poysaccharides in Si-Jun-Zi tang, 2005, british journal of nutrition
Effects of Si-Jun-Zi decoction polysaccharides on cell migration and gene expression in wounded rat intestinal epithelial cells
L.Liu 1,L.Han 2,Daisy Y.L.Wong 1,Patrick Y.K.Yue 1,W.Y.Ha 1,Y.H.Hu 3,P.X.Wang 2and Ricky N.S.Wong 1,3*
1Research and Development Division,School of Chinese Medicine,Hong Kong Baptist University,Hong Kong
2
Immunology Laboratory,Guangzhou University of Traditional Chinese Medicine,Guangzhou,People’s Republic of China 3
Department of Biology,Science Faculty,Hong Kong Baptist University,Kowloon Tong,Hong Kong
(Received 7October 2003–Revised 17August 2004–Accepted 4September 2004)
Si-Jun-Zi decoction (SJZD),a traditional Chinese herbal prescription,has been used clinically for treating patients with disorders of the digestive system.Previous studies indicated that the polysaccharides of SJZD (SJZPS)are the active components contributing towards its pharmacological effects in improv-ing gastrointestinal function and immunity.However,the protective and restitutive effects on intestinal epithelial cells remain unknown.In the present study,SJZPS were ?rst extracted and chemically characterized.Then their stimulatory and restitutive effects on intestinal epithelial cells (IEC-6cells)were elicited by different in vitro models including migration of wounded IEC-6cells and cell proliferation.Results indicated that SJZPS not only protects the cells against the harmful impairment of indomethacin but also enhances re-epithelialization of a wounded monolayer at an optimal dose of 100m g/ml at 24h incubation.To elucidate the modulatory effect of SJZPS on wounded IEC-6cells at the molecular level,an oligonucleotide microarray was employed to study differential gene expression of SJZPS-treated IEC-6cells and the candidate genes were validated by RT-PCR.There was increased expression of genes coding for ion channels and transporters,which are critical to cell migration and restoration of wounded intestinal cells,suggesting a possible mechanism for re-epithelialization.In conclusion,our data show for the ?rst time that SJZPS can enhance intestinal restitution and protect against indo-methacin-induced damage of intestinal epithelial cells.These ?ndings provide new insight into the mechanism of action of a traditional Chinese herbal prescription,SJZD,in intestinal wound restitution.
Si-Jun-Zi decoction:Polysaccharides:IEC-6cell:Microarray
The intestinal mucosal epithelium plays an important role in preventing translocation of deleterious substances or organisms present within the lumen of the gut and in preserving normal homeostasis (Dignass &Podolsky,1995).Impairment of the epithelial surface barrier may facilitate the penetration of harmful factors from the intestinal lumen,leading to acute and chronic in?ammatory changes caused by the initiation of a complex network of immune responses.The inability to repair damage to the intestinal mucosal epithelium is a common physiological event that may account for numerous gastrointestinal diseases or disorders,such as infectious diarrhoea,gastrointestinal ulcer and in?ammatory bowel disease.Indeed,the intestinal epithelium exerts enormous regenerative capabilities to allow rapid healing of injury by at least two different processes:migration and proliferation.Re-establishment from super?cial injury can be achieved initially within minutes to hours by the migration of viable epithelial cells adjacent to or just beneath the injured surface to cover the denuded area,a process termed epithelial restitution or re-epithelialization (Lacy,1988;Moore et al.1989;McCormack et al.1992).After sealing of the epithelial surface to regain its integrity,epithelial cell proliferation takes place to replenish the decreased cell pool (Dignass &Podolsky,1995).
Around 50%of the Western population relies on herbal medi-cines for the treatment or prevention of digestive disorders (Lang-mead &Rampton,2001).Si-Jun-Zi decoction (SJZD),a traditional Chinese herbal prescription,is well known for treating disorders of digestive function manifested by poor appetite,indi-gestion and watery stools or diarrhoea.It also has been used as a tonic supplement for health maintenance.It is formulated from four Chinese herbs:Dangshen (Codonopsis pilosula (Franch.)Nannf);Gancao (Glycyrrhizae uralensis Fisch.);Baizhu (Atracty-lodes macrocephala Koidz.);Fuling (Poria cocos (Schw.)Wolf),of which the polysaccharides are considered the main active components contributing towards its therapeutic effects.In vivo studies have shown that SJZD can improve intestinal mucosal immunofunction by counteracting the adverse effect of cyclophosphamide on intestinal mucosal-associated lymphoid
*Corresponding author:Dr Ricky N.S.Wong,fax t852********,email rnswong@https://www.wendangku.net/doc/3014377147.html,.hk
Abbreviations:ASIC1,acid-sensing ion channels,proton-gated cation channels;DMEM,Dulbecco’s modi?ed Eagle medium;FBS,fetal bovine serum;GAPDH,glyceraldehye-3-phosphate dehydrogenase;JUND ,JunD proto-oncogene;Kv4,neuronal delayed recti?er K tchannel;MTT,3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide;PBK,phosphorylase kinase catalytic subunit;RCK4,putative potassium channel subunit protein;RIIA,type I regulatory subunit of cAMP-dependent protein kinase;SJZD,Si-Jun-Zi decoction;SJZPS,polysaccharides of SJZD.
British Journal of Nutrition (2005),93,21–29DOI:10.1079/BJN20041295
q The Authors 2005
tissues of mice(Liu et al.2000);improve intestinal disturbances to alleviate diarrhoea in rats caused by radiation(Chen&Fu, 1996);and ameliorate intestinal injury in scalded rats and prevent the translocation of intestinal bacteria(Guo et al.2003).As SJZD is used clinically for treating patients with gastrointestinal disorders,some clinical studies have indicated that SJZD is effective in treating chronic gastritis and intestinal metaplasia (Zhong et al.1997;Langmead&Rampton,2001).
With the advancement of molecular medicine,more evidence-based and scienti?c support has accumulated to strengthen the scienti?c foundation and promote the modernization of traditional Chinese medicine.Genome-wide expression monitoring of genes by microarray analysis seems to provide a holistic approach to examine the biochemical effects of Chinese medicines.This func-tional genomic approach has successfully revealed the possible mechanisms of action for some herbal extracts such as Ginkgo biloba(Watanabe et al.2001),Coptidis rhizome(Iizuka et al. 2003),a preparation of eight different herbs(Bonham et al. 2002)and four other Chinese herbal extracts(Sadava et al. 2002).Recently,the mechanism of SJZD in human gastric cancer grafted onto nude mice was studied at the gene expression level(Zhao et al.2002).Since migration of intestinal epithelial cells is the principal force behind early restitution of mucosal injury and the mechanism of action of the polysaccharides of SJZD(SJZPS)on intestinal function has not been elucidated, the aim of the present study was to characterize the effects of SJZPS on intestinal epithelial wound healing(restitution)in vitro.The non-transformed rat small intestinal epithelial cell line IEC-6was used in this study because it is a common cell model for demonstrating the process of migration during early mucosal restitution(McCormack et al.1992;Dignass& Podolsky,1995;Cario et al.2000).The restitutive effect of SJZPS in the wound healing assay and its stimulatory and protec-tive effects against indomethacin-induced damage in a prolifer-ation assay were investigated.Furthermore,to elucidate the molecular mechanism of the modulatory effect of SJZPS on wounded IEC-6cells,gene expression pro?ling with an oligonu-cleotide microarray was employed.
Materials and methods
Sources of IEC-6cells and reagents
The intestinal epithelial cell line(IEC-6cells;ATCC CRL1592) was purchased from the American Type Culture Collection(Rock-ville,MD,USA)at passage14.This IEC-6cell line originated from intestinal crypt cells,as judged by morphological and immunologi-cal criteria.The stock culture was maintained in a T-150?ask in Dulbecco’s modi?ed Eagle medium(DMEM)supplemented with 10%fetal bovine serum(FBS),10m g insulin/ml and50m g genta-micin sulphate/ml.The stock culture was incubated at378C in a humidi?ed atmosphere of95%air and5%CO2.The cells were sub-cultured once per week at a dilution of1:20and the medium was changed three times per week.In the experiments,cells at pas-sage15–20were taken up with0·25%(w/v)trypsin/1m M-EDTA in Hanks’basic salt solution without Ca and Mg.All chemicals used in the study were purchased from Sigma(St.Louis,MO,USA)unless speci?ed otherwise.DMEM,FBS,PBS,antibiotics,insulin and sodium pyruvate were obtained from Gibco(Invitrogen Life Technologies,Carlsbad,CA,USA).Extraction and characterization of polysaccharides from Si-Jun-Zi decoction
SJZD was derived from four herbs,Gancao(G.uralensis Fisch.), Dangshen(C.pilosula(Franch.)Nannf),Baizhu(A.macrocephala Koidz.)and Fuling(P.cocos(Schw.)Wolf),in a ratio of1:2:2:2by weight.SJZD was used for the preparation of SJZPS as described previously(Ou,1993;Chun et al.2002)with modi?cation.Dried herbs(70g)were soaked in distilled water for30min at room tem-perature and then extracted twice in boiling distilled water at1:8w/ v for4h.The fractions were combined and concentrated by rotary evaporation.The supernatant was recovered by centrifugation and then dialysed against running water for2d.The extract was preci-pitated with ethanol to a?nal concentration of60%(v/v)at48C overnight and the precipitate was pelleted by centrifugation. Then,the supernatant was concentrated by rotary evaporation, another ethanol precipitation performed to a?nal concentration of80%(v/v)and the precipitate obtained by centrifugation.The pellets from the two ethanol precipitations were combined,dis-solved in distilled water and deproteinated by the Sevag method (Staub,1965).The crude polysaccharide precipitate was extracted by shaking vigorously with?ve volumes of chloroform–n-butanol (4:1,v/v)for20min.The extraction was repeated?ve more times and the polysaccharides were recovered by ethanol precipitation as stated earlier.Finally,the SJZPS were dissolved in distilled water and sterilized by?ltration through a0·2m m membrane.
The sugar content of the SJZPS was determined by colorimetry with3,5-dinitrosalicylic acid(Miller,1959).Glucose was used as standard.The total protein content of the deproteinated polysac-charides was assayed by the Lowry method using bovine serum albumin as protein standard(Lowry et al.1951).The lipopolysac-charide content of SJZPS was determined by the Limulus amebo-cyte lysate kinetic assay.The sugar composition of SJZPS was analysed by HPLC after acid hydrolysis.Brie?y,polysaccharides (200mg)were hydrolysed with2M-H2SO4at1008C for4h.The depolymerized polysaccharides containing the neutral sugars were neutralized with NaB(OH)4to pH7.The supernatant was col-lected by centrifugation and concentrated before HPLC analysis using an Alltech Prevail Amino column(0·3m m, 2·1mm￡150mm)and Alltech2000ELSD detector(Alltech Associates,Inc.,Deer?eld,IL,USA).Column temperature was maintained at258C.Degassed acetonitrile–distilled water(9:1v/ v)was used as the eluent at a?ow rate of0·3ml/min. Wounding assay
The wounding assay was performed as described previously (McCormack et al.1992).Brie?y,Matrigel was diluted with cool serum-free medium at a dilution of1:7(v/v)and coated onto six-well plates at room temperature for1h.Excess diluted Matrigel was discarded.After washing twice with cool serum-free medium, the coated plates were ready for wounding assay.Brie?y,IEC-6 cells were seeded at a density of6·25￡104cells in six-well plates and cultured in10%FBS-supplemented DMEM for24h.Then, the cells were washed with PBS and further cultured for another 24h in serum-free medium.To initiate migration,the cell mono-layer was scratched with a razor blade followed by washing with serum-free medium.Fresh serum-free medium containing SJZPS to a?nal concentration of50,100,200,400or800m g/ml was then added and recovery of the wounded monolayers due to cell migration towards the denuded area was evaluated at intervals of
L.Liu et al. 22
8,16and24h.The area covered by the migrating cells was measured with an inverted phase-contrast microscope(model TMS;Nikon Corporation,Tokyo,Japan)and the images analysed using Motic Images Plus2.0software(Motic Instruments Inc., Richmond,Canada).The time-dependent effect of SZJPS on wounded IEC-6cells expressed was as migration rate compared with the medium as control:
Migration rate??eTreatment area–Control areaT=Control area
￡100%:
Determination of IEC-6cell proliferation
The dose effect of SJZPS on IEC-6cell proliferation was deter-mined by the3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazo-lium bromide(MTT)assay as described by Mosmann(1983) with modi?cation.Brie?y,IEC-6cells were seeded at a density of1￡104cells/well in96-well plates and cultured in DMEM supplemented with10%FBS for24h.Then,cells were washed with PBS and cultured for a further24h in serum-free medium. The cultures were then supplemented with serum-free medium containing sterilized SJZPS at a?nal concentration of50,100, 200,400or800m g/ml and subsequently incubated at378C for 24h.Two sets of positive control experiments were performed in parallel:one using20ng epithelial growth factor/ml and the other10%FBS alone.Cell proliferation was measured using the Cell Proliferation Kit I(Roche Diagnostics Corp.,Indianapo-lis,IN,USA)according to the manufacturer’s instructions.The colour development from the tetrazolium compound,which is directly proportional to the number of viable cells in culture, was measured spectrophotometrically at550nm using an ELISA plate reader(Sunrise;Tecan Instruments,Maennedorf, Switzerland).
To examine the protective effect of SJZPS against indometha-cin-induced damage of IEC-6cells,indomethacin(40m M)and various concentrations of SJZPS(50,100and200m g/ml)were co-incubated with IEC-6cells that had been starved for24h in serum-free medium.After further incubation for24h,the extent of cell proliferation was determined using the Cell Pro-liferation Kit I(Roche Diagnostics Corp.).All data are expressed as percentage relative viability:Relative viabilitye%T?eOD experiment=OD controlT￡100%;where OD is optical density. Gene expression pro?ling analysis
IEC-6cells(6·25￡104cells)were seeded in10cm Matrigel-coated Petri dishes and incubated in DMEM supplemented with10%FBS in a humidi?ed atmosphere of95%air and5%CO2.After72h,the cells became con?uent,the medium was replaced by serum-free DMEM and incubation was continued for a further24h.Con?uent cells were wounded vertically,horizontally and diagonally using a multi-channel pipette with attached yellow tips(eight channels). The distance between each line of denuded area was about0·9cm. Cells were washed with PBS.SJZPS,at a?nal concentration of 100m g/ml,was added and the cells were incubated for another 24h.A control was given the same wounding but without SJZPS treatment.The same batch of IEC-6cells at passage18was used for three independent sets of microarray experiments.
Total cellular RNA was isolated from the control and SJZPS-treated IEC-6cells using the Atlas e Glass Total RNA Isolation Kit(Clontech,Palo Alto,CA,USA)according to the manufacturer’s instructions.The quantity and quality of RNA were determined on the basis of spectrometric measurements (260and280nm)and electrophoresis in1·0%Tris–boric acid–EDTA agarose gels,respectively.
cDNA probes for microarray analysis were prepared using the Atlas e PowerScript e Fluorescent Labeling Kit(Clontech). Brie?y,8m g total RNA isolated from control and SJZPS-treated cells were primed separately with2m l CDS(cDNA synthesis) primer mix and1m l cDNA synthesis control,incubated at708C for5min in a thermal cycler(PTC-100w;MJ Research,Inc., Watertown,MA,USA)and cooled to488C.Then,a master mix containing4m l5￡?rst-strand buffer,2m l10￡dNTP,2m l dithiothreitol(100m M),1m l deionized water and1m l Power-Script Reverse Transcriptase was mixed with the primed RNA and incubated at488C for1h.The reaction was stopped by heat-ing at708C for5min.Then,the tubes were cooled to378C and 0·2m l RNase H(10U/m l)was added,followed by an additional incubation at378C for15min.The reaction was then stopped by adding0·5m l0·5M-EDTA,pH8·0and the transcribed cDNA was puri?ed by QuickClean e Puri?cation Resin(Clon-tech).Newly synthesized cDNA was precipitated with sodium acetate–absolute ethanol at2208C for1h and collected by cen-trifugation at12000rpm for20min.cDNA pellets were washed once with70%ethanol and re-suspended in10m l2￡?uorescent labelling buffer(Clontech).Then the control and SJZPS-treated cDNAs were mixed with0·5m l coupling reaction control oligos (Clontech)and subsequently coupled with10m l5m M-Cy5and Cy3reactive dyes(Amersham Pharmacia,Piscataway,USA, NJ),respectively,at248C for1h.The labelled probes were precipitated and collected by the same procedure as mentioned earlier.Finally,the pellets were dissolved in100m l sterile water(Clontech).Unincorporated reactive dye and other impuri-ties were removed on a Microcon YM-30column(Millipore, Billerica,MA,USA).
Cy5(control)and Cy3(SJZPS-treated)labelled probes were combined and mixed with GlassHyb hybridization solution (Clontech)in the ratio suggested by the company.Finally,the probe mixture was hybridized at508C overnight to the Atlas e Glass Rat1.0Microarray(Clontech),which includes1081rat genes(80-mer oligo array),nine housekeeping genes,negative and positive controls.Thereafter,the microarrays were washed once in GlassHyb washing solution(Clontech),twice in GlassHyb washing solution diluted with1￡standard saline citrate and once in GlassHyb washing solution diluted with0·1￡standard saline citrate;each washing was for10min at room temperature. The hybridized arrays were spun dry prior to scanning. Hybridized arrays were scanned using the ScanArray5000con-focal laser scanner(Packard BioChip/GSI Lumonics,Billerica, MA,USA).To get the best-?t image,laser power and PMT (photomultiplier tube)voltage were adjusted manually to mini-mize background and saturated spots.Cy5and Cy3signal inten-sities of each spot were obtained using QuantArray Analysis Software(Packard BioChip/GSI Lumonics).Comparisons were made between the normal(Cy5)and SJZPS-treated IEC-6cells (Cy3)from the same microarray.The signal intensities of each spot were corrected by subtracting local background signals. Then the Cy3and Cy5signal intensities of each spot were further normalized by taking the total averaged signal intensities across all spots in the microarray to be equal for both channels(Hegde et al.2000).To eliminate any unreliable data,spots were screened by?rst setting a threshold of minimum acceptable signal
Effects of polysaccharides on intestinal epithelial cells23
(background plus two standard derivations)to eliminate expression ratios of extremely high or low spots due to an undetectable signal in either channel and,second,performing visual inspection to eliminate any irregular spots.The genuine spots were subjected to calculation for the fold change (Cy3:Cy5).Differential gene expression was considered up-or down-regulated if the averaged ratio(Cy3:Cy5)between the two conditions was.1·5or,21·5,respectively.
Validation of candidate genes by semi-quantitative RT-PCR Semi-quantitative RT-PCR was used to verify microarray data of the selected genes.Glyceraldehye-3-phosphate dehydrogenase (GAPDH)primers were used to compare and monitor the ef?-ciency of cDNA synthesis between different samples.The primers were as follows:GAPDH sense primer AAG ATT GTC AGC AAT GCA TCC,antisense primer ACA GTC TTC TGA GTG GCA GTG A;phosphorylase kinase catalytic subunit(PBK) sense primer TCA CAG AAA CCA GCC CTT TAC C,antisense primer CAG GCG GTG ATG GCA AGG;JunD proto-oncogene (JUND)sense primer ACC CTC AAA AGC CAG AAC ACC, antisense primer CGC TCG GGC TCA ATA CGC;putative pot-assium channel subunit protein(RCK4)sense primer ATG CTG AAG CCC GAG AGA GAC,antisense primer TCG TGT GCT GTT ATC TGT GTG C;type I regulatory subunit of cAMP-dependent protein kinase(RIIA)sense primer TGG TGA AAT TGC CCT GCT GAT G,antisense primer ACT GCT GGA TGT TTC GCT TGA G;neuronal delayed recti?er Ktchannel (Kv4)sense primer TTC ACA CTG CCA CCC TCT TCC,anti-sense primer TGG TCA CAC TGG AGC TAC ACA C.RT-PCR was carried out according to the manufacturer’s instructions. Brie?y,cDNA templates for RT-PCR were synthesized from 1m g total RNA using the ThermoScript e RT-PCR system(Invi-trogen)and oligo-dT primer.PCR mixtures(?nal volume50m l) contained100ng cDNA template,0·2m M of each primer, 0·2m M of each dNTP,1￡Taq buffer(Pharmacia),1·5m M-MgCl2and1U Taq DNA polymerase(Pharmacia).The reactions went through a touchdown cycle of948C for5min;then?fteen cycles at948C for1min,658C for1min(minus18C per cycle) and728C for1min;then?fteen to twenty cycles of948C for 30s,558C for30s and728C for1min;and a?nal extension at 728C for10min.Reactions were performed in a PTC-200w ther-mal cycler(MJ Research Inc.).PCR products were analysed by electrophoresis in1·5%agarose gel and Kodak1D Image Analyis Software(Rochester,NY,USA)was used for the densitometric analysis.Due to variations in abundance of the different mRNA in the cell,the PCR cycles(Tsien&Tsien,1990;McCormack et al.1998)and different titrations of the RT reaction(equal to 100ng,500ng and1m g total RNA)were performed in duplicate to ensure that the PCR reaction was in the linear range. Statistical analysis
Data are expressed as mean and standard deviation.Signi?cance of differences among groups(e.g.among different concentrations of SJZPS and the control)were tested by one-way ANOVA.Dun-nett’s post hoc procedure was used for post hoc testing;Dunnett’s test compares the mean of each treatment group(e.g.SJZPS-trea-ted cells)with that of a designated control.The effect of both incubation time intervals(time-dependent effect)and SJZPS con-centration(dose-dependent effect)on cell migration was analysed by two-way ANOVA,followed by a multiple post hoc test using least signi?cant difference.Differences were considered signi?-cant if P,0·05.SPSS10.0software(SPSS Inc.,Chicago,IL, USA)was used to perform all calculations.
Results
Extraction and characterization of polysaccharides from Si-Jun-Zi decoction
The total polysaccharides from SJZD were successfully extracted and chemically characterized.As shown in Table1,SJZPS were composed of51·82%total sugar and37·5%protein;no lipopoly-saccharides were detected.In terms of sugars,the composition of SJZPS was glucose(44·61%),galactose(33·96%),arabinose (10·85%),rhamnose(5·26%),xylose(4·84%),mannose (0·46%)and fructose(0·02%)as determined by HPLC analysis. These results reveal that glucose and galactose are the main com-ponents of SJZPS.
Effect of polysaccharides from Si-Jun-Zi decoction on cell migration of wounded IEC-6cells
A cell migration assay was used to mimic the early stage of intes-tinal epithelial restitution in vitro,in which the migration rate of IEC-6cells after wounding was determined(Table2).In addition, the dose effect of SJZPS and the incubation time for re-epithelia-lization of wounded IEC-6cells were evaluated.Concerning the time effect,signi?cant re-epithelialization of the wounded IEC-6cells was observed24h after the addition of medium as control or SJZPS.However,the migration rate in the control group was much lower than that in the SJZPS-treated groups.Concerning the dose effect,SJZPS at a?nal concentration of100m g/ml was the optimal dose for stimulation of epithelial cell restitution in terms of cell migration towards the denuded area in vitro. Stimulation of epithelial restitution was increased by38·45, 102·39and132·74%at8,16and24h,respectively,compared with the control.
Protective effect of polysaccharides from Si-Jun-Zi decoction on IEC-6cells
We examined the relative viability of IEC-6cells after treat-ment with various concentrations of SJZPS ranging from50 to800m g/ml in the proliferation assay(Fig.1).The results show that SJZPS signi?cantly enhanced the growth of IEC-6 cells by more than40%at the optimal dose of100–200m g/ ml.Indomethacin,a common non-steroidal anti-in?ammatory Table1.The composition and sugar content analysis of the
polysaccharides of Si-Jun-Zi decoction
Total sugar(wt%)51·82
Protein(wt%)37·50
Lipopolysaccharides ND
Sugar components(mol%)
Glucose44·61
Galactose33·96
Arabinose10·85
Rhamnose5·26
Xylose4·84
Mannose0·46
Fructose0·02 ND,not detected.
L.Liu et al. 24
drug that causes inhibition of re-epithelialization,was used to study the protective effect of SJZPS on IEC-6cells(Fig.2). Indomethacin alone induced nearly40%inhibition of cell growth in comparison with the control.However,addition of SJZPS at a concentration of100m g/ml produced30%more viable IEC-6cells than the indomethacin treatment.
Gene expression pro?ling of IEC-6cells upon induction with polysaccharides from Si-Jun-Zi decoction
To explore the differential gene expression upon induction with SJZPS,DNA microarray analysis was performed.Wounded IEC-6cells treated with100m g SJZPS/ml for24h were subjected to the Atlas Glass Rat 1.0Microarray,which included nine housekeeping genes,negative and positive controls and a total of1081rat genes.Three sets of microarray hybridization exper-iments were performed,and the genes up-and down-regulated in response to SJZPS induction are shown in Table3.These genes could be classi?ed into different categories,mainly related to ion channels such as voltage-gated ion channels,gradient-driven transporters,calcium-modulating ion channel and calcium-binding proteins,transcription factors,intracellular kinase members and G-proteins.In particular,six genes,those coding for JUND,phosphorylase B kinase,protein kinase type 1,Ktvoltage-gated channel,Ktinwardly-rectifying channel and Ktchannel protein CDRK,were considered as up-regulated (Cy3:Cy5.1·5).Five genes,those coding for g-aminobutyric acid transporter,solute carrier family6(serotonin transport), neural visinin-like Ca2t-binding protein type2and3,and Ras-related GTP-binding protein4b,were down-regulated(Cy3:Cy5 ,0·6)(Table3).Besides,three additional genes,those coding for Ktchannel1,acid-sensing ion channels,proton-gated cation channels(ASIC1)and metabotropic glutamate receptor2,with expression ratio of1·4-fold were also selected and considered as marginal candidate genes.The expression levels of Ktchannel 1and ASIC1proton-gated cation channels were con?rmed by RT-PCR.
Con?rmation of microarray data by RT-PCR
Among the up-regulated genes found in the microarray study,the following were chosen for con?rmation by semi-quantitative RT-PCR:PBK(accession no.M73808);JUND(accession no. D26307);RCK4(accession no.X16002);RIIA(accession no. M17086);and Kv4(accession no.M68880).Because of the vari-ations in abundance of mRNA of different genes in the cell,the
Table2.The restitutive effect of the polysaccharides of Si-Jun-Zi decoction(SJZPS)on the migration of IEC-6cells upon injury.Various concentrations of SJZPS were used to treat the wounded IEC-6cells and the migration area for each group of SJZPS-treated cells was monitored at8,16and24h.Values are means with their standard deviation from three separate experiments.Migration rate at each time interval is expressed as a percentage of the control
Migration area(mm2)at different intervals
8h16h24h
SJZPS concentration
(m g/ml)Mean SD Migration
rate(%)Mean SD
Migration
rate(%)Mean SD
Migration
rate(%)
Control141·916·5–223·125·0–708·6148·5–50136·623·23·8250·311·612·2890·1*130·525·7 100196·533·638·5451·5*111·8102·41649·2***307·1132·7 200172·144·921·3321·235·244·01293·0***116·882·5 400174·68·923·1350·262·057·01485·0***171·5109·6 800204·6***72·244·2443·4162·398·81266·5***237·678·7
Least signi?cant difference test between means(two-way ANOVA):*P,0·01,***P,0·001with regard to control
values.
Fig.1.Dose effect of the polysaccharides of Si-Jun-Zi decoction(SJZPS)on proliferation of IEC-6cells.‘Control’represents the medium as control;posi-tive controls are20ng epithelial growth factor(EGF)/ml and10%fetal bovine serum(FBS).Values are means and standard deviation shown by vertical bars for three separate experiments.Statistical analysis was performed by one-way factorial ANOVA(P,0·0001)with Dunnett’s post hoc test.Signi?-cant difference v.control medium:*P,0·05,**P,0·01,*P,0·001.SJZPS signi?cantly enhances the growth of IEC-6cells by more than40%at the optimal dose of100–200m
g/ml.
Fig.2.Mucosal restitution effect of the polysaccharides of Si-Jun-Zi decoc-
tion(SJZPS)on indomethacin(Indo)-treated IEC-6cells.Values are means
and standard deviation shown by vertical bars for three separate exper-
iments.Statistical analysis was performed by one-way factorial ANOVA
(P,0·0001)with Dunnett’s post hoc test.Signi?cant difference v.indometha-
cin treatment:**P,0·01,***P,0·001.SJZPS at the concentration of
100m g/ml shows the best restorative effect on IEC-6cells,resulting in
approximately86%relative viability.
Effects of polysaccharides on intestinal epithelial cells25
PCR cycles (Tsien &Tsien,1990;McCormack et al.1998)and titration of the RT reaction (equal to 100ng,500ng and 1·m g total RNA)were performed in duplicate to ensure that PCR was in the linear range (data not shown).The relative expression ratios of selected genes were normalized with regard to ribosomal gene S29(Fig.3).The results indicate that the expression patterns of the selected genes as determined by DNA microarray and RT-PCR were highly comparable.
Discussion
The use of herbal medicinal decoctions to modulate body homeo-stasis is a common practice among the Chinese.Si-Jun-Zi decoc-tion,a traditional Chinese herbal prescription formulated by four Chinese herbs (Dangshen,Gancao,Baizhu and Fuling)has been used to treat disorders of the digestive system with symptoms such as poor appetite,indigestion and diarrhoea.Previous studies showed that many of its active constituents,in particular the poly-saccharides,shared anti-tumour and immunostimulatory effects (Haranaka et al.1985;Lin,1988;Xu et al.1994;Liu et al.2000;Chang,2002;Kiyohara et al.2002).However,the protec-tive effect of the polysaccharides of SJZD on the gastrointestinal epithelium had not been studied previously.
To elicit the gastrointestinal restitution effect of SJZPS,we per-formed several lines of bioassay.First,the proliferative effect of SJZPS on IEC-6cells was demonstrated by MTT assay (Fig.1).It can be inferred from the observations that replacing lost cells through cell proliferation can enhance restitution of damaged epi-thelial cells.Second,SJZD was found to protect IEC-6cells from indomethacin-induced injury.Indomethacin,a cyclooxygenase inhibitor,is the most potent of the non-steroidal anti-in?ammatory drugs and can cause gastroduodenal mucosal injury,ulceration and delay ulcer healing (Larkai et al.1987).Previous studies showed that the ulcerogenic action of indomethacin could be attributed to the increase of intestinal permeability (Kimura et al.1998),in?ux of neutrophils (Bjarnason et al.1993)and prostaglandin depletion (Ding et al.1998).Indomethacin has a
Table 3.IEC-6cells seeded in a Matrigel-coated Petri dish were incubated in Dulbecco’s modi?ed Eagle medium supplemented with 10%fetal bovine serum in a humidi?ed atmosphere with 95%air and 5%CO 2for 72h,followed by 24h serum-free incubation.Con?uent cells were mechanically wounded with the tip of a multi-channel pipette.Cells were washed with PBS and incubated with 100m g/ml polysaccharides of Si-Jun-Zi decoction (SJZPS)for 24h.Total RNA was extracted,reverse-transcribed into cDNA labelled with Cy5(for control)and Cy3(for treatment)and hybridized to the Altas e Glass Rat 1·0Microarray (Clontech,Palo Alto,CA,USA).The average gene expression ratio of control medium and SJZPS-treated IEC-6cells from three independent microarray experiments was calculated.Genes with an expression ratio .1·5or ,0·6were selected as candidate genes.‘#’indicates that the gene is a marginal candidate.Genes marked with ‘*’had their expression level con?rmed by RT-PCR Genes expressed differentially in wounded IEC-6cells in response to SJZPS induction GenBank accession no.Cy3:Cy5
Transcription factors JunD proto-oncogene *
D263071·7Intracellular kinase network members Phosphorylase B kinase catalytic subunit *
M738081·8Protein kinase,cAMP-dependent,regulatory,type 1*
M170861·5Voltage-gated ion channels
Potassium voltage-gated channel,shaker-related subfamily,member 4*X160021·5Potassium inwardly-rectifying channel,subfamily J,member 5L357711·5Potassium channel protein CDRK M774821·5Potassium channel 1*?
M688801·4Gradient-driven transporters
ASIC1proton-gated cation channel ?U944031·4GABA transporter
U289270·6Solute carrier family 6(neurotransmitter transporter,serotonin),member 4(5-hydroxytryptamine (serotonin)transporter)M79450
0·4
Calcium-modulated ion channels
Metabotropic glutamate receptor 2(GRM2;MGLUR2)?M920751·4Calcium-binding proteins
Neural visinin-like Ca 2t-binding protein type 2D131250·6Neural visinin-like Ca 2t2binding protein type 3D131260·5G-protein
Ras-related GTP-binding protein 4b
X78605
0·5
GABA,g -aminobutryic acid.
*Genes had their expression level con?rmed by RT-PCR.?Gene is a marginal
candidate.
Fig.3.Con?rmation of microarray data by semi-quantitative RT-PCR.cDNA was synthesized from 1m g RNA in duplicate.The expression ratio for type I regulatory subunit of cAMP-dependent protein kinase (RIIA)was observed at 12cycles;JunD proto-oncogene (JUND )and phosphorylase kinase catalytic subunit (PBK )were observed at 25cycles;putative potassium channel sub-unit protein (RCK4)and neuronal delayed recti?er K tchannel (Kv4)were observed at 35cycles.Expression of all target genes was normalized by that of the gene coding for ribosomal protein S29(S29).All genes were found to be up-regulated and the expression patterns were comparable to the micro-array data.
L.Liu et al.
26
pronounced effect on human intestinal mucosal injury(Li et al. 1999;McCormack et al.1999),whereas SJZPS can restore the recovery of IEC-6cells after treatment with indomethacin. This suggests that SJZPS can exert a certain repairing ability in the early phase of mucosal restitution.Third,the gastrointestinal restitution effect of SJZPS was further demonstrated by the in vitro migration assay(McCormack et al.1993).Accordingly, replacement of lost cells or tissues is partly achieved by cell migration(Tarnawski et al.1995).In the current study,the con-?uent monolayer of IEC-6cells was scratched with a razor blade and the cells were allowed to migrate towards the denuded area.The results indicated that SJZPS could enhance migration of the IEC-6cells;the most effective dosage was100m g/ml.
To gain further insight into the modulating effect of SJZPS during intestinal epithelial restitution,DNA microarray technol-ogy was applied to decipher the biological pathways involved. Among the1081genes pro?led in the microarray experiments, the candidate genes that were signi?cantly up-regulated coded for voltage-gated ion channel,voltage-gated Ktchannel, inwardly-rectifying Ktchannel,Ktchannel protein CDRK (Circumvallate Papilla Delayed Recti?er Ktchannel),Ktchan-nel1and gradient-driven transporter(proton-gated cation channel, ASIC1).Previous reports have shown that Ktchannels play a very important role in the migration of intestinal epithelial cells through the modulation of membrane potential and Ca2tin?ux (Wang et al.2000;Rao et al.2002).In addition,Ca2tis a well-known secondary messenger in many cellular activities and bio-chemical pathways.Alteration of cytosolic Ca2tconcentration would determine the response of cells to the surrounding environ-ment,sequential signal transduction linked with the surface receptors and hence the?nal behaviours of the cells.Basically, in the present study,the expression of Ktchannel genes due to the effect of SJZPS acting on wounded IEC-6cells correlated positively with the cell migration assay.
The activities of the voltage-gated Ktchannels that regulate membrane potential determine the cytosolic free Ca2tconcen-tration by regulating Ca2tin?ux.IEC-6cells are a type of non-excitable cell that does not express L-type voltage-dependent Ca2tchannels(Himmel et al.1993;Nilius et al.1997).Thus the cytosolic free Ca2tconcentration is regulated by the in?ux of extracellular Ca2tthrough passive Ca2tleakage,receptor-operated Ca2tchannels,stored-operated Ca2tchannels and the Ca2treleased from intracellular organelles such as endoplas-mic and sarcoplasmic reticulum(Quaroni et al.1979;Tsien& Tsien,1990;Himmel et al.1993;Putney&Bird,1993;Bilato et al.1995;Pauly et al.1995;Nilius et al.1997;Moore et al. 1998).Moreover,the magnitude of Ca2tin?ux has been found to depend on both the transmembrane Ca2tgradient and the membrane potential(Fleischmann et al.1993;Bilato et al. 1995).Previous studies have also indicated that the membrane potential of eukaryotic cells is basically controlled by the number and functional ability of membrane Ktchannels (Fleischmann et al.1993;Bilato et al.1995).When the gene expression of Ktchannels is being induced,the sequential increase of available Ktchannels and their functional ability result in membrane hyperpolarizaton.Since membrane potential is the major driving force controlling Ca2tin?ux,this tendency increases the magnitude of Ca2tin?ux into the cells and?nally contributes to the elevation of cytosolic Ca2tconcentration.Rais-ing cytosolic free Ca2tconcentration stimulates cell contraction and migration through the initiation and participation of cytoskeleton reorganization,such as activation of the Ca2t/cal-modulin(Pauly et al.1995)and Ca2t/RhoA(Rao et al.2001)sig-nalling pathways,mediation of cadherin–catenin-associated interaction(Guo et al.2003)and the Ras superfamily of small GTP(guanosine triphosphate)-binding proteins.
Another candidate gene also up-regulated by the action of SJZPS was that coding for acid-sensing ion channels,proton-gated cation channels(ASIC1).This gene is a member of the ASIC subfamily(Waldmann&Lazdunski,1998)that shares cer-tain identity(approximately20–25%)with the subunit of the epi-thelial Na channel with similar topological domains and intracellular termini.Both of its subunits(ASIC1a and ASIC b) were shown to associate with the formation of Nat-selective ion channels(Fyfe et al.1998),but with different permeability for Ca2t(Chen et al.1998).ASIC might provide certain means for controlling the Ca2tentry pathway as well as modulating neuronal activity(Waldmann&Lazdunski,1998).Furthermore, ASIC was found to participate in the formation of mechanosensi-tive ion channels that were implicated to be associated with the cytoskeleton(Price et al.2000).
Surprisingly,a proto-oncogene(JUND)was found to be over-expressed in the SJZPS-induced IEC-6cell system.JunD is a member of the Jun family,which consists of three members (c-jun,JunB and JunD)and they are all cellular transcription fac-tors.They occupy a central role in cellular signal transduction and regulation of proliferation(Lamph et al.1988)through their bind-ing to speci?c DNA sequences as homodimers or heterodimers with Fos proteins,forming a complex called activator protein-1 (Curran&Vogt,1992).Previous studies showed that c-jun, JunB and JunD mRNA levels were elevated several times after vascular injury and such focal expression indicates the conse-quences of the initiation of DNA synthesis and migration after vascular injury.Increasing evidence has con?rmed that c-jun and JunB are involved in the initiation of cell cycle and prolifer-ation.Conversely,the role of JunD in cell proliferation is still unclear.Some data showed that JunD could slow down cell pro-liferation(Hirai et al.1989;Ryder et al.1989;Li et al.2002) while some reports still proposed that it could induce cell prolifer-ation.There are two processes involved in the achievement of mucosal restitution:the rapid removal of damaged cells by sloughing followed by the migration of adjacent cells and the replacement of lost cells through cell proliferation.The former occurs within1h while the latter process may begin around 16h after injury(Silen,1987).So the over-expression of JunD can be explained in the sense that it transiently slowed down the proliferation of IEC-6cells,while the cells were actively migrating under the in?uence of SJZPS by modulating the K channels and cytosolic Ca2tconcentration.
In the present study,we used IEC-6cells as a cellular model to elicit the protective and restitution effects of SJZPS on gastroin-testinal lining.Our results indicate that SJZPS not only exerts a stimulatory effect on the proliferation of IEC-6cells,but also restores the recovery of IEC-6cells after treatment with indo-methacin.In addition,we also found that SJZPS can increase the cell migration rate after wounding.In applying the DNA microarray technology to study differential gene expression of SJZPS-induced IEC-6cells,we discovered increased expression of a group of genes that are related to K and ion channels.The microarray data were con?rmed by RT-PCR.
In research on traditional Chinese medicine formulations,the pharmacological actions of polysaccharides are usually neglected
Effects of polysaccharides on intestinal epithelial cells27
probably owing to the complexity of their chemical nature.How-ever,with the introduction of DNA microarray technologies into the study of Chinese medicine,it is possible to correlate the effects of the herbal compound at the gene level(Watanabe et al.2001;Bonham et al.2002;Sadava et al.2002;Iizuka et al.2003).SJZD is a classic Chinese herbal formula with tonic properties on the gastrointestinal system.In this work we have demonstrated that polysaccharides of SJZD can signi?cantly enhance the in vitro migration and proliferation of IEC-6cells. These data provide further evidence to support the therapeutic value of SJZD in traditional Chinese medicine formulations. Acknowledgements
This work was supported by Hong Kong Baptist University (Faculty Research Grant FRG//00-01/II-75-P).The authors also thank Dr Zhou H.for assisting in statistical analysis;Dr Jiang Z.H.,Ms Wang J.R.,Ms Shen X.L.and Mr Fu I.J.for determi-nation of sugar composition and content.
References
Bilato C,Pauly RR,Melillo G,Monticone R,Gorelick-Feldman D, Gluzband YA,Sollott SJ,Ziman B,Lakatta EG&Crow MT(1995) Intracellular signaling pathways required for rat vascular smooth muscle cell migration.Interactions between basic?broblast growth factor and platelet-derived growth factor.J Clin Invest96,1905–1915. Bjarnason I,Hayllar J,MacPherson AJ&Russell AS(1993)Side effects of nonsteroidal anti-in?ammatory drugs on the small and large intestine in humans.Gastroenterology104,1832–1847.
Bonham M,Arnold H,Montgomery B&Nelson PS(2002)Molecular effects of the herbal compound PC-SPES:identi?cation of activity pathways in prostate carcinoma.Cancer Res62,3920–3924.
Cario E,Jung S,Harder D’Heureuse J,Schulte C,Sturm A, Wiedenmann B,Goebell H&Dignass AU(2000)Effects of exogenous zinc supplementation on intestinal epithelial repair in vitro.Eur J Clin Invest30,419–428.
Chang R(2002)Bioactive polysaccharides from traditional Chinese medi-cine herb as anticancer adjuvants.J Altern Complement Med8, 559–565.
Chen C,England S,Akopian AN&Wood JN(1998)A sensory neuron-speci?c,proton-gated ion channel.Neurobiology95,10240–10245. Chen GZ&Fu D(1996)Effect of jiawei sijunzi decoction on migrating myoelectric complex in8Gy irradiated rats.Zhongguo Zhong Xi Yi Jie He Za Zhi16,221–223.
Chun H,Shin DH,Hong BS,Cho WD,Cho HY&Yang HC(2002) Biochemical properties of polysaccharides from black pepper.Biol Pharm Bull25,1203–1208.
Curran T&Vogt P(1992)Transcriptional Regulation.New York:Cold Spring Harbor Press.
Dignass AU&Podolsky DK(1995)Growth factors and cytokinase in in?ammatory bowel disease:injury and healing in the epithelium.In In?ammatory Bowel Diseases,pp.375–383[GNJ Tygat,JFWM Bartelsman and SJH van Deventer,editors].Dordrecht:Kluwer Academic Publishers.
Ding SZ,Lam SK,Yuen ST,Wong BC,Hui WM,Ho J,Guo X&Cho CH(1998)Prostaglandin,tumor necrosis factor alpha and neutrophils: causative relationship in indomethacin-induced stomach injuries.Eur J Pharmacol348,257–263.
Fleischmann BD,Fleischmann BK,Punt JA,Gaulton G,Hashimoto Y &Kotlikoff MI(1993)Control of resting membrane potential by delayed recti?er potassium currents in ferret airway smooth muscle cells.J Physiol469,625–638.
Fyfe GK,Quinn A&Canessa CM(1998)Structure and function of the Mec-ENaC family of ion channels.Semin Nephrol18,138–151.Guo L,Dong ND,Xiong AB,Liu ZY,Liu CR&He XC(2003)An exper-imental study on the prevention and treatment of postburn intestinal injury and bacterial translocation by Sijunzi decoction in scalded rats.
Zhonghua Shao Shang Za Zhi19,89–93.
Haranaka K,Satomi N,Sakurai A,Haranaka R,Okada N&Kobay M (1985)Antitumor activities and tumor necrosis factor producibility of traditional Chinese medicine and crude drug.Cancer Immunol Immun-other20,1–5.
Hegde P,Qi R,Abernathy K,Gay C,Dharap S,Gaspard R,Hughes JE, Snesrud E,Lee N&Quackenbush J(2000)Concise guide to cDNA microarray analysis.Biotechniques29,548–562.
Himmel HM,Whorton AR&Strauss HC(1993)Intracellular calcium, currents,and stimulus-response coupling in endothelial cells.Hyperten-sion21,112–127.
Hirai SI,Ryseck RP,Mechta F,Bravo R&Yaniv M(1989)Characteriz-ation of junD:a new member of the jun proto-oncogene family.EMBO J8,1433–1439.
Iizuka N,Oka M,Yamamoto K,Tangoku A,Miyamoto K,Miyamoto T, Uchimura S,Hamamoto Y&Okita K(2003)Identi?cation of common or distinct genes related to antitumor activities of a medicinal herb and its major component by oligonucleotide microarray.Int J Cancer107, 666–672.
Kimura RE,Arango V&Lloyd-Still J(1998)Indomethacin and pancrea-tic enzymes synergistically damage intestine of rats.Dig Dis Sci43, 2322–2332.
Kiyohara H,Matsumoto T&Yamada H(2002)Intestinal immune system modulating polysaccharides in a Japanese herbal(Kampo)medicine, Juzen-Taiho-To.Phytomedicine9,614–624.
Lacy ER(1988)Epithelial restitution in the gastrointestinal tract.J Clin Gastroenterol10,72–77.
Lamph WW,Wamsley P,Sassone-Corsi P&Verma IM(1988)Induction of proto-oncogene JUN/AP-1by serum and TPA.Nature334, 629–631.
Langmead L&Rampton DS(2001)Herbal treatment in gastrointestinal and liver disease–bene?ts and dangers.Aliment Pharmacol Ther 15,1239–1252.
Larkai EN,Smith JL,Lidsky MD&Graham DY(1987)Gastroduodenal mucosa and dyspeptic symptoms in arthritic patients during chronic nonsteroidal anti-in?ammatory drug use.Am J Gastroenterol82, 1153–1158.
Li L,Li J,Rao JN,Li M,Bass BL&Wang JY(1999)Inhibition of poly-amine synthesis induces p53gene expression but not apoptosis.Am J Physiol Cell Physiol276,C946–C954.
Li L,Liu L,Rao JN,Esmaili A,Strauch ED,Bass BL&Wang JY(2002) JunD stabilization results in inhibition of normal intestinal epithelial cell growth through P21after polyamine depletion.Gastroenterology 123,764–779.
Lin PF(1988)Antitumor effect of Actinidia chinensis polysaccharide on murine tumor.Zhongghua Zhong Liu Za Zhi10,441–444.
Liu L,Zhou H,Wang PX&Hu YJ(2000)In?uence of total polysaccharide extracted from Sijunzi decoction on the intestinal mucosa associated lymphoid tissues of mice.Zhougguo Mianyixue Zazhi17,204–206.
Lowry O,Rosebrough NH,Farr AL&Randall RJ(1951)Protein measure-ment with the Folin phenol reagent.J Biol Chem193,265–275. McCormack SA,Viar MJ&Johnson LR(1992)Migration of IEC-6cells:
a model for mucosal healing.Am J Physiol263,G426–G435. McCormack SA,Viar MJ&Johnson LR(1993)Polysaccharides are necessary for cell migration by a small intestinal crypt cell line.Am J Physiol264,G367–G374.
McCormack SA,Blanner PM,Zimmerman BJ,Ray R,Poppleton HM,Patel TB&Johnson LR(1998)Polyamine de?ciency alters EGF receptor dis-tribution and signaling effectiveness in IEC-6cells.Am J Physiol274, C192–C205.
McCormack SA,Ray R,Blanner PM&Johnson LR(1999)Polyamine depletion alters the relationship of F-actin,G-actin,and thymosin beta4in migrating IEC-6cells.Am J Physiol276,C459–C468.
L.Liu et al. 28
Miller GL(1959)Use of dinitrosalicylic acid reagent for determination of reducing sugar.J Anal Chem31,426–430.
Moore R,Carlson S&Madara JL(1989)Rapid barrier restitution in an in vitro model of intestinal epithelial https://www.wendangku.net/doc/3014377147.html,b Invest60, 237–244.
Moore TM,Brough GH,Babal P,Kelly JJ,Li M&Stevens T(1998) Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1.Am J Physiol275,L574–L582. Mosmann T(1983)Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays.J Immunol Methods65,55–63.
Nilius B,Vianna F&Droogmans G(1997)Ion channels in vascular endo-thelium.Annu Rev Physiol59,145–170.
Ou M(1993)Chinese–English Manual of Common-used Prescription in TCM.Guangdong:Guangdong Science&Technology Publishing House.
Pauly RR,Bilato C,Sollott SJ,Monticone R,Kelly PT,Lakatta EG& Crow MT(1995)Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration.
Circulation91,1107–1115.
Price MP,Lewin GR,McIlwrath SL,et al.(2000)The mammalian sodium channel BNC1is required for normal touch sensation.Nature407, 1007–1011.
Putney JWJ&Bird GS(1993)The inositol phosphate–calcium signaling system in nonexcitable cells.Endocr Rev14,610–631.
Quaroni A,Wands J,Trelstad RL&Isselbacher KJ(1979)Epithelial cell cultures from rat small intestine:characterization by morphologic and immunologic criteria.J Cell Biol80,248–265.
Rao JN,Li L,Golovina VA,Platoshyn O,Strauch ED,Yuan JX&Wang JY(2001)CA2t-RhoA signalling pathway required for polyamine-dependent intestinal epithelial cell migration.Am J Physiol Cell Physiol 280,C993–C1007.
Rao JN,Platoshyn O,Li L,Guo X,Golovina VA,Yuan JX&Wang JY (2002)Activation of Ktchannels and increased migration of differentiated intestinal epithelial cells after wounding.Am J Physiol Cell Physiol282,C885–C898.
Ryder K,Lanahan A,Perez-Albuerne E&Nathans D(1989)Jun-D:a
third member of the jun gene family.Proc Natl Acad Sci USA86, 1500–1503.
Sadava D,Ahn J,Zhan M,Pang ML,Ding J&Kane SE(2002)Effects of four Chinese herbal extracts on drug-sensitive and multidrug-resistant small-cell lung carcinoma cells.Cancer Chemother Pharmacol49, 261–266.
Silen W(1987)Gastric mucosal defense and repair.In Physiology of the Gastrointestinal Tract,pp.1055–1069,[LR Johnson and J Chris-tense,editors].New York:Raven Press.
Staub AM(1965)Removal of proteins–Sevag method.Methods Carbo-hydr Chem5,5–6.
Tarnawski A,Tanoue K,Santos AM&Sarfen IJ(1995)Cellular and molecu-lar mechanisms of gastric ulcer healing.Is the quality of mucosal scar affected by treatment?Scand J Gastroenterol210,9–14.
Tsien RW&Tsien RY(1990)Calcium channels,stores,and oscillations.
Annu Rev Cell Biol6,715–760.
Waldmann R&Lazdunski M(1998)H(t)-gated cation channels:neur-onal acid sensors in the NaC/DEG family of ion channels.Curr Opin Neurobiol8,418–424.
Wang JY,Wang J,Golovina VA,Li L,Platoshyn O&Yuen JXJ (2000)Role of Ktchannel expression in polyamine-dependent intesti-nal epithelial cell migration.Am J Physiol Cell Physiol278, C303–C314.
Watanabe CM,Wolffram S,Ader P,Rimbach G,Packer L,Maguire JJ, Schultz PG&Gohil K(2001)The in vivo neuromodulatory effects of the herbal medicine Ginkgo biloba.Proc Natl Acad Sci USA98, 6577–6580.
Xu HM,Xie ZH&Zhang WY(1994)Immuomodulatory function of polysaccharide of Hericium erinaceus.Zhongguo Zhong Xi Yi Jie He Za Zhi14,427–428.
Zhao AG,Zhao HL,Jin XJ,Yang JK&Tang LD(2002)Effects of Chi-nese Jianpi herbs on cell apoptosis and related gene expression in human gastric cancer grafted onto nude mice.World J Gastroenterol 8,792–796.
Zhong WR,Huang YX&Cui JP(1997)Clinical study on modi?ed sijunzi decoction in treating intestinal metaplasia of gastric mucosa.Zhongguo Zhong Xi Yi Jie He Za Zhi17,462–464.
Effects of polysaccharides on intestinal epithelial cells29
数据管理系统之数据可视化设计
数据管理系统企业级数据可视化项目Html5 应用实践 项目经理：李雪莉 组员：申欣邹丽丹陈广宇陈思 班级：大数据&数字新媒体 一、项目背景 随着大数据、云计算和移动互联网技术的不断发展，企业用户对数据可视化的需求日益迫切。用户希望能够随时随地简单直观的了解企业生产经营、绩效考核、关键业务、分支机构的运行情况，即时掌握突发性事件的详细信息，快速反应并作出决策。随着企业信息化的不断推进，企业不断的积累基础信息、生产运行、经营管理、绩效考核、经营分析等以不同形式分布在多个系统或个人电脑文档内的业务数据。如何将大量的数据进行分析整理，以简单、直观、高效的形式提供给管理者作为经营决策的依据是当前企业数据应用的迫切需求。传统的企业数据可视化方案多基于Java Applet、Flash、Silverlight 等浏览器插件技术进行开发，在当前互联网和移动互联网技术高速发展的背景下，Web技术标准也随之高速发展，用户对互联网技术安全性和使用体验的要求越来越高。Java Applet、Flash、Silverlight 等浏览器插件技术因为落后和封闭的技术架构，以及高功耗、高系统资源占用，已经被微软、谷歌、苹果、火狐等主流操作系统和浏览器厂商逐步放弃，转而不断支持和完善基于HTML5的新一代Web技术标
准 对数据进行直观的拖拉操作以及数据筛选等，无需技术背景，人人都能实现数据可视化无论是电子表格，数据库还是 Hadoop 和云服务，都可轻松分析其中的数据。 数据可视化是科学、艺术和设计的结合，当枯燥隐晦的数据被数据科学家们以优雅、简明、直观的视觉方式呈现时，带给人们的不仅仅是一种全新的观察世界的方法，而且往往具备艺术作品般的强大冲击力和说服力。如今数据可视化已经不局限于商业领域，在社会和人文领域的影响力也正在显现。 数据可视化的应用价值，其多样性和表现力吸引了许多从业者，而其创作过程中的每一环节都有强大的专业背景支持。无论是动态还是静态的可视化图形，都为我们搭建了新的桥梁，让我们能洞察世界的究竟、发现形形色色的关系，感受每时每刻围绕在我们身边的信息变化，还能让我们理解其他形式下不易发掘的事物。 二、项目简介 目前，金融机构（银行，保险，基金，证劵等）面临着诸如利率汇率自由化，消费者行为改变，互联网金融崛起等多个挑战。为满足企业的发展需要，要求管理者运用大数据管理以更为科学的手段对企业进行精准管理，从而更好地把握市场在竞争中胜出。德昂BI商务智能解决方案基于业务的数据分析正是帮助企业实现科学化管理的关键，因而获得客户的高度重视与高频度使用。 激烈的市场竞争下，通过对金融机构业务数据的汇总与整理实现
图书管理系统数据流程图
图书管理系统数据流程图 2009-04-14 17:20 1.1 系统分析 1.1.1 图书馆管理信息系统的基本任务 该“图书馆管理信息系统”是一个具有万人以上的员工，并地理位置分布在大型企的图 书馆理系统，图书馆藏书 100 多万册，每天的借阅量近万册。在手工操作方式下，图书的编目和借阅等的工作量大，准确性低且不易修改维护，读者借书只能到图书馆手工方式查找书目，不能满足借阅需求。需要建立一套网络化的电子图书馆信息系统。 该图书馆管理信息系统服务对象有两部分人：注册用户和一般读者。一般读者经注册后成为注册用户，注册用户可以在图书馆借阅图书，其他人员只可查阅图书目录，但不能借阅图书。系统同时考虑提供电子读物服务，目前只提供电子读物的目录查询服务，不久的将来将提供电子读物全文服务。用户可通过网络方式访问读图书馆管理信息系统。 1.1.2 系统内部人员结构、组织及用户情况分析 为了对系统有一个全貌性的了解，首先要对系统内部人员结构、组织及用户情况有所了 解。图书馆系统的组织结构如图 1 － 1 所示。 图 1 － 1 图书馆管理信息系统的组织结构 图书馆由馆长负责全面工作，下设办公室、财务室、采编室、学术论文室、图书借阅室、电子阅览室、期刊阅览室和技术支持室。各部门的业务职责如下。
办公室：办公室协助馆长负责日常工作，了解客户需求，制定采购计划。 财务室：财务室负责财务方面的工作。 采编室：采编室负责图书的采购，入库和图书编目，编目后的图书粘贴标签，并送图书借阅室上架。 学术论文室：负责学术论文的收集整理。 图书借阅室：提供对读者的书目查询服务和图书借阅服务。 电子阅览室：收集整理电子读物，准备提供电子读物的借阅服务，目前可以提供目录查询和借阅。 期刊阅览室：负责情况的收集整理和借阅。 技术支持室：负责对图书馆的网络和计算机系统提供技术支持。 1.1.3 系统业务流程分析 系统的业务室系统要达到的业务目标，业务流程分析是系统分析的基础环节。图书馆管 理信息系统的业务流程如图 1 － 2 所示。
图书管理系统——数据字典
软件产品开发需求模型(DFD 和DD) 数据字典是关于数据的信息的集合，对数据流程图中的各个元素做完整的定义与说明，是 数据流程图的补充工具。数据流图和数据字典共同构成系统的逻辑模型。 数据字典由下列六类元素的定义组成： （1）数据流 （2）数据项：是“不可再分”的数据单位，是数据的最小组成单位。 （3）数据结构 （4）数据存储：数据存储是数据结构停留或保存的场所。 （5）处理逻辑 （6）外部实体 在第一层和第二层数据流图的定义之后，我们都已经详细定义了数据字典的各元素。 对于各数据项的详细符号描述，见实验二的《软件概要设计说明》中的“软件数据结构设计”。 一、 数据流图： 1． 网上购书电子商务系统数据流程图（第一层） DBMS1.1暂存订单 DBMS1.2书籍库存 DBMS1.3采购订单 DBMS1.4销售历史DBMS1.6应付款明细帐DBMS1.5应收款明细帐DBMS1.7总帐
数据流图说明：（DD） 1．1 E：外部项 1．2 P：处理逻辑 1．3 F：数据流 共有FBMS1.1～FBMS1.10这10个数据流，分别描述如下：（1）数据流名称：FBMS1.1 数据流说明：用户登入 （2）数据流名称：FBMS1.2 数据流说明：密码修改 （3）数据流名称：FBMS1.3 数据流说明：顾客的订单
（4）数据流名称：FBMS1.4 （5）数据流名称：FBMS1.5 （6）数据流名称：FBMS1.6 数据流说明：送货人给顾客的收据（发货票） （7）数据流名称：FBMS1.7
（8）数据流名称：FBMS1.8 （9）数据流名称：FBMS1.9 （10）数据流名称：FBMS1.10 1．4 D：数据存储 描述如下： （1）数据存储代号：DBMS1.1 数据存储名称：暂存订单
大数据管理系统技术
信息技术（选修4） 数据管理技术复习提纲 概要： 信息技术学科模块4——《数据管理技术》，全书以应用数据管理技术解决问题为主线，按照“分析问题——设计数据库——建立数据库——使用数据库——管理数据库”这一线索呈现学习容。全书分五章，下面介绍第一章至第五章的主要容： 第一章 认识数据管理技术 一、数据管理基本知识 1、数据管理技术的基本概念 数据：是人类社会的一种重要信息资源，是对现实世界中客观事物的符号。计算机中的数 据分为数值型数据与非数值型数据。 例题：如商品价格、销售数量等数据是（ ） A 、数值数据 B 、非数值数据 说明：数据是信息的符号表示或称为载体。即为了表达信息（抽象概念），必须使用某种符号，这些符号就叫数据，如字符、图表、图形、图像、声音、视频等都可以称为数据。信息依赖数据来表达，是数据的涵，是对数据语义的解释。 数据管理：是指对数据的收集、分类、组织、编码、存储、查询和维护等活动。 数据管理技术：指与数据管理活动有关的技术。 数据库（DB ）：是指按照某种模型组织起来的，可以被用户或应用程序共享的数据的集合。 数据库系统（DBS ）：是指采用的数据库技术的完整的计算机系统。 数据库管理系统（DBMS ）：是能够建立数据库、维护数据库及管理数据库的一个开发平台。 数据库应用系统：是应用了数据库的信息系统。 说明：数据库系统的核心为数据库管理系统，数据库管理系统的核心为数据库（或数据） 例题：下列软件中，不属于数据库应用系统的是（ ） A 、学籍管理系统 B 、中考成绩查询系统 C 、Linux 操作系统 D 、网络售票系统 例题：数据库管理系统英文简写是（ ） A 、D B B 、DBS C 、DBMS D 、Access 系统软件 应用软件 数据库系统结构示意图