ourexperiment

311 Effects of EESF Supplementation on the GastrocnemiusMuscle Immunohistochemistry Marked and significant (119901 lt001) increases in the apoptoticmarkers caspase-3 andPARP

immunoreactive fibers in the gastrocnemius muscle bun-dles were observed in NTX control mice however EESFdose-dependently and significantly (119901 lt 001) normal-ized these NTX-related changes (Table 10 and Figure 7)In addition oxymetholone also significantly (119901 lt 001)decreased these changes as compared with those in NTXcontrol mice in our experiment (Table 10 and Figure 6)EESF and oxymetholone also dose-dependently normalizedthe increases in oxidative stress markers such as nitroty-rosine and iNOS and the lipid peroxidation marker 4-HNE in the gastrocnemius muscle bundles of NTX con-trol mice Moreover the increase in inflammatory markersincluding COX-2 and TNF-120572 observed in the gastrocne-mius muscle bundles of NTX control mice was signifi-cantly attenuated in EESF-treated mice and oxymetholone-treated mice which was associated with normalizationof NTX-induced increase in myostatin immunoreactivefibers


Table 7 Changes in the gastrocnemius muscle cytokine content inmice with NTX-induced muscle atrophy

Groups Muscle cytokine content (pgmg protein)IL-1120573 TNF-120572

ControlsSham 164 plusmn 055 7567 plusmn 1606NTX 545 plusmn 151a 24476 plusmn 4321a

ReferenceOxymetholone 258 plusmn 056ab 13700 plusmn 3423ab

EESF treated500mgkg 270 plusmn 071ab 13643 plusmn 2785ab

250mgkg 327 plusmn 049ab 14986 plusmn 1501ab


Values are expressed as mean plusmn SD of 10 micea119901 lt 001 as compared with sham control mice by the MW test

b119901 lt 001 as compared with NTX control mice by the MW test

4 Discussion

Sciatic neurectomy denervation can induce muscle atro-phy characterized by a reduction in the protein contentorganelles cytoplasm the fiber diameter the productionof muscle strength and the resistance to fatigue [4 8 9]Denervation also causes numerous changes in contractileelectrical metabolic and molecular properties of the musclefiber membrane and sarcoplasm in hindlimb skeletal muscles[19 20] In the present study we assessed the possiblebeneficial skeletalmuscle preserving effects of EESF on sciaticNTX-induced hindlimb muscle atrophy in mice Accord-ingly decreases in calf thicknesses and muscle strengths andin gastrocnemius muscle thicknesses and weights at sacrificewere induced by sciatic NTX as a result of calf muscleatrophy However EESF dose-dependently diminished thesedecreases which can be considered as one of the directevidences that EESF as did oxymetholone ameliorated theNTX-induced calf muscle atrophic changes

Creatine is a nitrogenous organic acid that occurs nat-urally and helps to supply energy to all cells in the bodyprimarily muscle and plasma creatine levels can be used as avaluable serum biochemical marker for assessing the skeletalmuscle activity or amounts [56 57] LDH is of medical signif-icance because it is found extensively in body tissues such asblood cells and heart muscle and CK an enzyme expressedby various tissues and cell types catalyses the conversionof creatine and consumes adenosine triphosphate Becausethey are released during tissue damage they are markersof common injuries and disease especially muscle damageTherefore the plasma activities of CK and LDH have beencommonly used as markers of muscle tissue damage [58 59]They are also markedly elevated in disuse muscle atrophy inanimals [60] In the present studymarked decreases in serumcreatine levels were demonstrated along with NTX-induceddecreases in muscle masses and activities as in a previousstudy [60] but treatment with EESF dose-dependently andsignificantly diminished these NTX-induced decreases inthe serum creatine level Significant elevations in serum

CK and LDH levels indicating muscle damage were alsodetected in NTX control mice however significant and dose-dependent decreases in serum CK and LDH levels wereobserved in EESF-treated mice which can be considered asthe indirect evidences suggesting that EESF has favorable andpotent muscle preserving effects

Various toxic substances from lipid peroxidation destroythe surrounding tissues [61] and the oxidative stress is alsoan important inducer of muscle atrophy in both disuse aswell as the muscle cachexia [10] GSH is a representativeendogenous antioxidant prevents tissue damage by keepingthe ROS at low levels at certain cellular concentrationsand is accepted as a protective antioxidant factor in tissues[62] SOD is one of the antioxidant enzymes that contributeto enzymatic defense mechanisms and CAT is an enzymewhich catalyzes the conversion of H

2O2to H2O [63] Hence

the inhibition of increased lipid peroxidation and ROSwith increases in GSH content SOD and CAT activitiesin the damaged muscle tissue is secondarily important forprotection against muscle atrophy changes [44 64 65]4-HNE is an 120572 120573-unsaturated hydroxyalkenal which isproduced by lipid peroxidation in cells and it has been usedas a valuable tissue lipid peroxidation marker Also it isconsidered as a potential causal agent in numerous diseasessuch as chronic inflammation neurodegenerative diseasesadult respiratory distress syndrome atherogenesis diabetesand different types of cancer [66ndash68] Nitrotyrosine is aproduct of tyrosine nitration mediated by reactive nitrogenspecies (RNSs) such as peroxynitrite anion and nitrogendioxide It is detected in large number of pathologicalconditions and it is considered to be a marker of iNOS-dependentRNS-inducednitrative stress [69 70] In our studySF dose-dependently protected NTX-induced gastrocnemiusmuscle oxidative stresses the increases in lipid peroxidationand ROS formation decreases in GSH content SOD andCAT activities and increases in nitrotyrosine and 4-HNE-immunolabeled muscle fibers Oxymetholone also showedpotent antioxidant effects against NTX-induced depletionof antioxidant defense systems which corresponded well tothose of the previous anabolic steroids [71 72] Oxidativestress-induced apoptosis of muscle fiber is also involved inthe early phase of muscle atrophy regardless of its etiology[11 12] and caspase-3 and PARP are the key executionersof apoptosis [73 74] Therefore the increase in caspase-3and PARP immunoreactive muscle fibers in muscle bundlesrepresents their apoptosis and related damage [75 76]Therefore the dose-dependent inhibition of the caspase-3and PARP immunoreactivities in the NTX gastrocnemiusmuscle bundles by treatment with EESF as observed in thisstudy is considered as the direct evidence which indicatesthat it can preserve themusclemass through inhibition effectson NTX-induced muscle fiber apoptosis

The increased production of proinflammatory cytokinesmainly IL-1120573 and TNF-120572 seems to be involved in inflamma-tory cachexia and induces the production of prostaglandin E

2

(PGE2) which plays an important role in the inflammatory

response COXs are the key enzymes in regulating thebiosynthesis of PGsThere are twoCOX isoforms encoded bydifferent genes COX-1 which is constitutively expressed and


Table 8 Changes in the gastrocnemius muscle mRNA expression levels in mice with NTX-induced muscle atrophy

TargetsGroups

Controls Reference EESF treated mice (mgkg)Sham NTX Oxymetholone 500 250 125

Atrogin-1 105 plusmn 011 441 plusmn 106d 224 plusmn 028df 229 plusmn 059df 285 plusmn 061df 319 plusmn 078df

MuRF 1 104 plusmn 013 381 plusmn 069a 221 plusmn 043ac 220 plusmn 055ac 237 plusmn 042ac 295 plusmn 061ac

PI3K p85120572 095 plusmn 011 074 plusmn 012d 154 plusmn 064ef 158 plusmn 073f 105 plusmn 013f 093 plusmn 012f

Akt 1 097 plusmn 011 060 plusmn 018a 088 plusmn 014c 088 plusmn 010c 080 plusmn 010ac 078 plusmn 010ac

Adenosine A1R 098 plusmn 012 057 plusmn 010a 097 plusmn 013c 098 plusmn 014c 084 plusmn 015bc 079 plusmn 011ac

TRPV4 099 plusmn 009 045 plusmn 012a 077 plusmn 013ac 077 plusmn 011ac 069 plusmn 011ac 064 plusmn 012ac

Myostatin 100 plusmn 011 765 plusmn 153d 325 plusmn 099df 320 plusmn 071df 416 plusmn 078df 536 plusmn 049df

SIRT1 103 plusmn 014 683 plusmn 107d 380 plusmn 081df 383 plusmn 095df 435 plusmn 110df 522 plusmn 079df

Values are expressed as mean plusmn SD of six mice relative expressions18s ribosomal RNAa119901 lt 001 and b

119901 lt 005 as compared with intact control mice by the LSD testc119901 lt 001 as compared with NTX control mice by the LSD test

d119901 lt 001 and e

119901 lt 005 as compared with intact control mice by the LSD testf119901 lt 001 as compared with NTX control mice by the LSD test

Table 9 Changes in the gastrocnemius muscle histomorphometrical analysis in mice with NTX-induced muscle atrophy

GroupsHistomorphometry of the gastrocnemius muscle bundles

Mean muscle fiberdiameter (120583mfiber)

Number of infiltratedinflammatory cells (cellsmm2)

Regions occupied bycollagen fibers (mm2)

ControlsSham 5323 plusmn 721 590 plusmn 152 598 plusmn 306NTX 1481 plusmn 314a 32710 plusmn 9604a 4011 plusmn 634a

ReferenceOxymetholone 2964 plusmn 230ab 8660 plusmn 4121ab 2304 plusmn 266ab

EESF treated500mgkg 2994 plusmn 302ab 8900 plusmn 2267ab 2350 plusmn 411ab

250mgkg 2990 plusmn 374ab 12400 plusmn 1561ab 2750 plusmn 530ab




COX-2 which is inducible in response to proinflammatorycytokines and other stimuli COX-2-induced synthesis of PGshas been associated with chronic inflammation and is alsoincreased in disuse muscle atrophy [77] Proinflammatorycytokine IL-1120573 exhibits a wide variety of biological actionsincluding induction of the expression of various growthfactors chemokines and adhesion molecules [78] TNF-120572a 17-kDa protein which was first identified as a product ofactivated macrophages [79] is also one of the well-knownproinflammatory cytokines [80 81] It is well establishedthat the serum levels of proinflammatory cytokines partic-ularly IL-1120573 and TNF-120572 are elevated in cachectic patientscontributing to muscle atrophy [4 8 9] In the presentstudy marked elevations in the gastrocnemius muscle IL-1120573 and TNF-120572 levels with increases in COX-2 and TNF-120572 immunoreactive cells were demonstrated in NTX controlmice as compared with sham vehicle control mice but theseelevations in expression levels of proinflammatory cytokineswere dose-dependently diminished by treatment with EESF

suggesting that the muscle protective effects of EESF may bemediated by its anti-inflammatory activity at least partially

]Muscle mass and structure are determined by the balance

between protein degradation and synthesis In the proteindegradation pathway ATP-ubiquitin-dependent proteolysisis the process most probably responsible for muscle wasting[4 15] Recent studies have established that muscle-specificE3 ubiquitin ligases such as Atrogin-1 andMuRF1 play criticalroles in muscle atrophy [4] and their expression levels areincreased in atrophic calf muscles [15 82] and mice deficientin either Atrogin-1 or MuRF1 are resistant to muscle atrophy[3 83 84] Accordingly marked elevations in the gastrocne-mius muscle Atrogin-1 and MuRF1 mRNA expression levelswere demonstrated in NTX control mice as compared withsham vehicle control mice but these elevations were dose-dependently inhibited by treatment with EESF which are thedirect evidences suggesting that EESF exerts potent muscleprotective effects through downregulation of Atrogin-1 and


HampE stain Sirius red stain

(a)

(b)

(c)

(d)

(e)

(f)

Figure 6 Representative gastrocnemius muscle histology in samples taken from sham mice or mice with NTX-induced muscle atrophySamples from gastrocnemius muscles were separated and fixed in 10 neutral buffered formalin then embedded in paraffin sectioned andstained with HampE for general histopathology or Sirius red for collagen fiber and after that the histopathological profiles of each samplewere observed under light microscope ((a) intact vehicle control mice (b) NTX control mice (c) 50mgkg oxymetholone-treated mice(d) 500mgkg EESF orally treated mice (e) 250mgkg EESFmgkg orally treated mice (f) 125mgkg EESF orally treated mice scale bars =40 120583m)


Table 10 Changes in the gastrocnemius muscle immunohistomorphometrical analysis in mice with NTX-induced muscle atrophy

AntibodyGroups


Caspase-3 (fibersmm2) 370 plusmn 149 4070 plusmn 585c 1940 plusmn 409cd 1960 plusmn 250cd 2310 plusmn 390cd 3140 plusmn 375cd

PARP (fibersmm2) 610 plusmn 160 7350 plusmn 1133c 4460 plusmn 1117cd 4370 plusmn 1227cd 5390 plusmn 1050cd 6080 plusmn 643cd

NT (fibersmm2) 870 plusmn 200 6670 plusmn 1401c 3060 plusmn 1109cd 3350 plusmn 1142cd 4490 plusmn 1048cd 5086 plusmn 1119ce

4-HNE (fibersmm2) 570 plusmn 216 7460 plusmn 1238c 4290 plusmn 1034cd 4250 plusmn 1225cd 5240 plusmn 1046cd 6130 plusmn 1079ce

iNOS (fibersmm2) 640 plusmn 201 7190 plusmn 1213a 3630 plusmn 1147ab 3590 plusmn 1035ab 4570 plusmn 1053ab 5700 plusmn 1181ab

COX-2 (cellsmm2) 1080 plusmn 478 25980 plusmn 3090c 19410 plusmn 3050cd 15420 plusmn 2518cd 18510 plusmn 2063cd 22620 plusmn 2513ce

TNF-120572 (cellsmm2) 3240 plusmn 1320 23120 plusmn 4187a 17110 plusmn 1839ab 14820 plusmn 2995ab 18370 plusmn 2207ab 19130 plusmn 2154ab

Myostatin (fibersmm2) 330 plusmn 134 4530 plusmn 1065c 1740 plusmn 427cd 1850 plusmn 695cd 2720 plusmn 512cd 3210 plusmn 1092cd




d119901 lt 001 and e


Caspase-3 PARP Nitrotyrosine 4-HNE COX-2 MyostatiniNOS TNF-120572

(a)

(b)

(c)

(d)

(e)

(f)

Figure 7 Representative gastrocnemius muscle caspase-3 PARP nitrotyrosine 4-HNE iNOS COX-2 TNF-120572 and myostatin immunoreac-tivities in samples taken from intact mice or mice with NTX-induced muscle atrophy After deparaffinization of prepared gastrocnemiusmuscle histological paraffin sections citrate buffer antigen (epitope) retrieval pretreatment was conducted Thereafter the sectionswere immunostained using ABC methods for caspase-3 PARP nitrotyrosine 4-HNE iNOS COX-2 TNF-120572 and myostatin ((a) intactvehicle control mice (b) NTX control mice (c) 50mgkg oxymetholone-treated mice (d) 500mgkg EESF orally treated mice (e)250mgkg EESFmgkg orally treated mice (f) 125mgkg EESF orally treated mice Scale bars = 40120583m)


MuRF1 which are involved in muscle protein degradationMoreover the insulin-like growth factor 1 (IGF-1)PI3KAktsignaling pathway is known to play a pivotal role in activatingprotein synthesis [4] PI3K which is activated by insulinor IGF in turn activates Akt a serinethreonine kinaseand its downstream phosphorylates glycogen synthase kinase3120573 and mammalian target of rapamycin thereby inducinghypertrophy [8] In our study marked downregulation ofAkt1 and PI3K mRNA expressions was observed in NTXcontrol mice as denervation-related disuse However EESFdose-dependently upregulated the Akt1 and PI3K mRNAexpressions as compared with that in NTX control micewhich is the direct evidence suggesting that EESF can activatemuscle protein synthesis and confer resistance to NTX-induced disuse muscle atrophy similar to the effects ofoxymetholone

On the other hand adenosine is known to modulatevarious physiological functions of the cardiovascular system[85 86] and of most tissues including skeletal muscle [87]It has been proposed that adenosine is involved in boththe regulation of blood flow to skeletal muscle [88] and inthe synergistic effect of contraction and insulin stimulatedglucose uptake in skeletal muscle [89] The majority ofthe physiological effects of adenosine are believed to bemediated via specific adenosine receptors [90] Among themadenosine A1R has been shown to have cytoprotective effectson skeletal muscles [91] TRPV4 is a member of the TRPchannel superfamily [92 93] TRPV4 is known to be aCa2+-permeable nonselective cation channel and it appearsto play a mechanosensory or osmosensory role in severalmusculoskeletal tissues and prevents muscle atrophy or boneloss [93 94] NTX significantly decreased the adenosineA1R and TRPV4 mRNA expression levels in the gastrocne-mius muscle also as denervation-related disuse proteolysishowever EESF upregulated the adenosine A1R and TRPV4mRNA expression levels in a dose-dependent manner ascompared with those in NTX control mice which can beconsidered as direct evidences suggesting that it can increasethe muscle growth and confer resistance to NTX-induceddisusemuscle atrophy similar to the effects of oxymetholone

Furthermore myostatin a secreted growth differentia-tion factor is a member of the TGF-120573 protein family thatinhibits muscle differentiation and growth during the processknown as myogenesis Myostatin a potent negative regulatorof muscle growth [15] is produced primarily in skeletalmuscle cells circulates in the blood and acts on the muscletissue by binding to a cell-bound receptor called the activintype II receptor [95 96] The sirtuin family of proteinspossesses NAD+-dependent deacetylase activity andor ADPribosyltransferase activity The seven mammalian sirtuinsSIRT1-7 are localized differentially within the cell and havea variety of functions [97] Among them SIRT1 is the mostextensively studied member of the family and regulatesdiverse biological processes ranging from cell proliferationdifferentiation apoptosis and metabolism [98] It controlsthe transcription of the peroxisome proliferator-activatedreceptor-120574 coactivator 1 120572 in the skeletal muscle [99] andinduces cachexia through inhibiting muscle regeneration[100] In disuse muscle atrophy the expressions of myostatin

and SIRT1 mRNA have been detected along with decreasesin muscle mass [15 82] and also after NTX in this studyHowever these elevations in myostatin and SIRT1 mRNAexpression levels were dose-dependently inhibited by treat-ment with EESF which can be considered as the directevidence suggesting that it exerts potent muscle protectiveeffects through downregulation of myostatin and SIRT1

5 Conclusion

Thepresent results suggested that EESF exerts favorable ame-liorating effects onmuscle atrophy induced by NTX throughanti-inflammatory and antioxidant effects related to musclefiber protective effects and via an increase in protein synthesisand a decrease in protein degradation These effects of EESFmay help improve various muscle atrophies due to variousetiologiesThe administration of 500mgkg EESF led to quitesimilar favorable muscle preserving effects on NTX-inducedmuscle atrophy to those of 50mgkg oxymetholone a 17120572-alkylated anabolic-androgenic steroid which has been usedfor treating various muscular disorders but EESF showedmore favorable antioxidant effects in this experiment

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Joo Wan Kim and Sae-Kwang Ku contributed equally

Acknowledgment

This research was supported by RampD program of MOTIEKEIT (10040391 Development of Functional Food MaterialsandDevice for Prevention of Aging-AssociatedMuscle Func-tion Decrease)

References

[1] S V Brooks and J A Faulkner ldquoSkeletal muscle weakness inold age underlying mechanismsrdquoMedicine amp Science in Sportsamp Exercise vol 26 no 4 pp 432ndash439 1994

[2] E J Metter L A Talbot M Schrager and R Conwit ldquoSkeletalmuscle strength as a predictor of all-cause mortality in healthymenrdquo The Journals of Gerontology Series A Biological Sciencesand Medical Sciences vol 57 no 10 pp B359ndashB365 2002

[3] S C Bodine E Latres S Baumhueter et al ldquoIdentification ofubiquitin ligases required for skeletal Muscle Atrophyrdquo Sciencevol 294 no 5547 pp 1704ndash1708 2001

[4] D J Glass ldquoSignaling pathways perturbing muscle massrdquoCurrent Opinion in Clinical Nutrition and Metabolic Care vol13 no 3 pp 225ndash229 2010

[5] C Ramırez T L Russo M C Sandoval et al ldquoJoint inflamma-tion alters gene and protein expression and leads to atrophy inthe tibialis anterior muscle in ratsrdquoAmerican Journal of PhysicalMedicine amp RehabilitationAssociation of Academic Physiatristsvol 90 no 11 pp 930ndash939 2011


[6] R W Jackman and S C Kandarian ldquoThe molecular basis ofskeletal muscle atrophyrdquo The American Journal of PhysiologymdashCell Physiology vol 287 no 4 pp C834ndashC843 2004

[7] S Schiaffino K A Dyar S Ciciliot B Blaauw and M SandrildquoMechanisms regulating skeletal muscle growth and atrophyrdquoThe FEBS Journal vol 280 no 17 pp 4294ndash4314 2013

[8] M Sandri ldquoSignaling in muscle atrophy and hypertrophyrdquoPhysiology vol 23 no 3 pp 160ndash170 2008

[9] R Bassel-Duby and E N Olson ldquoSignaling pathways in skeletalmuscle remodelingrdquoAnnual Review of Biochemistry vol 75 pp19ndash37 2006

[10] S K Powers A N Kavazis and J M McClung ldquoOxidativestress and disuse muscle atrophyrdquo Journal of Applied Physiologyvol 102 no 6 pp 2389ndash2397 2007

[11] T Arakawa A Katada H Shigyo et al ldquoElectrical stimulationprevents apoptosis in denervated skeletal musclerdquo NeuroReha-bilitation vol 27 no 2 pp 147ndash154 2010

[12] J-Y Lim and T R Han ldquoEffect of electromyostimulation onapoptosis-related factors in denervation and reinnervation ofrat skeletal musclesrdquo Muscle amp Nerve vol 42 no 3 pp 422ndash430 2010

[13] B Leger R Senese AWAl-Khodairy et al ldquoAtrogin-1MuRF1and FoXo as well as phosphorylated GSK-3beta and 4E-BP1are reduced in skeletal muscle of chronic spinal cord-injuredpatientsrdquoMuscle amp Nerve vol 40 no 1 pp 69ndash78 2009

[14] T Hofer E Marzetti J Xu et al ldquoIncreased iron content andRNA oxidative damage in skeletal muscle with aging and disuseatrophyrdquo Experimental Gerontology vol 43 no 6 pp 563ndash5702008

[15] A Onda Q Jiao Y Nagano et al ldquoAcupuncture amelioratedskeletal muscle atrophy induced by hindlimb suspension inmicerdquo Biochemical and Biophysical Research Communicationsvol 410 no 3 pp 434ndash439 2011

[16] F W Booth ldquoPhysiologic and biochemical effects of immobi-lization onmusclerdquo Clinical Orthopaedics and Related Researchvol 219 pp 15ndash20 1987

[17] D RThomas ldquoLoss of skeletalmusclemass in aging examiningthe relationship of starvation sarcopenia and cachexiardquoClinicalNutrition vol 26 no 4 pp 389ndash399 2007

[18] F Kawano J Tanihata S Sato et al ldquoEffects of dexamethasoneon the expression of 120573

1

- 1205732

- and 1205733

-adrenoceptor mRNAsin skeletal and left ventricle muscles in ratsrdquo The Journal ofPhysiological Sciences vol 59 no 5 pp 383ndash390 2009

[19] E A Connor and U J McMahan ldquoCell accumulation in thejunctional region of denervated musclerdquo The Journal of CellBiology vol 104 no 1 pp 109ndash120 1987

[20] P DellrsquoEra L Coco R Ronca B Sennino andM Presta ldquoGeneexpression profile in fibroblast growth factor 2-transformedendothelial cellsrdquoOncogene vol 21 no 15 pp 2433ndash2440 2002

[21] M AMurray andN Robbins ldquoCell proliferation in denervatedmuscle time course distribution and relation to disuserdquoNeuro-science vol 7 no 7 pp 1817ndash1822 1982

[22] M AMurray andN Robbins ldquoCell proliferation in denervatedmuscle identity and origin of dividing cellsrdquo Neuroscience vol7 no 7 pp 1823ndash1833 1982

[23] T Ogawa T Nikawa H Furochi et al ldquoOsteoactivin upregu-lates expression of MMP-3 andMMP-9 in fibroblasts infiltratedinto denervated skeletal muscle in micerdquoThe American Journalof PhysiologymdashCell Physiology vol 289 no 3 pp C697ndashC7072005

[24] V Salonen M Lehto H Kalimo R Penttinen and H AroldquoChanges in intramuscular collagen and fibronectin in dener-vation atrophyrdquoMuscle amp Nerve vol 8 no 2 pp 125ndash131 1985

[25] A Panossian and G Wikman ldquoPharmacology of Schisandrachinensis Bail an overview of Russian research and uses inmedicinerdquo Journal of Ethnopharmacology vol 118 no 2 pp183ndash212 2008

[26] Y Lu and D-F Chen ldquoAnalysis of Schisandra chinensis andSchisandra sphenantherardquo Journal of Chromatography A vol1216 no 11 pp 1980ndash1990 2009

[27] C-H Ma L Yang Y-G Zu and T-T Liu ldquoOptimization ofconditions of solvent-free microwave extraction and study onantioxidant capacity of essential oil from Schisandra chinensis(Turcz) Baillrdquo Food Chemistry vol 134 no 4 pp 2532ndash25392012

[28] C J Liu S Q Zhang J S Zhang Q Liang and D S LildquoChemical composition and antioxidant activity of essentialoil from berries of Schisandra chinensis (Turcz) Baillrdquo NaturalProduct Research vol 26 no 23 pp 2199ndash2203 2012

[29] X Chen Y Zhang Y Zu and L Yang ldquoChemical compositionand antioxidant activity of the essential oil of Schisandrachinensis fruitsrdquo Natural Product Research vol 26 no 9 pp842ndash849 2012

[30] L Song J-Y Ding C Tang and C-H Yin ldquoCompositionsand biological activities of essential oils of Kadsura longepedun-culata and Schisandra sphenantherardquo The American Journal ofChinese Medicine vol 35 no 2 pp 353ndash364 2007

[31] H I Jang G M Do HM Lee H M Ok J Shin and O KwonldquoSchisandra chinensis Baillon regulates the gene expression ofphase II antioxidantdetoxifying enzymes in hepatic damageinduced ratsrdquo Nutrition Research and Practice vol 8 no 3 pp272ndash277 2014

[32] S Park S M Hong I S Ahn Y J Kim and J B Lee ldquoHuang-Lian-Jie-Du-Tang supplemented with Schisandra chinensisBailland Polygonatum odoratum Druce improved glucose toler-ance by potentiating insulinotropic actions in islets in 90pancreatectomized diabetic ratsrdquo Bioscience Biotechnology andBiochemistry vol 73 no 11 pp 2384ndash2392 2009

[33] J Zhang L L Shi and Y N Zheng ldquoDibenzocyclooctadienelignans from fructus Schisandrae chinensis improve glucoseuptake in vitrordquo Natural Product Communications vol 5 no 2pp 231ndash234 2010

[34] D Y Kwon D S Kim H J Yang and S Park ldquoThe lignan-richfractions of Fructus Schisandrae improve insulin sensitivity viathe PPAR-120574 pathways in in vitro and in vivo studiesrdquo Journal ofEthnopharmacology vol 135 no 2 pp 455ndash462 2011

[35] J Young Park J Wook Yun Y Whan Choi et al ldquoAntihy-pertensive effect of gomisin A from Schisandra chinensis onangiotensin II-induced hypertension via preservation of nitricoxide bioavailabilityrdquo Hypertension Research vol 35 no 9 pp928ndash934 2012

[36] J-M Yang P S P Ip C-T Che and J H K Yeung ldquoRelaxanteffects of Schisandra chinensis and itsmajor lignans on agonists-induced contraction in guinea pig ileumrdquo Phytomedicine vol18 no 13 pp 1153ndash1160 2011

[37] P Nagpal P J Plant J Correa et al ldquoThe ubiquitin ligaseNedd4-1 participates in denervation-induced skeletal muscleatrophy in micerdquo PLoS ONE vol 7 no 10 Article ID e464272012

[38] H J Ringold E Batres O Halpern and E NecoechealdquoSteroids CV1 2-Methyl and 2-hydroxymethylene-androstane


derivativesrdquo Journal of the American Chemical Society vol 81no 2 pp 427ndash432 1959

[39] A M Pavlatos O Fultz M J Monberg and A VootkurldquoReview of oxymetholone a 17alpha-alkylated anabolic-androgenic steroidrdquo Clinical Therapeutics vol 23 no 6 pp789ndash801 2001

[40] J Isaacs K Loveland S Mallu S Adams and RWodicka ldquoTheuse of anabolic steroids as a strategy in reversing denervationatrophy after delayed nerve repairrdquoHand vol 6 no 2 pp 142ndash148 2011

[41] D Del Rio A J Stewart and N Pellegrini ldquoA review ofrecent studies on malondialdehyde as toxic molecule andbiological marker of oxidative stressrdquo Nutrition Metabolism ampCardiovascular Diseases vol 15 no 4 pp 316ndash328 2005

[42] O H Lowry N J Rosenbrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[43] I S Jamall and J C Smith ldquoEffects of cadmium on glutathioneperoxidase superoxide dismutase and lipid peroxidation in therat heart a possible mechanism of cadmium cardiotoxicityrdquoToxicology and Applied Pharmacology vol 80 no 1 pp 33ndash421985

[44] H J He G Y Wang Y Gao W H Ling Z W Yu and T R JinldquoCurcumin attenuates Nrf2 signaling defect oxidative stress inmuscle and glucose intolerance in high fat diet-fedmicerdquoWorldJournal of Diabetes vol 3 no 5 pp 94ndash104 2012

[45] J Sedlak and R H Lindsay ldquoEstimation of total protein-bound andnonprotein sulfhydryl groups in tissuewith Ellmanrsquosreagentrdquo Analytical Biochemistry vol 25 no 1 pp 192ndash2051968

[46] H Aebi ldquoCatalaserdquo in Methods in Enzymatic Analysis H UBergmeyer Ed pp 673ndash686 Academic Press New York NYUSA 1974

[47] Y Sun L W Oberley and Y Li ldquoA simple method for clinicalassay of superoxide dismutaserdquo Clinical Chemistry vol 34 no3 pp 497ndash500 1988

[48] B D Clark I Bedrosian R Schindler et al ldquoDetection ofinterleukin 1120572 and 1120573 in rabbit tissues during endotoxemia usingsensitive radioimmunoassaysrdquo Journal of Applied Physiologyvol 71 no 6 pp 2412ndash2418 1991

[49] G L Tipoe T M Leung E C Liong T Y H Lau M L Fungand A A Nanji ldquoEpigallocatechin-3-gallate (EGCG) reducesliver inflammation oxidative stress and fibrosis in carbontetrachloride (CCl

4

)-induced liver injury in micerdquo Toxicologyvol 273 no 1ndash3 pp 45ndash52 2010

[50] S-R Shi B Chaiwun L Young R J Cote and C R TaylorldquoAntigen retrieval technique utilizing citrate buffer or ureasolution for immunohistochemical demonstration of androgenreceptor in formalin-fixed paraffin sectionsrdquo The Journal ofHistochemistry and Cytochemistry vol 41 no 11 pp 1599ndash16041993

[51] S H Ki J H Yang S K Ku S C Kim YW Kim and I J CholdquoRed ginseng extract protects against carbon tetrachloride-induced liver fibrosisrdquo Journal of Ginseng Research vol 37 no1 pp 45ndash53 2013

[52] H S Lee S H Choi and S K Ku ldquoRegional distribution andrelative frequency of gastrointestinal endocrine cells in the ddNmice an immunohistochemical studyrdquo Anatomia HistologiaEmbryologia vol 39 no 6 pp 521ndash528 2010

[53] M Y Song S K Ku H J Kim and J S Han ldquoLow molecularweight fucoidan ameliorating the chronic cisplatin-induced

delayed gastrointestinal motility in ratsrdquo Food and ChemicalToxicology vol 50 no 12 pp 4468ndash4478 2012

[54] A Levene ldquoPathological factors influencing excision oftumours in the head and neck Part Irdquo Clinical Otolaryngologyand Allied Sciences vol 6 no 2 pp 145ndash151 1981

[55] J Ludbrook ldquoUpdate microcomputer statistics packages Apersonal reviewrdquo Clinical and Experimental Pharmacology andPhysiology vol 24 no 3-4 pp 294ndash296 1997

[56] A Sala M Tarnopolsky C Webber G Norman and R BarrldquoSerum creatinine a surrogate measurement of lean body massin children with acute lymphoblastic leukemiardquo Pediatric Bloodamp Cancer vol 45 no 1 pp 16ndash19 2005

[57] S A Stimpson S M Turner L G Clifton et al ldquoTotal-body creatine pool size and skeletal muscle mass determinationby creatine-(methyl-d3) dilution in ratsrdquo Journal of AppliedPhysiology vol 112 no 11 pp 1940ndash1948 2012

[58] Y Zhang J-J Huang Z-Q Wang N Wang and Z-Y WuldquoValue of muscle enzyme measurement in evaluating differentneuromuscular diseasesrdquoClinica Chimica Acta vol 413 no 3-4pp 520ndash524 2012

[59] M Choi H Park S Cho and M Lee ldquoVitamin D3 supple-mentation modulates inflammatory responses from the muscledamage induced by high-intensity exercise in SD ratsrdquoCytokinevol 63 no 1 pp 27ndash35 2013

[60] I Cohen E Bogin A Chechick and V Rzetelny ldquoBiochemicalalterations secondary to disuse atrophy in the ratrsquos serum andlimb tissuesrdquo Archives of Orthopaedic and Trauma Surgery vol119 no 7-8 pp 410ndash417 1999

[61] M Comporti ldquoLipid peroxidation and cellular damage in toxicliver injuryrdquo Laboratory Investigation vol 53 no 6 pp 599ndash6231985

[62] F Odabasoglu A Cakir H Suleyman et al ldquoGastroprotectiveand antioxidant effects of usnic acid on indomethacin-inducedgastric ulcer in ratsrdquo Journal of Ethnopharmacology vol 103 no1 pp 59ndash65 2006

[63] K H Cheeseman and T F Slater ldquoAn introduction to freeradical biochemistryrdquo British Medical Bulletin vol 49 no 3 pp481ndash493 1993

[64] M Gore R Fiebig J Hollander C Leeuwenburgh H Ohnoand L L Ji ldquoEndurance training alters antioxidant enzymegene expression in rat skeletal musclerdquo Canadian Journal ofPhysiology and Pharmacology vol 76 no 12 pp 1139ndash1145 1998

[65] H Suleyman E Cadirci A Albayrak et al ldquoComparativestudy on the gastroprotective potential of some antidepressantsin indomethacin-induced ulcer in ratsrdquo Chemico-BiologicalInteractions vol 180 no 2 pp 318ndash324 2009

[66] N Zarkovic ldquo4-hydroxynonenal as a bioactive marker ofpathophysiological processesrdquo Molecular Aspects of Medicinevol 24 no 4-5 pp 281ndash291 2003

[67] E E Dubinina and V A Dadali ldquoRole of 4-hydroxy-trans-2-nonenal in cell functionsrdquo Biochemistry (Moscow) vol 75 no9 pp 1069ndash1087 2010

[68] R L Smathers J J Galligan B J Stewart and D R PetersenldquoOverview of lipid peroxidation products and hepatic proteinmodification in alcoholic liver diseaserdquo Chemico-BiologicalInteractions vol 192 no 1-2 pp 107ndash112 2011

[69] J-H Chen G L Tipoe E C Liong et al ldquoGreen teapolyphenols prevent toxin-induced hepatotoxicity in mice bydown-regulating inducible nitric oxide-derived prooxidantsrdquoThe American Journal of Clinical Nutrition vol 80 no 3 pp742ndash751 2004


[70] P Pacher J S Beckman and L Liaudet ldquoNitric oxide andperoxynitrite in health and diseaserdquo Physiological Reviews vol87 no 1 pp 315ndash424 2007

[71] J Delgado A Saborido and A Megıas ldquoProlonged treat-ment with the anabolic-androgenic steroid stanozolol increasesantioxidant defences in rat skeletal musclerdquo Journal of Physiol-ogy and Biochemistry vol 66 no 1 pp 63ndash71 2010

[72] Y-E Yoo and C-P Ko ldquoDihydrotestosterone amelioratesdegeneration in muscle axons and motoneurons and improvesmotor function in amyotrophic lateral sclerosis model micerdquoPLoS ONE vol 7 no 5 Article ID e37258 2012

[73] G Nunez M A Benedict Y Hu and N Inohara ldquoCaspasesthe proteases of the apoptotic pathwayrdquo Oncogene vol 17 no25 pp 3237ndash3245 1998

[74] K L Barrett J M Willingham A J Garvin and M CWillingham ldquoAdvances in cytochemical methods for detectionof apoptosisrdquoThe Journal of Histochemistry and Cytochemistryvol 49 no 7 pp 821ndash832 2001

[75] A D Dam A S Mitchell J W E Rush and J QuadrilateroldquoElevated skeletal muscle apoptotic signaling following glu-tathione depletionrdquo Apoptosis vol 17 no 1 pp 48ndash60 2012

[76] Y-P Yen K-S Tsai Y-WChen C-FHuang R-S Yang and S-H Liu ldquoArsenic induces apoptosis in myoblasts through a reac-tive oxygen species-induced endoplasmic reticulum stress andmitochondrial dysfunction pathwayrdquoArchives of Toxicology vol86 no 6 pp 923ndash933 2012

[77] B A Bondesen S T Mills and G K Pavlath ldquoThe COX-2 pathway regulates growth of atrophied muscle via multiplemechanismsrdquo American Journal of PhysiologymdashCell Physiologyvol 290 no 6 pp C1651ndashC1659 2006

[78] K Matsushima and J J Oppenheim ldquoInterleukin 8 andMCAFnovel inflammatory cytokines inducible by IL 1 and TNFrdquoCytokine vol 1 no 1 pp 2ndash13 1989

[79] L J Old ldquoTumor necrosis factor (TNF)rdquo Science vol 230 no4726 pp 630ndash632 1985

[80] R Assuma T Oates D Cochran S Amar and D T GravesldquoIL-1 and TNF antagonists inhibit the inflammatory responseand bone loss in experimental periodontitisrdquo The Journal ofImmunology vol 160 no 1 pp 403ndash409 1998

[81] B J R Whittle C Varga A Berko et al ldquoAttenuation ofinflammation and cytokine production in rat colitis by a novelselective inhibitor of leukotriene A4 hydrolaserdquo British Journalof Pharmacology vol 153 no 5 pp 983ndash991 2008

[82] D L Allen B J Greybeck A M Hanson A S Cleary and SF Lindsay ldquoSkeletal muscle expression of bone morphogeneticprotein-1 and tolloid-like-1 extracellular proteases in differentfiber types and in response to unloading food deprivation anddifferentiationrdquoThe Journal of Physiological Sciences vol 60 no5 pp 343ndash352 2010

[83] M D Gomes S H Lecker R T Jagoe A Navon and A LGoldberg ldquoAtrogin-1 a muscle-specific F-box protein highlyexpressed during muscle atrophyrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 98 no25 pp 14440ndash14445 2001

[84] S Cohen J J Brault S P Gygi et al ldquoDuring muscle atrophythick but not thin filament components are degraded byMuRF1-dependent ubiquitylationrdquo The Journal of Cell Biologyvol 185 no 6 pp 1083ndash1095 2009

[85] RM Berne RMKnabb SW Ely andR Rubio ldquoAdenosine inthe local regulation of blood flow a brief overviewrdquo FederationProceedings vol 42 no 15 pp 3136ndash3142 1983

[86] S S Segal and D T Kurjiaka ldquoCoordination of blood flow con-trol in the resistance vasculature of skeletal musclerdquoMedicine ampScience in Sports amp Exercise vol 27 no 8 pp 1158ndash1164 1995

[87] P Hespel and E A Richter ldquoRole of adenosine in regulation ofcarbohydrate metabolism in contracting musclerdquo Advances inExperimental Medicine and Biology vol 441 pp 97ndash106 1998

[88] J G Dobson Jr R Rubio and R M Berne ldquoRole of adeninenucleotides adenosine and inorganic phosphate in the regula-tion of skeletal muscle blood flowrdquo Circulation Research vol 29no 4 pp 375ndash384 1971

[89] L Vergauwen PHespel and EA Richter ldquoAdenosine receptorsmediate synergistic stimulation of glucose uptake and transportby insulin and by contractions in rat skeletal musclerdquo TheJournal of Clinical Investigation vol 93 no 3 pp 974ndash981 1994

[90] J Lynge and Y Hellsten ldquoDistribution of adenosine A1

A2119860

and A2119861

receptors in human skeletal musclerdquo Acta PhysiologicaScandinavica vol 169 no 4 pp 283ndash290 2000

[91] J Zheng R Wang E Zambraski D Wu K A Jacobsonand B T Liang ldquoProtective roles of adenosine A

1

A2119860

andA3

receptors in skeletal muscle ischemia and reperfusioninjuryrdquo American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 293 no 6 pp H3685ndashH3691 2007

[92] M J Caterina M A Schumacher M Tominaga T A RosenJ D Levine and D Julius ldquoThe capsaicin receptor a heat-activated ion channel in the pain pathwayrdquoNature vol 389 no6653 pp 816ndash824 1997

[93] F Guilak H A Leddy and W Liedtke ldquoTransient receptorpotential vanilloid 4 the sixth sense of the musculoskeletalsystemrdquo Annals of the New York Academy of Sciences vol 1192pp 404ndash409 2010

[94] F Mizoguchi A Mizuno T Hayata et al ldquoTransient receptorpotential vanilloid 4 deficiency suppresses unloading-inducedbone lossrdquo Journal of Cellular Physiology vol 216 no 1 pp 47ndash53 2008

[95] N F Gonzalez-Cadavid W E Taylor K Yarasheski et alldquoOrganization of the human myostatin gene and expressionin healthy men and HIV-infected men with muscle wastingrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 95 no 25 pp 14938ndash14943 1998

[96] G Carnac S Ricaud B Vernus and A Bonnieu ldquoMyostatinbiology and clinical relevancerdquo Mini-Reviews in MedicinalChemistry vol 6 no 7 pp 765ndash770 2006

[97] E Michishita J Y Park J M Burneskis J C Barrett and IHorikawa ldquoEvolutionarily conserved and nonconserved cel-lular localizations and functions of human SIRT proteinsrdquoMolecular Biology of the Cell vol 16 no 10 pp 4623ndash4635 2005

[98] M C Haigis and L P Guarente ldquoMammalian sirtuinsmdashemerging roles in physiology aging and calorie restrictionrdquoGenes amp Development vol 20 no 21 pp 2913ndash2921 2006

[99] R Amat A Planavila S L Chen R Iglesias M Giralt andF Villarroya ldquoSIRT1 controls the transcription of the perox-isome proliferator-activated receptor-120574 co-activator-1120572 (PGC-1120572) gene in skeletal muscle through the PGC-1120572 autoregulatoryloop and interaction with MyoDrdquo The Journal of BiologicalChemistry vol 284 no 33 pp 21872ndash21880 2009

[100] M Toledo S Busquets E Ametller F J Lopez-Soriano and JM Argiles ldquoSirtuin 1 in skeletal muscle of cachectic tumour-bearing rats a role in impaired regenerationrdquo Journal ofCachexia Sarcopenia and Muscle vol 2 no 1 pp 57ndash62 2011


process The disuse of the muscles will also lead to disuseatrophy while neurogenic atrophy is muscle atrophy thatresults from damage to the nerve that stimulates the muscle[4 6 7]

There is a common genetic program involved in muscleproteolysis regardless of its etiology however the signalingpathways that modulate this system are distinct [6 8] It iswell established that the serum levels of proinflammatorycytokines particularly interleukin- (IL-) 1 and tumor necrosisfactor- (TNF-) 120572 are elevated in cachectic patients contribut-ing to muscle atrophy [8 9] Moreover the oxidative stressis an important inducer of muscle atrophy in both disuseand muscle cachexia [10] In addition apoptosis of musclefibers is also involved in the early phase of muscle atrophyregardless of its etiology [11 12] Up till now various animalmodels of skeletal muscle atrophy have been used in researchfields including those of denervation [13] unloading [1415] immobilization [16] starvation [17] and administrationof dexamethasone [18] Among them denervation (sciaticneurectomy NTX) causes numerous changes in contractileelectrical metabolic and molecular properties of the musclefiber membrane and sarcoplasm in hindlimb skeletal muscles[19 20] Previous studies have shown that denervation altersinterstitial spaces between muscle fibers mononucleatedcells are infiltrated into interstitial spaces of muscle fibersand fibrosis occasionally occurs in the muscle [21ndash24]These findings suggest that NTX-induced hindlimb muscleatrophy is a useful and rapid model in vivo for screeningthe agents that can reverse the abnormalities in muscleatrophy

Schisandrae Fructus the dried fruits of Schisandra chi-nensis (Turcz) Baill (Magnoliaceae) one of the most famousherbal medicines has been extensively used in Asia includingKorea China and Japanwith Russia [25 26] It was originallyused as a tonic and has been traditionally used for thetreatment of many uncomfortable symptoms such as coughdyspnea dysentery insomnia and amnesia for a long time[25 26] Previous reports have shown that SchisandraeFructusand its related compounds possess various biologicalactivities such as antioxidant anti-inflammatory antimicro-bial antiseptic antiaging hepatoprotective immunostim-ulating and anticancer effects [27ndash31] In addition recentreports have suggested that Schisandrae Fructus has favorableeffects on diabetes and it is related [32ndash34] with smoothmus-cle relaxant effects [35 36] However the effect of SchisandraeFructus on muscle atrophy has not yet been evaluated Inthe present study we assessed the possible beneficial skeletalmuscle preserving effects of ethanol extract of SchisandraeFructus (EESF) on sciatic NTX-induced hindlimb muscleatrophy in mice In this experiment calf muscle atrophy wasinduced by unilateral right sciatic NTX according to previousstudies [23 37] In order to investigate whether adminis-tration of EESF prevents or improves sciatic NTX-inducedmuscle atrophy with possible action mechanisms 125 250and 500mgkg of EESF were administered orally once a dayfor 28 days from2weeks afterNTX inmice for the therapeuticstudy

2 Materials and Methods

21 Test Substance Schisandrae Fructuswere collected aroundMungyeong- (Gyeongsangbuk-do Republic of Korea) andwashed three times with tap water before storage at minus20∘CThe frozen samples were lyophilized and homogenized usinga grinder before extractionThematerials were extractedwith20 ethanol (EESF) at room temperature for 24 h filteredand concentrated using a rotary vacuum evaporator (BuchiRotavapor R-144 BUCHI Labortechnik Flawil Switzerland)The extract was dissolved in dimethyl sulfoxide (DMSOSigma-Aldrich) as a 50mgmL stock solution and stored at4∘C until use In addition oxymetholone 17120573-hydroxy-2-hydroxymethylidene-17120572-methyl-3-androstanone (CelltrionPharm Inc Jincheon Republic of Korea) which is anorally active 17120572-alkylated anabolic-androgenic steroid firstdescribed by Ringold et al [38] was used as the referencedrug it was also stored in a refrigerator at 4∘C to protect fromlight and degeneration until use

22 Animals and Experimental Design Total 60 adultSPFVAF Outbred CrljOriCD1 (ICR) mice (Orient Bio IncSeongnam Republic of Korea) weighing 29sim32 g were usedin this experiment after allowing for 7 days of acclimati-zation The animals were allocated five per polycarbonatecage in a temperature (20ndash25∘C) and humidity (40ndash45)controlled room The light dark cycle was 12 hr 12 hr andnormal rodent pellet diet (Samyang Co Seoul Republicof Korea) and water were supplied during acclimatizationad libitum After acclimatization unilateral sciatic NTX wasperformed to induce hindlimb muscle atrophy according tothe previously established method [23 37] Briefly all of themice were anesthetized with 2 to 3 isoflurane (Hana PharmCo Hwasung Republic of Korea) in the mixture of 70N2O and 285 O

2 and anesthesia was maintained with 1

to 15 isoflurane in the mixture of 70 N2O and 285

O2 Body temperature was maintained at 370 plusmn 1∘C and

mice were subjected to operation Right sciatic neurectomywas performed by removing a 05 cm section of the nerve inthe mid thigh and a sham operation (nerve identification)was performed in the case of the sham groups as followsanimals were randomly divided into 6 groups of 10 mice eachat 2 weeks after NTX treatment sham control NTX controloxymetholone-treated group and EESF-treated groups (125250 and 500mgkg) based on the body weights (3761 plusmn163 g range 3570sim4040 g in the vehicle sham control group3724 plusmn 220 g range 3400sim4390 g in the NTX group) andcalf thicknesses (286 plusmn 011mm range 271sim300mm in thevehicle sham control group 230 plusmn 006mm range 218sim241mm in the NTX group) The dosage of oxymetholonewas selected as 50mgkg based on the previous efficacy testin mice [39 40] Prepared and stored EESF stock solution(500mgmL in DMSO) was 10- 20- or 40-fold diluted as 5025 and 125mgmL by distilled water and then orally admin-istered in a volume of 10mLkg of body weight as matchedas 500 250 and 125mgkg of body weight by oral gavagesonce a day for 28 days from2weeks afterNTX respectively Inaddition oxymetholone was also dissolve in distilled water as5mgmL concentrations and orally administered in a volume













































2O2




3 Results











b119901 lt 001 and c



Body

wei

ghts

(g)

28

30

32

34

36

38

40

42

44

46


a


minus1





Calf

thic

knes

ses (

mm

)

18

20

22

24

26

28

30

32

34

36

ab

abab

a

minus1





















Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

Gas

trocn

emiu

s mus

cle th

ickn

ess (

mm

)

00

05

10

15

20

25

30

35

a

ab abab ab




astro

cnem

ius m

uscle

wei

ghts

00

01

02

03

04

05

06

07


ab ab

ab aba

ccd cd cd cd

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)


119901 lt 001





Calf

mus

cle st

reng

th (N

)

0

2

4

6

8

10

ab abab

ab

a

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

















d119901 lt 001 and e




MDA(nMmg protein)


GSH(nMmg protein)


CAT(Umg protein)









d119901 lt 001 and e















4 Discussion








2





TargetsGroups












d119901 lt 001 and e




















(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3























































2O2




3 Results











b119901 lt 001 and c



Body

wei

ghts

(g)

28

30

32

34

36

38

40

42

44

46


a


minus1





Calf

thic

knes

ses (

mm

)

18

20

22

24

26

28

30

32

34

36

ab

abab

a

minus1





















Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

Gas

trocn

emiu

s mus

cle th

ickn

ess (

mm

)

00

05

10

15

20

25

30

35

a

ab abab ab




astro

cnem

ius m

uscle

wei

ghts

00

01

02

03

04

05

06

07


ab ab

ab aba

ccd cd cd cd

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)


119901 lt 001





Calf

mus

cle st

reng

th (N

)

0

2

4

6

8

10

ab abab

ab

a

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

















d119901 lt 001 and e




MDA(nMmg protein)


GSH(nMmg protein)


CAT(Umg protein)









d119901 lt 001 and e















4 Discussion








2





TargetsGroups












d119901 lt 001 and e




















(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3




















b119901 lt 001 and c



Body

wei

ghts

(g)

28

30

32

34

36

38

40

42

44

46


a


minus1





Calf

thic

knes

ses (

mm

)

18

20

22

24

26

28

30

32

34

36

ab

abab

a

minus1





















Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

Gas

trocn

emiu

s mus

cle th

ickn

ess (

mm

)

00

05

10

15

20

25

30

35

a

ab abab ab




astro

cnem

ius m

uscle

wei

ghts

00

01

02

03

04

05

06

07


ab ab

ab aba

ccd cd cd cd

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)


119901 lt 001





Calf

mus

cle st

reng

th (N

)

0

2

4

6

8

10

ab abab

ab

a

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

















d119901 lt 001 and e




MDA(nMmg protein)


GSH(nMmg protein)


CAT(Umg protein)









d119901 lt 001 and e















4 Discussion








2





TargetsGroups












d119901 lt 001 and e




















(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3




























Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

Gas

trocn

emiu

s mus

cle th

ickn

ess (

mm

)

00

05

10

15

20

25

30

35

a

ab abab ab




astro

cnem

ius m

uscle

wei

ghts

00

01

02

03

04

05

06

07


ab ab

ab aba

ccd cd cd cd

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)


119901 lt 001





Calf

mus

cle st

reng

th (N

)

0

2

4

6

8

10

ab abab

ab

a

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

















d119901 lt 001 and e




MDA(nMmg protein)


GSH(nMmg protein)


CAT(Umg protein)









d119901 lt 001 and e















4 Discussion








2





TargetsGroups












d119901 lt 001 and e




















(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3












astro

cnem

ius m

uscle

wei

ghts

00

01

02

03

04

05

06

07


ab ab

ab aba

ccd cd cd cd

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)


119901 lt 001





Calf

mus

cle st

reng

th (N

)

0

2

4

6

8

10

ab abab

ab

a

Controls

Sham

NTX

Oxy

met

holo

ne 500

250

125

EESF treated (mgkg)

















d119901 lt 001 and e




MDA(nMmg protein)


GSH(nMmg protein)


CAT(Umg protein)









d119901 lt 001 and e















4 Discussion








2





TargetsGroups












d119901 lt 001 and e




















(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3






















d119901 lt 001 and e




MDA(nMmg protein)


GSH(nMmg protein)


CAT(Umg protein)









d119901 lt 001 and e















4 Discussion








2





TargetsGroups












d119901 lt 001 and e




















(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3





















4 Discussion








2





TargetsGroups












d119901 lt 001 and e




















(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3













TargetsGroups












d119901 lt 001 and e




















(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3













(a)

(b)

(c)

(d)

(e)

(f)




AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3













AntibodyGroups













d119901 lt 001 and e



(a)

(b)

(c)

(d)

(e)

(f)







5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3
















5 Conclusion






Acknowledgment


References




















1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3

























1

- 1205732

- and 1205733



































4













































A2119860

and A2119861



1

A2119860

andA3
























4













































A2119860

and A2119861



1

A2119860

andA3

































A2119860

and A2119861



1

A2119860

andA3











Schisandrae Fructus Supplementation Ameliorates Sciatic ......2.8.4. GSH Content Measurement....

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Transcript of Schisandrae Fructus Supplementation Ameliorates Sciatic ......2.8.4. GSH Content Measurement....