Research Article Rhubarb Tannins Extract Inhibits the...

Research ArticleRhubarb Tannins Extract Inhibits the Expression of Aquaporins2 and 3 in Magnesium Sulphate-Induced Diarrhoea Model

Chunfang Liu1 Yanfang Zheng12 Wen Xu12 Hui Wang12 and Na Lin1

1 Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences No 16 Nanxiaojie DongzhimenneiBeijing 100700 China

2 Fujian University of Traditional Chinese Medicine No 1 Huatuo Road Minhoushangjie Fuzhou 350122 China

Correspondence should be addressed to Na Lin linna888163com

Received 19 May 2014 Revised 14 July 2014 Accepted 21 July 2014 Published 18 August 2014

Academic Editor Paul M Tulkens

Copyright copy 2014 Chunfang Liu 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

Tannins a group of major active components of Chinese rhubarb and widely distributed in nature have a significant antidiarrhoealactivity Aquaporins (AQPs) 2 and 3 play important roles in regulating water transfer during diarrhoea The present study aims todetermine the effect of the total tannins extract of rhubarb on aquaporins (AQPs) 2 and 3 in diarrhoea mice and HT-29 cells bothinduced bymagnesium sulphate (MgSO

4) Our results showed that rhubarb tannins extract (RTE) significantly decreased the faecal

water content in colon and evaluation index of defecation of diarrhoea mice Interestingly RTE could markedly reduce the mRNAand protein expression levels of AQPs 2 and 3 in apical and lateral mucosal epithelial cells in the colons of diarrhoeamice andHT-29cells both induced byMgSO

4in a dose-dependentmanner Furthermore RTE suppressed the production of cyclicmonophosphate-

(cAMP-) dependent protein kinase A catalytic subunits 120572 (PKAC-120572) and phosphorylated cAMP response element-binding protein(p-CREB Ser133) in MgSO

4-induced HT-29 cells Our data showed for the first time that RTE inhibit AQPs 2 and 3 expression in

vivo and in vitro via downregulating PKAp-CREB signal pathway which accounts for the antidiarrhoeal effect of RTE

1 Introduction

Diarrhoea is the frequent passage of unformed loose orwatery stools usually three ormore times in 24 hours [1 2] Inthe pathogenesis of diarrhea transepithelial hypersecretionof fluid in the gastrointestinal (GI) tract and defects in waterabsorption in the colon are both important factors Theregulation of transepithelial fluid transport in the GI tractis based on ion transport and also on water transport byaquaporin- (AQP-) type water channels [3] In particular achange in expression of AQPs in the colon appears to be cor-related with the severity of diarrhoea in animals and patients[4] AQPs constitute a family of small integral membraneproteins that are selectively permeable to water and drivenby osmotic gradients [5ndash8] It has been reported that a defector an increase in the expression andor function of AQPs2 3 4 and 8 underlies many kinds of diarrhoea [9ndash15]Of these extensive research has been conducted on AQPs2 and 3 which are considered to play an important rolein regulating water transfer in the colon during diarrhoea

[9 16 17] Noteworthily increasing knowledge about thestructure and function of AQPs led to new approaches usingAQPs as drug targets [18 19] Thus AQPs 2 and 3 are likelyto prove central to the pathophysiology of diarrhoea andultimately they could be a target for therapy in diarrhoea

Diarrhoea is a common cause of death in developingcountries and the second most common cause of infantdeaths worldwide About four billion cases occur each yearand 3-4 million individuals die as a result annually [20]In many cases of diarrhoea the treatments utilized are oralrehydration therapy and pharmacological intervention suchas antibiotics Although oral rehydration salt has greatlyimproved clinical outcome in cholera and other diarrheasthere remains significant mortality from infectious diar-rheas with recurrent major outbreaks Antibiotics used asantidiarrheal drugs sometimes provoke adverse effects andmicroorganisms tend to develop resistance toward them [21]In this regard use of traditionalmedicines to combat the con-sequences of diarrhea has been emphasized by World HealthOrganization in its Diarrhoea Control Programme [22 23]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 619465 14 pageshttpdxdoiorg1011552014619465

2 BioMed Research International

It is therefore important to identify and evaluate availablenatural drugs as alternatives to current antidiarrheal drugswhich are not always free from adverse effects

Rhubarb an old and well-known traditional Chineseherbal medicine has been used as a folk remedy for gas-trointestinal disease including diarrhea and constipation forover two thousand years in China Today it is alreadyofficially listed in Chinese European and Japanese Pharma-copoeia [24ndash26] Rhubarb has many pharmacological activi-ties including purgation [27] antidiarrhoeal effect [28] anti-inflammation [28] and immunoregulation [29] On the otherhand rhubarb contains lots of compounds mainly includ-ing anthraquinones (anthraquinone derivatives) tanninspolysaccharides and other substances Among these tan-nins widely distributed in nature and present in almostall plant foods and some beverages are the major activecompounds of rhubarb moreover they are often the activecompounds of the other medicinal plants in which theyoccur [30 31] It is demonstrated that the antidiarrhoealactivity of many medicinal plants which are rich in tanninsis related to the existence of tannins [2 32] In particularthe purgative activity of rhubarb extract could be significantlyincreased by reducing the content of tannins [33] and someprocessed products from crude rhubarb with decrease ofpurgative anthraquinones and relative increase of tanninseven exhibited an antidiarrhoeal activity [34] The first directconvincing evidence of antidiarrhoeal activity of rhubarbtannins is that successive administration of the total tanninsextract of rhubarb produces the antidiarrhoeal effect onmag-nesium sulphate- (MgSO

4-) and castor oil-induced diarrhoea

mice by protein-precipitating reaction to the GI mucosa [35]Besides previous studies have demonstrated that tanninsreduce significantly intraluminal fluid accumulation by theantisecretory activity and promoting reabsorption of waterand decrease intestinal motility [36ndash38] However the effectof tannins on AQPs 2 and 3 remains unknown which playimportant roles in regulating water transfer in the colonduring diarrhoea Therefore we here investigated the effectof the total tannins extract of rhubarb on intestinal fluidaccumulation and the expression of AQPs 2 and 3 inMgSO

4-

induced diarrhoeamousemodel and the results were furtherconfirmed in HT-29 cells In addition the mechanism ofthe changes induced by rhubarb tannins in the expression ofAQPs 2 and 3 was examined

2 Materials and Methods

The study was approved by Research Ethics Committee ofChina Academy of Chinese Medical Sciences in accordancewith the National Institutes of Health Guidelines for the Careand Use of Laboratory Animals All animals were treated inaccordance with the guidelines and regulations for the useand care of animals of theCenter for LaboratoryAnimalCareChina Academy of Chinese Medical Sciences

21 Preparation of RTE The dried root and rhizoma ofRheum palmatum produced in Yushu county of Qinghaiprovince of China were purchased from the Yushu Pharma-ceuticals Company and were identified by Professor Shilin

Hu a taxonomist at China Academy of Chinese MedicalSciences A voucher specimen (Rh201005Z) was depositedin the institute RTE was prepared according to the reportedmethod with some modified steps [35] 1 kg of rhubarb waspowdered and extractedwith 5 L of 60 ethanol for 30min ina reflux condenser for three times and then was filtrated Thefiltrate was merged and concentrated to 500mL in vacuo ina rotary evaporator at 40∘C Then the concentrated solutionwas added into D-101 macroporous resin with 25 ethanolas the eluent The solution was concentrated continually andthen precipitated with 1 gelatinThe sediment was dissolvedin acetone recovered acetone and dried in a vacuum oven asthe total tannins extract of rhubarb The total tannins extractwas free of anthraquinones according to the color reactionsof anthraquinones with magnesium acetate methanol solu-tion and tannins with ferric chloride solution The yield ofthe total tannins from rhubarb was 340 The total tanninsextract of rhubarb was diluted with distilled water or cellculture medium to the proper concentration before use invivo and in vitro experiments as follows

22 High-Performance Liquid Chromatography (HPLC) Fin-gerprint of RTE The authentic standards of gallic acid andepigallocatechin gallate were purchased from Jingchun Bio-logical Technology Co Ltd (Shanghai China)The standardsof procyanidin-B1 and procyanidin-B2 were obtained fromTauto Biotech Co Ltd (Shanghai China) Standards of cate-chin and epicatechin were supplied by National Institute forFood and Drug Control (Beijing China) RTE and standardsubstance were dissolved in 50 methanol and then filtratedthrough 045 120583m filter membrane HPLC was performed ona ShimadzuHPLC system (Shimadzu Corporation ShimaneJapan) equipped with an LC-20AT binary pump an SPD-M20Adiode array detector a CBM-20Alite system controlleran SIL-20A autosampler a DGU-20A5 degasser and a CTO-10ASvp column oven An Ultimate XB-C18 column (150times 46mm 5 120583m particle size) was maintained at 30∘CDetection wave length was set at 280 nm The mobile phasefor fingerprint analysis consisted of solvent A (acetonitrileMerck NJ USA) and solvent B (01 formic acidwater vv)at a flow rate of 08mLmin A gradient program was usedas follows 27ndash10 B at 0ndash25min The injected volume was10 120583L Online UV spectra were recorded in the range of 190ndash800 nmTheHPLC fingerprint profile of RTEwas establishedby optimizing the chromatographic separate conditions Thechromatogram of RTE at 280 nm could exhibit the wholechromatographic characteristic in Figure 1(a) By comparisonof the retention time and online UV spectra of the referencestandards 6 compounds of tannins were identified as previ-ously reported in rhubarb [39] The structure of gallic acidprocyanidin-B1 catechin procyanidin-B2 epicatechin andepigallocatechin gallate were shown in Figure 1(b)

23 Quantitative Analysis of RTE The content of the RTEwas determined using Shimadzu Visible Ultraviolet Spec-trophotometer UV-1800 (Shimadzu Technologies) accordingto themethod inChinese Pharmacopoeia (Committee for thePharmacopoeia of China 2010) with gallic acid as reference

BioMed Research International 3

00 25 50 75 100 125 150 175 200 225 250minus5000

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Epicatechin(F)

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OH

OH

HO

Epigallocatechin gallate

(b)

Figure 1 HPLC profile (UV chromatograms at 280 nm) of rhubarb tannins extract (RTE) (a) HPLC fingerprint of RTE (1)mdashRTE (2)mdashstandards (b) Structure of identified components of RTE (A)mdashgallic acid (B)mdashprocyanidin-B1 (C)mdashcatechin (D)mdashprocyanidin-B2 (E)mdashepicatechin (F)mdashepigallocatechin gallate

substance The mass of tannins in extracts was 5569equivalent of gallic acid

24 Induction of MgSO4-Induced Diarrhoea Mouse Model

and RTE Treatment Thirty male ICR mice (18ndash22 g) wereobtained from Laboratory Animal Center of Academy ofMilitary Medical Sciences Beijing China (License NoSCXK 2007-004)They were kept in a temperature controlledenvironment (22 plusmn 2∘C) 55 plusmn 5 relative humidity with a12 h 12 h light-dark cycle and fed with standard chow forat least one week before any manipulations ICR mice were

divided separately into 5 groups randomly with the equalnumber (119899 = 6) the normal control group (Control) theMgSO

4-induced diarrhoea model group (Vehicle) and the

RTE- (125 250 and 500mgkg resp) treated diarrhoea micegroups Five groups of mice were orally administrated bysyringe feeding with distilled water (20mLkg) or RTE dailyfor three days respectively Eachmouse except for the normalgroup was orally administrated with MgSO

4sdot7H2O (2 gkg

Jinhuitaiya Chemical Industries Tianjin China) 1 h afterthe last administration to induce diarrhoea as previouslyreported [35] The assay was repeated 3 times


25 Fecal Water Content Detailed procedures have beenpreviously described [12] Briefly fecal samples from themicewere collected for up to 5 h after the administration of theMgSO

4solutions and were placed in silica gel followed by

drying for 48 h in a desiccator The fecal water content pergram of feces was calculated based on the difference betweenthe wet and dry fecal weights This was then used to calculatethe percentage of water in the fecal samples These percent-ages were averaged among six animals for each group andthe assay was repeated 3 times

26 Evacuation Index of Mice Defecation The method aspreviously reported [35] was used to assess the defecationfunction of mice A numerical score based on stool consis-tency was assigned 1 = normal stool 2 = semisolid stool and3 = watery stool Each mouse received an evacuation index(EI) expressed according to the formula EI = 1 times (number ofnormal stool) + 2 times (number of semi-solid stool) + 3 times (num-ber of watery stool) The data were expressed as mean EI foreach group (119899 = 6) The assay was repeated 3 times

27 Cell Culture Human colon cancer HT-29 cells werepurchased from the Type Culture Collection of the ChineseAcademy of Sciences HT-29 cells were maintained in sterileDulbeccorsquos Modified Eagle Medium (DMEM) supplementedwith 10 FBS 100UmL penicillin 100120583gmL streptomycinand 2mM Gln-glutamine at 37∘C Cells were plated on a24-well plate 96-well plate or 100-mm dish at a densityof 2 times 105 cellscm2 incubated in a CO

2incubator at 37∘C

for 24 h and then treated with compounds dissolved inculture medium Experiments were done using cells that hadpreviously been passaged five to fifteen times

28 Cell Viability Assay HT-29 cells were seeded in 96-well plates and incubated in serum free sterile DMEM(supplemented with 100UmL penicillin 100 120583gmL strepto-mycin and 2mM Gln-glutamine) for 24 h Cells were thenincubated with media containing MgSO

4(50mM) andor

RTE (20 40 and 80 120583gmL resp) for 24 h After treatmentcells were washed twice with phosphate-buffered saline(PBS pH 74) and then cell viability was determined by 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) method using Cell Titer 96 Non-Radioactive CellProliferation Assay (Promega Madison USA) according tothe manufacturerrsquos instructions All absorbance at 570 nmwas measured with a microplate reader The experimentswere carried out 3 times in triplicate measurements

29 Immunolocalization Mice were sacrificed by cervicaldislocation under ether anesthesia at 2 h after the administra-tion of the MgSO

4solutions and the colons were removed

After washing the colons with PBS the samples were fixedimmediately for 3 h in 4 paraformaldehyde and embeddedin paraffin and tissue sections (5120583m) of colon were preparedfor immunofluorescence analysis

For in vitro experiments HT-29 cells were cultured in24-well plates with medium containing MgSO

4andor RTE

for 24 h The control was treated with the original medium

After washing with PBS twice cells were fixed immedi-ately for 15min in 4 paraformaldehyde and prepared forimmunofluorescence analysisThe experiments were done intriplicate for three times

Immunofluorescence analyses were carried out followingthe protocol of previous study [9 40] Tissue sections (5 120583m)of colon and HT-29 cells were treated with 02 Triton inPBS prior to blocking with 5 normal goat serum (NGS)in TPBS-BSA (PBS containing 005 Tween-20 and 01bovine serumalbumin)Theprimary rabbit anti-AQP2 (1 50Abcam Cambridge MA UK) antibody or rabbit anti-AQP3antibody (1 50 Abcam) in TPBS-BSA supplemented with1NGS was incubated on the tissue and cells overnight at4∘C Then the tissue and cells were washed extensively withthe TPBS-BSA After that the sections and cells were exposedfor 2 h at room temperature to a secondary goat anti-rabbitantibody conjugated with Alexa Fluor 488 (1 200 Zhong-shanjinqiao Beijing China) Then the tissue and cells werereacted with 46-diamidino-2-phenylindole (DAPI) solution(Beyotime Jiangsu China) in PBS at room temperature for5min washed 3 more times with PBS and then cover-slipped with vectashield (Zhongshanjinqiao) and visualizedusing a Zeiss Lsm710 confocal microscope (Carl Zeiss AGOberkochen German)

210 Preparation of Tissue and Cell Extraction forWestern BlotAnalysis The colons were removed frommice Large intesti-nal mucosa scraped with a slide glass was homogenized usingdissecting buffer (03M sucrose 25mM imidazole 1mMEDTA 85mM leupeptin and 1mM phenylmethylsulfonylfluoride pH 72) on ice The resulting suspension was cen-trifuged (800timesg at 4∘C for 15min) and the supernatant wascentrifuged (200000timesg at 4∘C for 1 h) The precipitate wasresuspended using dissecting buffer Protein concentrationsweremeasured by the Lowrymethod [41] usingBSAas a stan-dardThe protein expression levels of AQPs 2 and 3 in colonswere determined

AQPs 2 and 3 were expressed in the plasma membraneand in intracellular vesicles Thus the crude fraction domi-nantly containing these fractions in HT-29 cells was preparedas previously reported [10] Briefly HT-29 cells were culturedin 100-mmdishes withmedia containingMgSO

4andor RTE

for further culture at 37∘C for different times (3 6 12 and24 h resp)The controlwas treatedwith the originalmediumAfter washing with PBS cells were recovered and suspendedin dissecting buffer The cell suspension was homogenizedand the homogenate was centrifuged (4000timesg for 10min)The supernatant was centrifuged (200000timesg for 1 h) and theresulting supernatant was discarded Dissecting buffer wasadded to the pellets and the suspension was homogenizedusing an ultrasonic homogenizer to make a crude fraction[42] All procedures were carried out at 4∘C

The phosphorylation of cyclic monophosphate (cAMP)response element-binding protein (CREB) occurred in thenucleus Thus the nuclear fraction dominantly containingthe nucleus was prepared as previously reported [10] andthe protein expression level of phosphorylated CREB (p-CREB) was determined Briefly isolated HT-29 cells werecultured with media containing MgSO

4andor RTE for


VehicleRTE (mgkg)

Control 125 250 500

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)

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RTE 500mgkg

200120583m

Control

(d)

Figure 2 Rhubarb tannins extract (RTE) has the antidiarrhoeal activity in magnesium sulphate- (MgSO4-) induced diarrhoea mice Mice

were orally administrated with RTE (125 250 and 500mgkg resp) or water daily for 3 days before MgSO4-induced diarrhoea During the

experiment fecal water content and evacuation index (EI) of mice defecation were evaluated (a) Doses of 125sim500mgkg RTE significantlydecreased the fecal water content dose-dependently compared with vehicle-treated diarrhoea mice (b) Doses of 125sim500mgkg RTEsignificantly decreased the EI of defecation in a dose-dependent manner compared with vehicle-treated diarrhoea mice (c) Macroscopicevidence of watery stool in colon was markedly observed in vehicle-treated diarrhoea mice while dose of 125sim500mgkg RTE significantlyalleviated the watery stool of diarrhoea mice (d) Histological structure of colon in mice induced by MgSO

4appeared edematous while RTE

treatment seemed to reduce edema Data are represented as the mean plusmn SD 119875 lt 0001 versus normal control group lowast119875 lt 005 lowastlowast119875 lt 001and lowastlowastlowast119875 lt 0001 versus vehicle group respectively 119899 = 6 in each group and each assay was repeated 3 times

24 h After that the cells were treated with extraction buffer(phosphosafe extraction reagent with 85 120583M leupeptin and1mM phenylmethylsulfonyl fluoride) The suspension wasleft on ice for 5min After detaching and recovering cellswith a cell scraper the cells were then homogenized andcentrifuged (4000timesg for 10min) After the supernatant wasdiscarded extraction buffer was added to the pellets Thesuspension was centrifuged (16000timesg for 30min) to obtainthe supernatant as a nuclear fraction [43 44] All procedureswere carried out at 4∘C

Meanwhile HT-29 cells were treated with MgSO4andor

RTE and then dissociated with lysis buffer (20mM MOPS50mM 120573-glycerophosphate 50mM sodium fluoride 1mM

sodium vanadate 5mM EGTA 2mM EDTA 1 NP-401mM DTT 1mM benzamidine 1mM PMSF 10120583gmL leu-peptin and 10 120583gmL aprotinin) After that the cells wererecovered and centrifuged (16000timesg for 15min at 4∘C) Thesupernatant was recovered and the protein expression ofcAMP-dependent protein kinaseA (PKA)was determined bywestern blot

211 Western Blot Analysis The western blot protocol andsemiquantitative analysis were carried out following the pro-tocol of previous study [10]The following primary antibodieswere used AQP2 (rabbit polyclonal antibody dilution 1 200Abcam) AQP3 (rabbit polyclonal antibody dilution 1 200


Negative control

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Figure 3 Continued


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Figure 3 Rhubarb tannins extract (RTE) reduces the protein expression of aquaporins (AQPs) 2 and 3 in the colons of magnesium sulphate-induced diarrhoea mice by fluorescent immunohistochemistry ((a) and (b)) Localization of AQPs 2 and 3 in both the apical and lateralmucosal epithelial cells in the proximal colons of normal control vehicle-treated diarrhea and RTE- (500mgkg) treated diarrhoea miceNegative controls in which the AQPs 2 and 3 antibodies were replaced with normal rabbit immunoglobulin (IgG) at identical concentrationsresulted in a lack of specific staining Tissue is colocalized with DAPI to demonstrate the location of cell nuclei (blue) The images in mergersquopanel are the big magnification in merge panel respectively ((c) and (d)) Positive expression levels of AQPs 2 and 3 in the colons of normalcontrol vehicle-treated diarrhea and RTE- (125 250 and 500mgkg) treated diarrhoea mice 10 microscopic fields were selected randomlyand AQPs 2 and 3 positive cells were counted The AQPs 2 and 3 positive expression levels of the control were taken as 100 Data arerepresented as the mean plusmn SD 119875 lt 0001 versus normal control group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus vehicle grouprespectively 119899 = 6 in each group and each assay was repeated 3 times

Abcam) and PKA C-120572 (rabbit polyclonal antibody dilution1 1000 Cell Signaling Boston USA) p-CREB (ser133 rabbitpolyclonal antibody dilution 1 500 Thermo Fisher Scien-tific Waltham UK) and GAPDH (internal control rabbitpolyclonal antibody dilution 1 1000 Beyotime China) Agoat-anti-rabbit or goat-anti-mouse antibody conjugated tohorseradish peroxidase (1 2000 Thermo Fisher Scientific)was used as the second antibody AQP2 AQP3 PKA andp-CREB protein levels were normalized against GAPDH Allprotein expressions are given as percentages compared to thecontrol group (100) All experiments were done for threetimes

212 RNA Isolation and TaqManTM Real-Time RT-PCR Thecolons were dissected from mice at 2 h after the administra-tion of the MgSO

4solutions snap-frozen in liquid nitrogen

ground into powder and homogenized This procedure wasdone under RNase-free conditions The RNA isolation andreal-time PCR assay were carried out following the protocolof previous study [11] Briefly total RNA was extractedwith TRIzol reagent (Invitrogen USA) from the tissuehomogenates according to the manufacturerrsquos instructionsThe total RNA (1 120583g) was reverse transcribed to cDNA usingthe QuantiTect Reverse Transcription Kit (QIAGEN Japan)according to the instruction manual The specific transcriptswere quantified by quantitative real-time PCR using TaqManUniversal Master Mix II (ABI USA) and analyzed with ABI7500 real-time PCR system (ABI) Gene-specific primerswere used for AQP2 (Mm00437575 m1 Cat 431182 ABI)

AQP3 (Mm01208559 m1 Cat 431182 ABI) and 18S rRNA(Mm00835095 g1 Cat 431182 ABI) The mRNA levels ofAQP2 and AQP3 were normalized to 18S rRNA level PCRwas performed as 40 cycles at 95∘C for 15 s 56∘C for 30 s and72∘C for 30 s The relative mRNA expression was calculatedwith comparative 119862

119879method All experiments were done in

triplicate for three times

213 Statistical Analysis Thesoftware of SPSS version 160 forWindows (SPSS Chicago IL USA) was used for statisticalanalysis Values were expressed as means plusmn SD Statisticaldifferences were analyzed using One-Way-ANOVA methodfollowed by Turkeyrsquos multiple comparison test Results with119875 lt 005 were considered to be significant

3 Results

31 RTE Has the Antidiarrhoeal Effect in MgSO4-Induced

DiarrhoeaMice To investigate the effect of RTE on diarrheathe MgSO

4-induced diarrhoea model in mice was used As

shown in Figure 2(a) fecal water content in mice up to 5 hafter MgSO

4administration increased significantly versus

that in normal mice (119875 lt 001) and severe diarrhoea wasobserved RTE dose-dependently interfered with the fecalwater content in MgSO

4-induced diarrhoea mice (all 119875 lt

005) Consistent with the fecal water content the assessmentof EI of mice defecation also showed RTE to be highly effec-tive (all 119875 lt 005 Figure 2(b)) Additionally macroscopicevidence of watery stool in colon was markedly observed in


GAPDH

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(a)

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Figure 4 Rhubarb tannins extract (RTE) inhibits the protein and gene expression of aquaporins (AQPs) 2 and 3 in the colons of magnesiumsulphate-induced diarrhoea mice by western blot and real-time Reverse Transcription-Polymerase Chain Reaction ((a) and (b)) Expressionlevels of AQPs 2 and 3 protein in the colons of normal control vehicle-treated diarrhea and RTE- (125 250 and 500mgkg) treated diarrhoeamice ((c) and (d)) Expression levels of AQPs 2 and 3 mRNA in the colons of mice Data are represented as the mean plusmn SD 119875 lt 0001versus normal control group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus vehicle group respectively 119899 = 6 in each group and each assaywas repeated 3 times

vehicle-treated diarrhoea mice while dose of 125ndash500mgkgRTE significantly alleviated the watery stool of diarrhoeamice (Figure 2(c)) Meanwhile the histological structureof colon in mice induced by MgSO

4appeared edematous

while RTE treatment seemed to reduce edema (Figure 2(d))Besides we also investigated the effect of RTE on diarrhoeamice induced by castor oil (03mLmouse po) and theresults are similar to above (data not shown)

Nextly apparent adverse effects including weight lossalterations of physical appearance and behavior changeswere not noted in mice treated with RTE (results not shown)Taken together these observations indicate that systemicadministration of RTE in mice has the antidiarrhoeal activityby inhibiting intestinal fluid accumulation without seriousside effects

32 RTE Inhibits the Production and Gene Expression of AQPs2 and 3 Both InVivo and InVitro AQPs 2 and 3mainly local-ized in the colon play a very important role in transepithelialfluid transport and involved in many kinds of diarrhoea [9ndash11] As shown in Figure 3 significant amounts of AQPs 2 and3 positive staining were present in both the apical and lateralmucosal epithelial cells in the colon tissues from vehicle-treated mice while there was evident depression of AQPs 2and 3 in the colon tissues from RTE-treated mice As shownin Figures 4(a) and 4(b) compared with vehicle-treated diar-rhoea mice doses of 125sim500mgkg RTE markedly reducedthe protein levels of AQPs 2 and 3 in the colon tissues ofdiarrhoea mice by western blot analysis (all 119875 lt 005)Additionally findings were similar to the gene expressionlevels of AQPs 2 and 3 (all 119875 lt 005 Figures 4(c) and 4(d))


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RTE80120583gmL

(a)

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(d)

Figure 5 Rhubarb tannins extract (RTE) decreases the protein expression of aquaporins (AQPs) 2 and 3 inHT-29 cells induced bymagnesiumsulphate (MgSO

4) by fluorescent immunohistochemistry Cells were placed in 24-well plate or 100-mm dishes for 24 h in the presence of

MgSO4with or without RTE (control MgSO

4and RTE- (20 40 and 80 120583gmL) treatment groups resp) HT-29 cells were fixed stained

by immunofluorescence and scanned in 10 random fields ((a) and (b)) Immunolocalization of AQPs 2 (green) and 3 (red) in HT-29 cellsNegative controls in which the AQPs 2 and 3 antibodies were replaced with normal rabbit immunoglobulin (IgG) at identical concentrationsresulted in a lack of specific staining Nuclei were stained with DAPI (blue) ((c) and (d)) Positive expression levels of AQPs 2 and 3 in HT-29cells 10 microscopic fields were selected randomly and AQPs 2 and 3 positive cells were countedThe AQPs 2 and 3 positive expression levelsof the control were taken as 100 Data are represented as the mean plusmn SD 119875 lt 0001 versus normal control group lowast119875 lt 005 lowastlowast119875 lt 001and lowastlowastlowast119875 lt 0001 versus vehicle group respectively 119899 = 3 in each group and each assay was repeated 3 times

These results suggest that RTE reduces the protein and geneexpression of AQPs 2 and 3 in vivo

HT-29 cells have been widely used in the study of themechanisms of diarrhoea and laxative actions since HT-29 cells represent the normal physiological condition of thecolon despite the fact that they are cancer cell lines derivedfrom human colon cancer [45 46] In the study the proteinand gene expression of AQPs 2 and 3 were also investigatedin HT-29 cells As shown in Figure 5 significant amounts ofAQPs 2 and 3 staining were present in MgSO

4-treated HT-

29 cells while there was evident depression of AQPs 2 and 3

in HT-29 cells treated by RTE Western blot analysis demon-strated that the expression levels of AQPs 2 and 3 proteinsin HT-29 cells were reduced by RTE at a time-dependentmanner (all 119875 lt 005 Figure 6(a) for 6 12 and 24 h all119875 lt 005 Figure 6(b)) and a dose-dependent manner (all119875 lt 005 Figures 6(c) and 6(d)) respectively These resultssuggest that RTE reduces the protein expression of AQPs 2and 3 in vitro

33 RTEDownregulatesMgSO4-Induced Activation of PKAp-

CREB Downstream Pathway In order to investigate whether


lowast lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP2

prot

ein

( o

f0h)

(a)

lowastlowast


AQP3

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP3

prot

ein

( o

f0h)

(b)

lowast

lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

100

200

300

VehicleControl 20 40 80

RTE (120583gmL)

AQP2

prot

ein

( o

f con

trol)

(c)

lowast


AQP3

GAPDH

0

100

200

300

400


RTE (120583gmL)

AQP3

prot

ein

( o

f con

trol)

(d)

Figure 6 Rhubarb tannins extract (RTE) reduces the protein expression of aquaporins (AQPs) 2 and 3 in magnesium sulphate- (MgSO4-)

induced HT-29 cells by western blot Cells were placed in 24-well plate or 100-mm dishes for 24 h in the presence of MgSO4with or without

RTE (control MgSO4 and RTE- (20 40 and 80 120583gmL) treatment groups resp) ((a) and (b)) Expression levels of AQPs 2 and 3 protein in

HT-29 cells treated by RTE (80 120583gmL) for different time (0 3 6 12 and 24 h resp) ((c) and (d)) Expression levels of AQPs 2 and 3 proteins inHT-29 cells treated by RTE at the dosages of 20 40 and 80 120583gmL for 24 h respectively Data are represented as the mean plusmn SD 119875 lt 0001versus normal control group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus vehicle group respectively 119899 = 3 in each group and each assaywas repeated 3 times

the activation of MgSO4-induced PKAp-CREB signal path-

way was modulated by RTE we further detected the expres-sion levels of PKA and p-CREB protein in MgSO

4-induced

HT-29 cells As shown in Figures 7(a) and 7(b) RTEmarkedlydiminished the activation of MgSO

4-induced PKA (all 119875 lt

005) and p-CREB (all 119875 lt 005) which are associated withthe production of AQPs 2 and 3 in HT-29 cells

Nextly we examinedwhether the above suppressive effectof RTE was due to its cytotoxicity When confluent HT-29cells were treated with RTE andor MgSO

4for 24 h the cyto-

toxicity was monitored by MTT assay Our results showedthat RTE did not exert any cytotoxic effects on HT-29 cellsunder the experimental conditions used in the present study(Figure 7(c)) suggesting that RTEmight specifically suppressAQPs 2 and 3 PKA and p-CREB expression in vitro

4 Discussion

Tannins a unique group of phenolic metabolites with theproperty of precipitating proteins are commonly found inplants such as apple fruit pine bark grape seed tea oakand medicinal plants and possess a variety of biologicaleffects including anticarcinogenic antimutagenic antimi-crobial and antioxidative activities [30 47] Besides tanninsextracted from many medicinal plants also have exhibitedan antidiarrhoeal activity (Table 1) The mechanisms oftheir antidiarrhoeal activity are those by inhibiting cysticfibrosis transmembrane conductance regulator protein chlo-ride channels [39] and by generating protein-precipitatingreaction to the gastrointestinal mucosa due to the protein-precipitating action [48] However their effects on AQPsare still unclear In 2011 the total RTE has proved the


lowast


GAPDH

0

100

200

300


RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

200

300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)

Figure 7 Rhubarb tannins extract (RTE) downregulates the expression of magnesium sulphate- (MgSO4-) activated PKA and p-CREB in

HT-29 cells without effect on cell viability Cells were placed in 100-mmdishes for 24 h in the presence ofMgSO4with orwithout RTE (control

MgSO4and RTE- (20 40 and 80 120583gmL) treatment groups resp) HT-29 cells were collected to detect the expression levels of PKA (a) and

p-CREB (b) protein by western blot analysis (c) No effect of RTE (20 40 80 120583gmL resp) on the cell viability by 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromidemethod Cell viability of the control was taken as 100 Data are represented as themean plusmn SD 119875 lt 0001versus normal control group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus vehicle group respectively 119899 = 3 in each group and each assaywas repeated 3 times

Table 1 Traditional antidiarrhoeal plants with the composition of tannins

Scientific name Part used Levels of tannins Diarrhoea models ReferencesLibidibia ferrea (Mart ex Tul) L P Queiroz (Caesalpinia ferreaMart) Bark 624 mdash [49]Caesalpinia pyramidalis Tul Bark 601 mdash [49]Croton blanchetianus Baill Bark 247 Castor oil [49]Eugenia uvalha Cambess Bark 168 mdash [49]Spondias tuberosa Arruda Bark 151 mdash [49]Cedrela odorata L Bark 209 mdash [49]Endopleura uchi Bark mdash mdash [50]Rhododendron arboreum Flower mdash Castor oil MgSO4 [51]Green tea Leaf mdash mdash [52]Heeria insignisO Ktze (Anacardiaceae) Leaf mdash Castor oil [53]Trilepisium madagascariense Bark mdash Castor oil [54]Ixora coccineaLinn (Rubiaceae) Flower mdash Castor oil [22]Capparis zeylanica Leaf mdash Castor oil [55]Rhubarb Root 1355 Castor oil MgSO4 [35]Note mdash not detected


antidiarrhoeal activity in different kinds of diarrhoea modelsfor the first time [35] In the study we further investigated theeffect of RTE on AQPs 2 and 3 in vivo and in vitro The resultswill help to elucidate the antidiarrhoeal action of tanninswidely distributed in nature

Wet stool is one of the major characteristics of diarrheawhich is usually caused by altered motility and fluid accumu-lation in the intestine lumen MgSO

4 an osmotic-acting lax-

ative has been reported to induce diarrhoea by increasing thevolume of intestinal content through prevention of reabsorp-tion of water In the present study diarrhea characterized byintestinal fluid accumulation was developed in mice after theadministration of MgSO

4 Obvious watery stool in the colon

increased fecal water content and increased EI of defecationwere observed in vehicle-treated mice While in RTE-treatedmice we noticed that RTE attenuated the severity of diarrheaby reducing the degree of watery stool in the colon thefecal water content and EI of defecation in a dose-dependentmanner which is consistent with the data of the previousstudy [35] The results suggest that RTE has an antidiar-rhoeal activity in MgSO

4-induced diarrhoea mice partly

via reducing the colonic water secretion induced by MgSO4

In healthy individuals about 15sim2 L water is absorbeddaily by the colon while the maximal capacity of the humanlarge intestine to absorb fluids may be as high as 5-6 L per day[56] Theoretically water crosses the colonic epithelia eitherby paracellular or transcellular routes driven by an osmoticgradient Because the colon is a tight epithelium character-ized by high electrical resistance there is an underlyingmech-anism by which water can pass the colonic epithelium layeragainst considerable osmotic gradients [57] The discoveryof specific water channels called AQPs on epithelial cells inthe gastrointestinal tract may give a reasonable explanationAQPs are a family of highly conserved transmembrane chan-nel proteins mainly responsible for rapid water movementsThere are currently thirteen known types of AQPs in humansAQP0 through AQP12 that are expressed in a variety oftissues [58 59] In intestine various subtypes of AQP arelocalized (AQPs 1 2 3 4 5 6 7 8 9 and 10) [60ndash63] Ofthese the main types expressed in mucosal epithelial cellsin colon are AQPs 2 3 4 and 8 [9 62] In this study wefocused on AQPs 2 and 3 because the mice deficient inAQPs 4 and 8 demonstrated no alteration in stool watercontent [64 65] We found that AQPs 2 and 3 were obviouslyexpressed in both the apical and lateral mucosal epithelialcells in the colons of MgSO

4-induced diarrhoea mice but

when mice were treated with RTE the expression levelsof AQPs 2 and 3 were significantly decreased which wasconfirmed by immunofluorescence Similarly RTE could alsoreduce both themRNA and protein expression levels of AQPs2 and 3 in the colons of MgSO

4-induced diarrhoea mice by

real-time RT-PCR and western blot analysis Furthermoreto confirm these findings the HT-29 cells which have beenwidely used in the study of the mechanisms of diarrhoeaand laxative actions were employed Compared with HT-29cells treated with MgSO

4 we found that the protein levels

of AQPs 2 and 3 were markedly decreased in HT-29 cellscotreated with MgSO

4and RTE These results indicate that

RTE has the ability to decrease the expression of AQPs 2

and 3 which partly accounts for reducing the colonic watersecretion induced by MgSO

4involved in the antidiarrhoeal

activity of RTEIn addition to these findings we further explored the

precise mechanism involved in the inhibition of AQPs 2 and3 of RTE in diarrhoea induced by MgSO

4 Previous study

has demonstrated that MgSO4as a laxative may increase

the AQP3 expression level in HT-29 cells via the followingPKAp-CREB signal pathway [10] First an increase in theintracellular Mg2+ concentration causes the activation ofadenylate cyclase which leads to an increase in cAMPproduction Then the increase in the cAMP concentra-tion in turn causes PKA activation which promotes CREBphosphorylation Meanwhile the increased expression ofAQP3 in HT-29 cells by vasoactive intestinal polypeptide andnatriuretic peptide is correlated with the activation of PKA[16 30] Another study has reported that the AQP2 expres-sion induced by arginine-vasopressin is also mediated by theincrease of cAMP [66] although the relationship betweenAQP2 expression and the PKAp-CREB pathway in MgSO

4-

induced HT-29 cells is still unclear In the present study ourdata showed that RTE significantly reduced the expressionlevels of PKA andCREB phosphorylation inMgSO

4-induced

HT-29 cells in a dose-dependent manner suggesting that theinhibitory effect of RTE on AQPs 2 and 3 is partly by thedownregulation PKAp-CREB signal pathway

In conclusion our data offered the convincing evidencefor the first time that RTE inhibit AQPs 2 and 3 expressionin vivo and in vitro via downregulating PKAp-CREB signalpathwaywhich partially accounts for the antidiarrhoeal effectof RTEThis observation disclosed a novel function of RTEonthe management of diarrhoea

Conflict of Interests

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

Authorsrsquo Contribution

Chunfang Liu and Yanfang Zheng contributed equally to thiswork

Acknowledgment

This studywas supported by grants from theNational NaturalScience Foundation of China (30902004 and 30730111)

References

[1] I M Mandomando E V Macete J Ruiz et al ldquoEtiology ofdiarrhea in children younger than 5 years of age admitted in arural hospital of Southern MozambiquerdquoThe American Journalof Tropical Medicine and Hygiene vol 76 no 3 pp 522ndash5272007

[2] E A Palombo ldquoPhytochemicals from traditional medicinalplants used in the treatment of diarrhoea modes of action andeffects on intestinal functionrdquo Phytotherapy Research vol 20no 9 pp 717ndash724 2006


[3] J A Hardin L E Wallace J F K Wong et al ldquoAquaporin exp-ression is downregulated in a murine model of colitis and inpatients with ulcerative colitis Crohns disease and infectiouscolitisrdquo Cell and Tissue Research vol 318 no 2 pp 313ndash3232004

[4] K Kunzelmann and M Mall ldquoElectrolyte transport in themammalian colon mechanisms and implications for diseaserdquoPhysiological Reviews vol 82 no 1 pp 245ndash289 2002

[5] L S King and P Agre ldquoPathophysiology of the aquaporin waterchannelsrdquo Annual Review of Physiology vol 58 pp 619ndash6481996

[6] T Ma and A S Verkman ldquoAquaporin water channels in gas-trointestinal physiologyrdquo Journal of Physiology vol 517 no 2pp 317ndash326 1999

[7] K Takata TMatsuzaki and Y Tajika ldquoAquaporins water chan-nel proteins of the cell membranerdquo Progress in Histochemistryand Cytochemistry vol 39 no 1 pp 1ndash83 2004

[8] A S Verkman ldquoAquaporin water channels and endothelial cellfunctionrdquo Journal of Anatomy vol 200 no 6 pp 617ndash627 2002

[9] J A Guttman F N Samji Y Li W Deng A Lin and B BFinlay ldquoAquaporins contribute to diarrhoea caused by attachingand effacing bacterial pathogensrdquo Cellular Microbiology vol 9no 1 pp 131ndash141 2007

[10] N Ikarashi T Mochiduki A Takasaki et al ldquoA mechanism bywhich the osmotic laxative magnesium sulphate increases theintestinal aquaporin 3 expression in HT-29 cellsrdquo Life Sciencesvol 88 no 3-4 pp 194ndash200 2011

[11] N Ikarashi TUshiki TMochizuki et al ldquoEffects ofmagnesiumsulphate administration on aquaporin 3 in rat gastrointestinaltractrdquo Biological and Pharmaceutical Bulletin vol 34 no 2 pp238ndash242 2011

[12] N Ikarashi R Kon T Iizasa et al ldquoInhibition of aquaporin-3 water channel in the colon induces diarrheardquo Biological andPharmaceutical Bulletin vol 35 no 6 pp 957ndash962 2012

[13] H Sakai A Sagara K Matsumoto et al ldquo5-Fluorouracilinduces diarrhea with changes in the expression of inflamma-tory cytokines and aquaporins in mouse intestinesrdquo PLoS ONEvol 8 no 1 Article ID e54788 2013

[14] T Yamamoto H Kuramoto and M Kadowaki ldquoDownregula-tion in aquaporin 4 and aquaporin 8 expression of the colonassociated with the induction of allergic diarrhea in a mousemodel of food allergyrdquo Life Sciences vol 81 no 2 pp 115ndash1202007

[15] Y Zhang XWang S Sha et al ldquoBerberine increases the expres-sion of NHE3 and AQP4 in sennosideA-induced diarrhoeamodelrdquo Fitoterapia vol 83 no 6 pp 1014ndash1022 2012

[16] A Itoh T Tsujikawa Y Fujiyama andT Bamba ldquoEnhancementof aquaporin-3 by vasoactive intestinal polypeptide in a humancolonic epithelial cell linerdquo Journal of Gastroenterology andHepatology vol 18 no 2 pp 203ndash210 2003

[17] T Tsujikawa A Itoh T Fukunaga J Satoh T Yasuoka andY Fujiyama ldquoAlteration of aquaporin mRNA expression aftersmall bowel resection in the rat residual ileum and colonrdquo Jour-nal of Gastroenterology and Hepatology vol 18 no 7 pp 803ndash808 2003

[18] L S King M Yasui and P Agre ldquoAquaporins in health anddiseaserdquo Molecular Medicine Today vol 6 no 2 pp 60ndash652000

[19] J von Bulow ldquoAquaporinsmdashwater channels in the cell mem-brane and therapeutic targetsrdquo Medizinische Monatsschrift furPharmazeuten vol 36 no 3 pp 86ndash94 2013

[20] M J G Farthing ldquoNovel targets for the control of secretorydiarrhoeardquo Gut vol 50 no 3 pp III15ndashIII18 2002

[21] J R Soberon M A Sgariglia D A Sampietro E N Quirogaand M A Vattuone ldquoAntibacterial activity of plant extractsfrom northwestern Argentinardquo Journal of Applied Microbiologyvol 102 no 6 pp 1450ndash1461 2007

[22] Y Maniyar P Bhixavatimath and N V Agashikar ldquoAntidiar-rheal activity of flowers of Ixora Coccinea Linn in ratsrdquo Journalof Ayurveda and Integrative Medicine vol 1 no 4 pp 287ndash2912010

[23] J D Snyder and M H Merson ldquoThe magnitude of the globalproblem of acute diarrhoeal disease a review of active surveil-lance datardquo Bulletin of the World Health Organization vol 60no 4 pp 605ndash613 1982

[24] Pharmacopoeia European Strasbourg 2004[25] Japanese Pharmacopoeia Pharmaceutical and Food Safety

Bureau Ministry of Health Labour and Welfare 2006[26] Pharmacopoeia of the Peoples Republic of China First Division

China Chemical Industry Press Beijing China 2010[27] R KonN Ikarashi CNagoya et al ldquoRheinanthrone ametabo-

lite of sennoside A triggers macrophage activation to decreaseaquaporin-3 expression in the colon causing the laxative effectof rhubarb extractrdquo Journal of Ethnopharmacology vol 152 pp190ndash200 2014

[28] L Liu Z Guo Z Lv et al ldquoThe beneficial effect of Rheumtanguticum polysaccharide on protecting against diarrheacolonic inflammation and ulceration in rats with TNBS-induced colitis the role of macrophage mannose receptor ininflammation and immune responserdquo International Immuno-pharmacology vol 8 no 11 pp 1481ndash1492 2008

[29] L Liu S Yuan Y Long et al ldquoImmunomodulation of Rheumtanguticum polysaccharide (RTP) on the immunosuppressiveeffects of dexamethasone (DEX) on the treatment of colitis inrats induced by 246-trinitrobenzene sulfonic acidrdquo Interna-tional Immunopharmacology vol 9 no 13-14 pp 1568ndash15772009

[30] J Serrano R Puupponen-Pimia A Dauer A Aura and FSaura-Calixto ldquoTannins current knowledge of food sourcesintake bioavailability and biological effectsrdquo Molecular Nutri-tion and Food Research vol 53 no 2 pp 310ndash329 2009

[31] M Ye J Han H Chen J Zheng and D Guo ldquoAnalysis ofphenolic compounds in rhubarbs using liquid chromatographycoupled with electrospray ionization mass spectrometryrdquo Jour-nal of the American Society for Mass Spectrometry vol 18 no 1pp 82ndash91 2007

[32] G J Amabeoku ldquoAntidiarrhoeal activity of Geranium incanumBurm f (Geraniaceae) leaf aqueous extract in micerdquo Journal ofEthnopharmacology vol 123 no 1 pp 190ndash193 2009

[33] H Y ZhongM Zhang YDai andC F Zhang ldquoEffect of tannincontained in Radix et Rhizoma Rhei and Radix Polygoni Mul-tiflori on small intestinal propulsionrdquo Lishizhen Medicine andMateria Medica Research vol 17 pp 2478ndash2479 2006

[34] S T Zhu P Lei X Z Li S Liu andQ L Li ldquoComparative studyof different processed products of rhubarb on purgative andhemostatic activitiesrdquo Journal of Chinese Medicinal Materialsvol 31 pp 199ndash201 2008

[35] Y Qin J Wang W Kong et al ldquoThe diarrhoeogenic andantidiarrhoeal bidirectional effects of rhubarb and its potentialmechanismrdquo Journal of Ethnopharmacology vol 133 no 3 pp1096ndash1102 2011


[36] M J G Farthing ldquoDiarrhoea a significant worldwide problemrdquoInternational Journal of Antimicrobial Agents vol 14 no 1 pp65ndash69 2000

[37] A M Mujumdar A V Misar and A S Upadhye ldquoAntidiar-rhoeal activity of ethanol extract of the bark of Dalbergialanceolariardquo Journal of Ethnopharmacology vol 102 no 2 pp213ndash216 2005

[38] G N Teke J R Kuiate O B Ngouateu and D GatsingldquoAntidiarrhoeal and antimicrobial activities of Emilia coccinea(Sims) G Don extractsrdquo Journal of Ethnopharmacology vol 112no 2 pp 278ndash283 2007

[39] H Sun C Zhu H Y Zhang Y Y Wang G A Luo and P HuldquoComparative analysis of main constituents of Radix et Rhi-zoma Rhei and processed Radix et Rhizoma Rhei by HPLC-ESI-TOF-MSrdquo Chinese Traditional Patent Medicine vol 31 pp420ndash423 2009

[40] N Wongsamitkul L Sirianant C Muanprasat and V Chat-sudthipong ldquoA plant-derived hydrolysable tannin inhibitsCFTR chloride channel a potential treatment of diarrheardquoPharmaceutical Research vol 27 no 3 pp 490ndash497 2010

[41] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquoThe Jour-nal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[42] D Marples M A Knepper E I Christensen and S NielsenldquoRedistribution of aquaporin-2 water channels induced byvasopressin in rat kidney inner medullary collecting ductrdquoTheAmerican Journal of PhysiologymdashCell Physiology vol 269 no 3pp C655ndashC664 1995

[43] G Hawcroft C W S Ko and M A Hull ldquoProstaglandin E2-EP4 receptor signalling promotes tumorigenic behaviour ofHT-29 human colorectal cancer cellsrdquoOncogene vol 26 no 21pp 3006ndash3019 2007

[44] C D Toscano J L McGlothan and T R Guilarte ldquoLeadexposure alters cyclic-AMP response element binding proteinphosphorylation and binding activity in the developing ratbrainrdquo Developmental Brain Research vol 145 no 2 pp 219ndash228 2003

[45] A Itoh T Tsujikawa T Yasuoka T Nakahara M Sasaki andY Fujiyama ldquoNatriuretic peptides up-regulate aquaporin 3 in ahuman colonic epithelial cell linerdquo International journal ofmolecular medicine vol 14 no 4 pp 621ndash626 2004

[46] G Parnaud D E Corpet and L Gamet-Payrastre ldquoCytostaticeffect of polyethylene glycol on human colonic adenocarcinomacellsrdquo International Journal of Cancer vol 92 no 1 pp 63ndash692001

[47] K-T Chung T Y Wong C-I Wei Y-W Huang and Y LinldquoTannins and human health a reviewrdquo Critical Reviews in FoodScience and Nutrition vol 38 no 6 pp 421ndash464 1998

[48] J A Guttman Y Li M E Wickham W Deng A W VoglandB B Finlay ldquoAttaching and effacing pathogen-induced tightjunction disruption in vivordquo Cellular Microbiology vol 8 no 4pp 634ndash645 2006

[49] C F D Q Siqueira D L V Cabral T J D S PeixotoSobrinho et al ldquoLevels of tannins and flavonoids in medicinalplants evaluating bioprospecting strategiesrdquo Evidence-BasedComplementary and Alternative Medicine vol 2012 Article ID434782 7 pages 2012

[50] F A Politi J C de Mello K F Migliato A L NepomucenoR R Moreira and R C Pietro ldquoAntimicrobial cytotoxic andantioxidant activities and determination of the total tannincontent of bark extracts Endopleura uchirdquo International Journalof Molecular Sciences vol 12 pp 2757ndash2768 2011

[51] N Verma A P Singh A Gupta P K Sahu and C V RaoldquoAntidiarrheal potential of standardized extract of Rhododen-dron arboreum Smith flowers in experimental animalsrdquo IndianJournal of Pharmacology vol 43 no 6 pp 689ndash693 2011

[52] W Namkung J R Thiagarajah P Phuan and A S VerkmanldquoInhibition of Ca2+-activated Clminus channels by gallotannins asa possible molecular basis for health benefits of red wine andgreen teardquo The FASEB Journal vol 24 no 11 pp 4178ndash41862010

[53] A Agunu A A Ahmadu S O Afolabi A U Yaro J O Ehin-midu and Z Mohammed ldquoEvaluation of the antibacterial andantidiarrhoeal activities of heeria insignis O Ktzerdquo Indian Jour-nal of Pharmaceutical Sciences vol 73 no 3 pp 328ndash332 2011

[54] G N Teke J R Kuiate V Kuete R B Teponno L A Tapond-jou and G Vilarem ldquoAntidiarrheal activity of extracts andcompound from Trilepisium madagascariense stem barkrdquoIndian Journal of Pharmacology vol 42 no 3 pp 157ndash163 2010

[55] K R Sini B N Sinha and A Rajasekaran ldquoAntidiarrhealactivity of Capparis zeylanica leaf extractsrdquo Journal of AdvancedPharmaceutical Technology and Research vol 2 no 1 pp 39ndash422011

[56] J C Debongnie and S F Phillips ldquoCapacity of the human colonto absorb fluidrdquo Gastroenterology vol 74 no 4 pp 698ndash7031978

[57] U Laforenza G Gastaldi M Grazioli et al ldquoExpressionand immunolocalization of aquaporin-7 in rat gastrointestinaltractrdquo Biology of the Cell vol 97 no 8 pp 605ndash613 2005

[58] L S King D Kozono and P Agre ldquoFrom structure to diseasethe evolving tale of aquaporin biologyrdquo Nature Reviews Mole-cular Cell Biology vol 5 no 9 pp 687ndash698 2004

[59] U Laforenza ldquoWater channel proteins in the gastrointestinaltractrdquo Molecular Aspects of Medicine vol 33 no 5-6 pp 642ndash650 2012

[60] P Gallardo L P Cid C P Vio and F V Sepulveda ldquoAquaporin-2 a regulated water channel is expressed in apical membranesof rat distal colon epitheliumrdquo American Journal of PhysiologyGastrointestinal and Liver Physiology vol 281 no 3 pp G856ndashG863 2001

[61] U Laforenza E Cova G Gastaldi et al ldquoAquaporin-8 isinvolved in water transport in isolated superficial colonocytesfrom rat proximal colonrdquo Journal of Nutrition vol 135 no 10pp 2329ndash2336 2005

[62] TMatsuzaki Y Tajika A Ablimit T Aoki H Hagiwara and KTakata ldquoAquaporins in the digestive systemrdquo Medical ElectronMicroscopy vol 37 no 2 pp 71ndash80 2004

[63] U Laforenza G Gastaldi M Polimeni et al ldquoAquaporin-6 isexpressed along the rat gastrointestinal tract and upregulatedby feeding in the small intestinerdquo BMC Physiology vol 9 no 1article no 18 2009

[64] K S Wang T Ma F Filiz A S Verkman and J A BastidasldquoColon water transport in transgenic mice lacking aquaporin-4 water channelsrdquo The American Journal of PhysiologymdashGas-trointestinal and Liver Physiology vol 279 no 2 pp G463ndashG470 2000

[65] B Yang Y Song D Zhao and A S Verkman ldquoPhenotypeanalysis of aquaporin-8 null micerdquo The American Journal ofPhysiology Cell Physiology vol 288 no 5 pp C1161ndashC11702005

[66] P I Nedvetsky V Tabor G Tamma et al ldquoReciprocal regulationof aquaporin-2 abundance and degradation by protein kinaseA and p38-MAP kinaserdquo Journal of the American Society ofNephrology vol 21 no 10 pp 1645ndash1656 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


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OncologyJournal of



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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


It is therefore important to identify and evaluate availablenatural drugs as alternatives to current antidiarrheal drugswhich are not always free from adverse effects

Rhubarb an old and well-known traditional Chineseherbal medicine has been used as a folk remedy for gas-trointestinal disease including diarrhea and constipation forover two thousand years in China Today it is alreadyofficially listed in Chinese European and Japanese Pharma-copoeia [24ndash26] Rhubarb has many pharmacological activi-ties including purgation [27] antidiarrhoeal effect [28] anti-inflammation [28] and immunoregulation [29] On the otherhand rhubarb contains lots of compounds mainly includ-ing anthraquinones (anthraquinone derivatives) tanninspolysaccharides and other substances Among these tan-nins widely distributed in nature and present in almostall plant foods and some beverages are the major activecompounds of rhubarb moreover they are often the activecompounds of the other medicinal plants in which theyoccur [30 31] It is demonstrated that the antidiarrhoealactivity of many medicinal plants which are rich in tanninsis related to the existence of tannins [2 32] In particularthe purgative activity of rhubarb extract could be significantlyincreased by reducing the content of tannins [33] and someprocessed products from crude rhubarb with decrease ofpurgative anthraquinones and relative increase of tanninseven exhibited an antidiarrhoeal activity [34] The first directconvincing evidence of antidiarrhoeal activity of rhubarbtannins is that successive administration of the total tanninsextract of rhubarb produces the antidiarrhoeal effect onmag-nesium sulphate- (MgSO

4-) and castor oil-induced diarrhoea

mice by protein-precipitating reaction to the GI mucosa [35]Besides previous studies have demonstrated that tanninsreduce significantly intraluminal fluid accumulation by theantisecretory activity and promoting reabsorption of waterand decrease intestinal motility [36ndash38] However the effectof tannins on AQPs 2 and 3 remains unknown which playimportant roles in regulating water transfer in the colonduring diarrhoea Therefore we here investigated the effectof the total tannins extract of rhubarb on intestinal fluidaccumulation and the expression of AQPs 2 and 3 inMgSO

4-

induced diarrhoeamousemodel and the results were furtherconfirmed in HT-29 cells In addition the mechanism ofthe changes induced by rhubarb tannins in the expression ofAQPs 2 and 3 was examined

2 Materials and Methods

The study was approved by Research Ethics Committee ofChina Academy of Chinese Medical Sciences in accordancewith the National Institutes of Health Guidelines for the Careand Use of Laboratory Animals All animals were treated inaccordance with the guidelines and regulations for the useand care of animals of theCenter for LaboratoryAnimalCareChina Academy of Chinese Medical Sciences

21 Preparation of RTE The dried root and rhizoma ofRheum palmatum produced in Yushu county of Qinghaiprovince of China were purchased from the Yushu Pharma-ceuticals Company and were identified by Professor Shilin

Hu a taxonomist at China Academy of Chinese MedicalSciences A voucher specimen (Rh201005Z) was depositedin the institute RTE was prepared according to the reportedmethod with some modified steps [35] 1 kg of rhubarb waspowdered and extractedwith 5 L of 60 ethanol for 30min ina reflux condenser for three times and then was filtrated Thefiltrate was merged and concentrated to 500mL in vacuo ina rotary evaporator at 40∘C Then the concentrated solutionwas added into D-101 macroporous resin with 25 ethanolas the eluent The solution was concentrated continually andthen precipitated with 1 gelatinThe sediment was dissolvedin acetone recovered acetone and dried in a vacuum oven asthe total tannins extract of rhubarb The total tannins extractwas free of anthraquinones according to the color reactionsof anthraquinones with magnesium acetate methanol solu-tion and tannins with ferric chloride solution The yield ofthe total tannins from rhubarb was 340 The total tanninsextract of rhubarb was diluted with distilled water or cellculture medium to the proper concentration before use invivo and in vitro experiments as follows

22 High-Performance Liquid Chromatography (HPLC) Fin-gerprint of RTE The authentic standards of gallic acid andepigallocatechin gallate were purchased from Jingchun Bio-logical Technology Co Ltd (Shanghai China)The standardsof procyanidin-B1 and procyanidin-B2 were obtained fromTauto Biotech Co Ltd (Shanghai China) Standards of cate-chin and epicatechin were supplied by National Institute forFood and Drug Control (Beijing China) RTE and standardsubstance were dissolved in 50 methanol and then filtratedthrough 045 120583m filter membrane HPLC was performed ona ShimadzuHPLC system (Shimadzu Corporation ShimaneJapan) equipped with an LC-20AT binary pump an SPD-M20Adiode array detector a CBM-20Alite system controlleran SIL-20A autosampler a DGU-20A5 degasser and a CTO-10ASvp column oven An Ultimate XB-C18 column (150times 46mm 5 120583m particle size) was maintained at 30∘CDetection wave length was set at 280 nm The mobile phasefor fingerprint analysis consisted of solvent A (acetonitrileMerck NJ USA) and solvent B (01 formic acidwater vv)at a flow rate of 08mLmin A gradient program was usedas follows 27ndash10 B at 0ndash25min The injected volume was10 120583L Online UV spectra were recorded in the range of 190ndash800 nmTheHPLC fingerprint profile of RTEwas establishedby optimizing the chromatographic separate conditions Thechromatogram of RTE at 280 nm could exhibit the wholechromatographic characteristic in Figure 1(a) By comparisonof the retention time and online UV spectra of the referencestandards 6 compounds of tannins were identified as previ-ously reported in rhubarb [39] The structure of gallic acidprocyanidin-B1 catechin procyanidin-B2 epicatechin andepigallocatechin gallate were shown in Figure 1(b)

23 Quantitative Analysis of RTE The content of the RTEwas determined using Shimadzu Visible Ultraviolet Spec-trophotometer UV-1800 (Shimadzu Technologies) accordingto themethod inChinese Pharmacopoeia (Committee for thePharmacopoeia of China 2010) with gallic acid as reference


00 25 50 75 100 125 150 175 200 225 250minus5000

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AB

C

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F

(a)

(A)

OH

HO

HO COOH

Gallic acid(B)

O

O

OH

OH

OHOH

OH

OH

OH

OHHO

HO

Procyanidin-B1(C)

O

OH

OH

OH

OH

HO

Catechin

(D)

O

O

OH

OHOH

OH

OH

OH

OH

OH

HO

HO

Procyanidin-B2(E)

O

OH

OH

OH

OH

HO

Epicatechin(F)

O

O

O

OH

OH

OH

OH

OH

OH

OH

HO


(b)








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(d)












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(a)

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Vehicle

Merge998400

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(b)

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(c)

Figure 3 Continued


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(a)

GAPDH

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4appeared edematous





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RTE80120583gmL

(a)

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(b)

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API

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(c)

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GAPDH

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RTE (120583gmL)

AQP3

prot

ein

( o

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trol)

(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

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RTE (120583gmL)

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PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

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f con

trol)

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p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

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Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00 25 50 75 100 125 150 175 200 225 250minus5000

0

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1

2

AB

C

D E

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(a)

(A)

OH

HO

HO COOH

Gallic acid(B)

O

O

OH

OH

OHOH

OH

OH

OH

OHHO

HO

Procyanidin-B1(C)

O

OH

OH

OH

OH

HO

Catechin

(D)

O

O

OH

OHOH

OH

OH

OH

OH

OH

HO

HO

Procyanidin-B2(E)

O

OH

OH

OH

OH

HO

Epicatechin(F)

O

O

O

OH

OH

OH

OH

OH

OH

OH

HO


(b)








4sdot7H2O (2 gkg











4(50mM) andor






4andor RTE






4andor RTE



4andor RTE for


VehicleRTE (mgkg)

Control 125 250 500

Feca

l wat

er co

nten

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(c)

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200120583m

Control

(d)












Negative control

Control

Vehicle


RTE500mgkg

(a)

DAPI MergeAQP3

Negative control

Control

Vehicle

Merge998400

RTE500mgkg

(b)

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VehicleRTE (mgkg)

Control 125 250 500

lowast

lowastlowast

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AQP2

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API

+ce

lls (

of c

ontro

l)

(c)

Figure 3 Continued


0

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VehicleRTE (mgkg)

Control 125 250 500

lowast

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(d)










3 Results










GAPDH

0

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VehicleRTE (mgkg)

Control 125 250 500

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(a)

GAPDH

0

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(b)

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of c

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(d)



4appeared edematous





Control

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Negativecontrol

MergeDAPI AQP2

RTE80120583gmL

(a)

DAPI MergeAQP3

(b)

VehicleControl

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API

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of c

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lowast lowastlowast

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GAPDH

0

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0h 3h 6h 12h 24hRTE 80120583gmL

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AQP3

prot

ein

( o

f con

trol)

(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

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RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

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300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























4(50mM) andor






4andor RTE






4andor RTE



4andor RTE for


VehicleRTE (mgkg)

Control 125 250 500

Feca

l wat

er co

nten

t (

)

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200120583m

Control

(d)












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Control

Vehicle


RTE500mgkg

(a)

DAPI MergeAQP3

Negative control

Control

Vehicle

Merge998400

RTE500mgkg

(b)

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lowast

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AQP2

+D

API

+ce

lls (

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ontro

l)

(c)

Figure 3 Continued


0

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VehicleRTE (mgkg)

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lowast

lowastlowast

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3 Results










GAPDH

0

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prot

ein

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AQP2

(a)

GAPDH

0

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( o

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(b)

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mRN

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mRN

A (

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ontro

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4appeared edematous





Control

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Negativecontrol

MergeDAPI AQP2

RTE80120583gmL

(a)

DAPI MergeAQP3

(b)

VehicleControl

lowastlowastlowast

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+D

API

+ce

lls (

of c

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(c)

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lowast lowastlowast

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GAPDH

0

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0h 3h 6h 12h 24hRTE 80120583gmL

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f0h)

(a)

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GAPDH

0

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GAPDH

0

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RTE (120583gmL)

AQP3

prot

ein

( o

f con

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(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

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RTE (120583gmL)

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f con

trol)

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PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

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( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VehicleRTE (mgkg)

Control 125 250 500

Feca

l wat

er co

nten

t (

)

0

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(c)

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200120583m

Control

(d)












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Control

Vehicle


RTE500mgkg

(a)

DAPI MergeAQP3

Negative control

Control

Vehicle

Merge998400

RTE500mgkg

(b)

0

50

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VehicleRTE (mgkg)

Control 125 250 500

lowast

lowastlowast

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AQP2

+D

API

+ce

lls (

of c

ontro

l)

(c)

Figure 3 Continued


0

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350

VehicleRTE (mgkg)

Control 125 250 500

lowast

lowastlowast

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AQP3

+D

API

+ce

lls (

of c

ontro

l)

(d)










3 Results










GAPDH

0

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lowast

lowastlowast

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VehicleRTE (mgkg)

Control 125 250 500

AQP2

prot

ein

( o

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trol)

AQP2

(a)

GAPDH

0

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lowast


VehicleRTE (mgkg)

Control 125 250 500

AQP3

prot

ein

( o

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AQP3

(b)

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lowastlowastlowast


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mRN

A (

of c

ontro

l)

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lowastlowastlowast


AQP3

mRN

A (

of c

ontro

l)

(d)



4appeared edematous





Control

Vehicle

Negativecontrol

MergeDAPI AQP2

RTE80120583gmL

(a)

DAPI MergeAQP3

(b)

VehicleControl

lowastlowastlowast

lowastlowastlowast

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+D

API

+ce

lls (

of c

ontro

l)

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RTE (120583gmL)

(c)

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API

+ce

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of c

ontro

l)

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20 40 80

RTE (120583gmL)

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4-treated HT-






lowast lowastlowast

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AQP2

GAPDH

0

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0h 3h 6h 12h 24hRTE 80120583gmL

AQP2

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( o

f0h)

(a)

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AQP3

GAPDH

0

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0h 3h 6h 12h 24hRTE 80120583gmL

AQP3

prot

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( o

f0h)

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lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

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RTE (120583gmL)

AQP2

prot

ein

( o

f con

trol)

(c)

lowast


AQP3

GAPDH

0

100

200

300

400


RTE (120583gmL)

AQP3

prot

ein

( o

f con

trol)

(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

100

200

300


RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

200

300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Negative control

Control

Vehicle


RTE500mgkg

(a)

DAPI MergeAQP3

Negative control

Control

Vehicle

Merge998400

RTE500mgkg

(b)

0

50

100

150

200

250

300

350

VehicleRTE (mgkg)

Control 125 250 500

lowast

lowastlowast

lowastlowastlowast

AQP2

+D

API

+ce

lls (

of c

ontro

l)

(c)

Figure 3 Continued


0

50

100

150

200

250

300

350

VehicleRTE (mgkg)

Control 125 250 500

lowast

lowastlowast

lowastlowastlowast

AQP3

+D

API

+ce

lls (

of c

ontro

l)

(d)










3 Results










GAPDH

0

100

200

300

400

500

lowast

lowastlowast

lowastlowastlowast

VehicleRTE (mgkg)

Control 125 250 500

AQP2

prot

ein

( o

f con

trol)

AQP2

(a)

GAPDH

0

50

100

150

200

250

300

350

400

lowast


VehicleRTE (mgkg)

Control 125 250 500

AQP3

prot

ein

( o

f con

trol)

AQP3

(b)

0

100

200

300

400

500

600

700

lowastlowastlowast


AQP2

mRN

A (

of c

ontro

l)

(c)

0

100

200

300

400

500

600

lowastlowastlowast


AQP3

mRN

A (

of c

ontro

l)

(d)



4appeared edematous





Control

Vehicle

Negativecontrol

MergeDAPI AQP2

RTE80120583gmL

(a)

DAPI MergeAQP3

(b)

VehicleControl

lowastlowastlowast

lowastlowastlowast

AQP2

+D

API

+ce

lls (

of c

ontro

l)

0

50

100

150

200

250

300

20 40 80

RTE (120583gmL)

(c)

VehicleControl

lowast

lowastlowastlowast

lowastlowastlowast

AQP3

+D

API

+ce

lls (

of c

ontro

l)

0

50

100

150

200

250

300

350

400

20 40 80

RTE (120583gmL)

(d)








4-treated HT-






lowast lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP2

prot

ein

( o

f0h)

(a)

lowastlowast


AQP3

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP3

prot

ein

( o

f0h)

(b)

lowast

lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

100

200

300


RTE (120583gmL)

AQP2

prot

ein

( o

f con

trol)

(c)

lowast


AQP3

GAPDH

0

100

200

300

400


RTE (120583gmL)

AQP3

prot

ein

( o

f con

trol)

(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

100

200

300


RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

200

300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

300

350

VehicleRTE (mgkg)

Control 125 250 500

lowast

lowastlowast

lowastlowastlowast

AQP3

+D

API

+ce

lls (

of c

ontro

l)

(d)










3 Results










GAPDH

0

100

200

300

400

500

lowast

lowastlowast

lowastlowastlowast

VehicleRTE (mgkg)

Control 125 250 500

AQP2

prot

ein

( o

f con

trol)

AQP2

(a)

GAPDH

0

50

100

150

200

250

300

350

400

lowast


VehicleRTE (mgkg)

Control 125 250 500

AQP3

prot

ein

( o

f con

trol)

AQP3

(b)

0

100

200

300

400

500

600

700

lowastlowastlowast


AQP2

mRN

A (

of c

ontro

l)

(c)

0

100

200

300

400

500

600

lowastlowastlowast


AQP3

mRN

A (

of c

ontro

l)

(d)



4appeared edematous





Control

Vehicle

Negativecontrol

MergeDAPI AQP2

RTE80120583gmL

(a)

DAPI MergeAQP3

(b)

VehicleControl

lowastlowastlowast

lowastlowastlowast

AQP2

+D

API

+ce

lls (

of c

ontro

l)

0

50

100

150

200

250

300

20 40 80

RTE (120583gmL)

(c)

VehicleControl

lowast

lowastlowastlowast

lowastlowastlowast

AQP3

+D

API

+ce

lls (

of c

ontro

l)

0

50

100

150

200

250

300

350

400

20 40 80

RTE (120583gmL)

(d)








4-treated HT-






lowast lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP2

prot

ein

( o

f0h)

(a)

lowastlowast


AQP3

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP3

prot

ein

( o

f0h)

(b)

lowast

lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

100

200

300


RTE (120583gmL)

AQP2

prot

ein

( o

f con

trol)

(c)

lowast


AQP3

GAPDH

0

100

200

300

400


RTE (120583gmL)

AQP3

prot

ein

( o

f con

trol)

(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

100

200

300


RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

200

300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















GAPDH

0

100

200

300

400

500

lowast

lowastlowast

lowastlowastlowast

VehicleRTE (mgkg)

Control 125 250 500

AQP2

prot

ein

( o

f con

trol)

AQP2

(a)

GAPDH

0

50

100

150

200

250

300

350

400

lowast


VehicleRTE (mgkg)

Control 125 250 500

AQP3

prot

ein

( o

f con

trol)

AQP3

(b)

0

100

200

300

400

500

600

700

lowastlowastlowast


AQP2

mRN

A (

of c

ontro

l)

(c)

0

100

200

300

400

500

600

lowastlowastlowast


AQP3

mRN

A (

of c

ontro

l)

(d)



4appeared edematous





Control

Vehicle

Negativecontrol

MergeDAPI AQP2

RTE80120583gmL

(a)

DAPI MergeAQP3

(b)

VehicleControl

lowastlowastlowast

lowastlowastlowast

AQP2

+D

API

+ce

lls (

of c

ontro

l)

0

50

100

150

200

250

300

20 40 80

RTE (120583gmL)

(c)

VehicleControl

lowast

lowastlowastlowast

lowastlowastlowast

AQP3

+D

API

+ce

lls (

of c

ontro

l)

0

50

100

150

200

250

300

350

400

20 40 80

RTE (120583gmL)

(d)








4-treated HT-






lowast lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP2

prot

ein

( o

f0h)

(a)

lowastlowast


AQP3

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP3

prot

ein

( o

f0h)

(b)

lowast

lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

100

200

300


RTE (120583gmL)

AQP2

prot

ein

( o

f con

trol)

(c)

lowast


AQP3

GAPDH

0

100

200

300

400


RTE (120583gmL)

AQP3

prot

ein

( o

f con

trol)

(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

100

200

300


RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

200

300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

Vehicle

Negativecontrol

MergeDAPI AQP2

RTE80120583gmL

(a)

DAPI MergeAQP3

(b)

VehicleControl

lowastlowastlowast

lowastlowastlowast

AQP2

+D

API

+ce

lls (

of c

ontro

l)

0

50

100

150

200

250

300

20 40 80

RTE (120583gmL)

(c)

VehicleControl

lowast

lowastlowastlowast

lowastlowastlowast

AQP3

+D

API

+ce

lls (

of c

ontro

l)

0

50

100

150

200

250

300

350

400

20 40 80

RTE (120583gmL)

(d)








4-treated HT-






lowast lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP2

prot

ein

( o

f0h)

(a)

lowastlowast


AQP3

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP3

prot

ein

( o

f0h)

(b)

lowast

lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

100

200

300


RTE (120583gmL)

AQP2

prot

ein

( o

f con

trol)

(c)

lowast


AQP3

GAPDH

0

100

200

300

400


RTE (120583gmL)

AQP3

prot

ein

( o

f con

trol)

(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

100

200

300


RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

200

300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP2

prot

ein

( o

f0h)

(a)

lowastlowast


AQP3

GAPDH

0

20

40

60

80

100

120

0h 3h 6h 12h 24hRTE 80120583gmL

AQP3

prot

ein

( o

f0h)

(b)

lowast

lowastlowast

lowastlowastlowast

AQP2

GAPDH

0

100

200

300


RTE (120583gmL)

AQP2

prot

ein

( o

f con

trol)

(c)

lowast


AQP3

GAPDH

0

100

200

300

400


RTE (120583gmL)

AQP3

prot

ein

( o

f con

trol)

(d)







4-induced





4for 24 h the cyto-


4 Discussion



lowast


GAPDH

0

100

200

300


RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

200

300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast


GAPDH

0

100

200

300


RTE (120583gmL)

( o

f con

trol)

PKA

PKA

pro

tein

(a)

lowast


GAPDH


RTE (120583gmL)

0

100

200

300

( o

f con

trol)

p-CREB

p-CR

EB p

rote

in

(b)


RTE (120583gmL)

0

30

60

90

120

Cel

l via

bilit

y (

)

(c)





























4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































4 Previous study



4-


4-induced







Acknowledgment


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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