Hepatic fibrosis is a wound-healing process in the liver with acute or chronic injury and is charac-terized by the excess production and deposition of extracellular matrix（ECM）components[1]．Hepatic menchymal cells，such as stellate cell and hepatic myofibroblast，play a pivotal role in the liver fibroge-nesis as the main ECM-poroducing cells[2]．In respon to variety of liver injuries，the proliferation of hepatic stellate cells（HSCs）increas and HSCs undergo phenotypic change into myofibroblast-like cells（a process termed‘activation’）for synthesis of fibrotic matrix rich in type I collagen，which leads to the for-
形容微风的词语Bile acids enhance proliferation and motility of hepatic stellate cell through regulation of p38／JNK signaling
ZHANG Yi-ning1Tadashi Ikegami2DU Hong-wei1Yasushi Matsuzaki2 1．First Hospital of Jilin University（Changchun130021，Jilin，China）；2．Division of Gastroenterology and Hepatology，Tokyo Medical University Kasumigaura Hospital（Tsukuba300－0395，Japan）
Abstract：Objective Bile acids（BA）facilitate cholesterol hepatic fibrosis．Although hepatic stellate cell（HSC）is one of the most important cells during liver fibrogenesis，the effect of bile acids on HSC is rarely mentioned．Therefore，bile acids facilitate liver fibrosis through regulating activated H
SC should be tested．Methods The amount of BrdU incor-poration was determined to asss the proliferation of HSC treated by bile acid．Wound-healing assay was ud to determine the cellular motility．Meanwhile，the phosphorylation of p38and JNK in HSC was detected by Western blotting．Results 50μmol／L GCDCA enhanced the HSC proliferation significantly（152．0％±7．1％，P＜0．05）；50μmol／L GCDCA also induced phosphorylation of p38and JNK（450．0％±12．2％of control in p38（P＜0．01），210．0％±15．2％of control in JNK（P＜0．05））．50μmol／L GCDCA aided wound healing（remaining wound area is75．4％±5．8％of original area，P ＜0．05），but this effect was inhibited by JNK（SP600125）or p38（SB294002）inhibitor，respectively．Conclusions Bile acids enhance HSC proliferation and facilitate cellular motility through inducing phosphorylation of p38and JNK，in-dicating bile acid aid liver fibrosis through regulation of p38and JNK signaling．（J Clin Pediatr，2010，28（4）：301－306）Key words：liver fibrosis；bile acid；hepatic stellate cell
胆汁酸对肝星状细胞增殖及细胞动能的调控作用张一宁1，池上正2，杜红伟1，松崎靖司2（1．吉林大学第一医院，长春130021，吉林，中国；2．东京医科大学茨城医疗中心，筑波300-0395，日本）
摘要：目的探讨胆汁酸如何通过调控肝星状细胞促进肝纤维化发生。方法采用BrdU掺入法测定胆汁酸和TGFβ-1对GFSC-2G细胞增殖的影响；利用Wound-healing检测法判定细胞的移动能力；同时利用
Western blotting法检测与胆汁酸共同孵育的GFSC-2G细胞的p38和JNK的表达。结果与50μmol／L甘氨鹅脱氧胆酸（GCDCA）共同孵育后，CFSC-2G细胞增殖明显增加，为对照组的152．0％±7．1％（P＜0．05）。50μmol／L GCDCA诱导p38及JNK磷酸化的作用[在p38为对照组的450．0％±12．2％（P＜0．01），在JNK为对照组的210．0％±15．2％（P＜0．05）]。50μmol／L GCDCA促进培养细胞的伤痕愈合（剩余面积为原来的75．4％±5．8％，P＜0．05），而JNK （SP600125）或p38（SB294002）的抑制因子能够抑制胆汁酸的这种作用。结论胆汁酸具有促进肝星状细胞增殖的作用，还能提高该细胞的运动能力，这种作用是通过促进p38和JNK的磷酸化而实现的。提示胆汁酸可能通过调控p38和JNK的信号转导而促进肝纤维化的形成。[临床儿科杂志，2010，28（4）：301－306]关键词：肝纤维化；胆汁酸；肝星状细胞
中图分类号：R725文献标志码：A文章编号：1000－3606（2010）04－0301－06
·Original Article in English·Corresponding author：ZHANG Yi-ning E-mail：Yining＠jlu．edu．cn
mation of scar tissue and ultimately，liver cirrhosis．It has been well investigated that elevated bile acid concentration（no matter in intrahepatic chole-stasis or in extrahepatic cholestasis）leads to the damage of hepatocytes and cholangiocytes[3－5]．The sustained injuries of parenchymal cells have been thought to be a cau of hepatic fibrosis in cholestatic liver dias．On the other
hand，the direct role of bile acid on menchymal cells，including HSCs，has not been fully elucidated yet．
Svegliati et al[6]indicated that HSCs are resistant to bile acid-induced cell apoptosis and can survive even in the prence of toxic concentration of bile acids．Moreover，they found that glycochenodeoxy-cholic acid（GCDCA）can induce HSCs proliferation via activation of the epidermal growth factor receptor （EGFR），thus aiding the development of the hepatic fibrosis[7]．
Besides its proliferative character，the activated HSC shows veral different behaviors in the process of hepatic fibrosis．The include（1）enhanced con-tractility which promotes portal hypertension，and （2）incread cell motility which contributes to the effective distribution of HSC at the injured sites．The effect of bile acids on the characters in the indivi-dual cells has not been clarified yet．Therefore，pre-nt study was focud on the effect of bile acids in the motility of activated HSC．
MATERIALS AND METHODS
Immortalization and Culture of Myofibro-blast-like Cell
The rat myofibroblast-like cell clone（CFSC-2G），being considered as‘activated’HSC，ob-tained from a liver with CCl4-induced cirrhosis after spontaneous immortalization in culture[8]，was placed on plastic culture dishes（Corning Japan，Tokyo，Japan）in DMEM containing10％fetal bovine rum and100U／ml penicillin／streptomycin．CFSC-2G cells were cultured at37°C in a humidified atmosphere of 5％CO2．The medium was changed24hours after plating and then every48hours．Protein Extraction and Western Blotting Cells cultured on35mm-diameter culture dishes were rind twice with PBS（4°C）and lyd with ly-sis buffer containing150mmol／L NaCl，1．5mmol／L MgCl2，5mmol／L EDTA，1％Triton X-100，1％NP40，10mmol／L NaF，1mmol／L Na3VO4，and protea inhibitor cocktail（Roche，Indianapolis，IN）．The protein concentration of the cell lysates and tissue homogenates was determined by the BCA pro-tein assay kit（Pierce Biotechnology，Rockford，IL）．SDS-PAGE（10％acrylamide gel）and Western blotting were performed as previously described[9]．Cell Proliferation Assay
Proliferation of CFSC-2G cells（control and co-cultured with bile acids）was determined by ELISA with a bromodeoxyuridine（BrdU）colorimetric kit （Roche）．The cells were treated according to the manufacturer＇s instruction．The amount of BrdU in-corporation was determined at a wavelength of405nm by an Emax TM precision microplate reader（Molecular Devices Corporation，Menlo Park，CA）．
Wound-healing Assay
The wound-healing assay was performed as pre-viously described by Voulet-Craviari et al[10]．Plastic dishes（n＝20）containing confluent cells were scraped with a pipette tip（10μl）．Following wound-ing，the culture medium was changed to remove detached and damaged cells，and the extent of wound closure was determined microscopically12hours lat-er．Three fields of the wound were monitored from the beginning to the end of the experiment．Wound images were captured by a microscope（IX71）equipped with a DP70digital camera（Olympus），and the area of the wound was calculated by Image J software．The ratios before and after wound closure were determined and expresd as a percentage of the remaining wound area．
Reagents
As for bile acids，50micromollar glycocheno-deoxycholic acid（GCDCA，Sigma，St．Louis，MO），a non-toxic concentration confirmed by MTT assay，has been ud throughout the following study．The inhibitor of JNK（SP600125）or p38（SB294002）were pur-
Figure 1GCDCA enhanced cell proliferation
chad from Calbiochem （San Diego ，Calif．），anti-p38MAPK and anti-phosphop38MAPK from Cell Signaling （Beverly ，Mass．）．
Statistics
All of the experiments were repeated more than 3times．Statistical analysis was performed either by one-way analysis of variance or the two-tailed Stu-dent＇s t test using Prism software （Graphpad Software Inc ，SanDiego ，CA ）．Results were expresd as mean ±SD ，and P values less than 0．05were con-sidered statistically significant．All of the experiments were repeated 6times．
RESULTS
Bile Acid Enhanced CFSC-2G Proliferation
CFSC-2G cells were incubated with GCDCA and ／or TGF-β1and the incorporation of BrdU was de-tected at the indicating time．As shown in Figure1，After 24hours of co-incubation ，50μmol ／L GCDCA induced the cell proliferation in the prence of 10％FCS-containing medium （152．0％±7．1％compared to CTL ，P ＜0．05），but this proliferative effect of GCDCA was not en in the cells pre-incubated over 24hours with 1％FCS-containing medium （106．0％±7．3％compared to
小学成语CTL ，not statistically different ），whereas TGF-β1（10ng ／ml ）and GCDCA together incread the BrdU incorporation （140．0％±6．1％，P ＜0．05）at the prence of 10％FCS ，though TGF-β1itlf did not show effect on the cell proliferation．
To exclude the influence of the cell proliferation ，the cells pre-incubated with 1％FCS-containing medi-um for 24hours were ud in the following experi-ments．
Bile Acid Facilitated CFSC-2G Cell Migration
To determine the effect of bile acids on the cell migration ，an in vitro wound-healing model was uti-lized．The monolayer cell sheet on the plastic dish was wounded by pipette tip ，and then cultured at the prence or abnce of 50μmol ／L of GCDCA．The closure of the wound was monitored over time under a pha-contrast microscope．As shown in Figure 2A ，12hours after scraping the cells ，the wound was par-tially filled with migrated cells treated with bile acid ，while migration was more limited by 12hours in the untreated cells．For quantitative determination ，the area of the wound was measured at the beginning and 12hours after the scraping （Figure 2B ）．In the bile acid treated group ，the remaining wound area after 12hours was 75．4％±5．8％（Figure 2C ）of the original wound （P ＜0．05，compared to controls ），whereas in control group ，it was 97．5
％±3．8％of the original wound （NS compared to controls ），show-ing the closure was enhanced by GCDCA．Further-more ，this effect of bile acid was inhibited by either of the JNK or p38inhibitor．The remaining wound area is 98．2％±7．8％（SP600125）and 95．3％±6．5％（SB294002）of the original wound ，respectively．
MAP Kina Involved in the Effect of Bile Acid on Cellular Function of CFSC-2G Cell
To investigate the possible mechanisms of action ，MAP kina signaling was then focud since previous studies had demonstrated the alteration of MAP kina signaling by bile acids．Especially ，in the MAP kina family members ，p38as well as JNK was focud ，so called stress activate protein kina ，which had been also suggested to be involved in the cellular motility．
TGF-β1，which has been considered as the most potent fibrogenic cytokine ，induced p38and JNK phosphorylation dramatically．As shown in Figure 3A ／3B ，the phosphorylation of p38（1h ，650．0％±14．3％of CTL ，P ＜0．01）and JNK （10min ，150．％±18．9％of CTL ，P ＜0．05）reached their peak at 1h or 10minutes after being treated with TGF-β1．This pattern of phosphorylation was also ob-rved at the prence of 50μmol ／L GCDCA．After 1h of exposure to 50μmol ／L of GCDCA ，the phos-
Figure 3B Bile acid induced phosphorylation of JNK
Figure 3A Bile acid induced phosphorylation of p38
Figure 2A The effect of bile acide on cellular motility Figure 2B The effect of bile
acid on cellular motility was
霸道总裁言情小说
inhibited by JNK ／p38
inhibitor
Figure 2C The effect of bile acid on cellular motility
phorylation of p38augmented dramatically （1h ，450．0％±15．6％of control ，P ＜0．01）．Likewi ，the phosphorylation of JNK was also determined under the same condition．Although TGF-βonly induced JNK phosphorylation slightly and transiently ，the in-duction of GCDCA was greater and the effect re-mained until 24hours after the exposure （10min ，210．0％±15．2％of control ，24h ，160．0％±7．9％of control ，P ＜0．05）．
DISCUSSION
It is well investigated that damage of hepatocytes induced by toxic bile acid contributes to liver fibroge-nesis due to cholestasis．But how does HSC involve in hepatic fibrosis is under investigation．It is reported that synthetic FXR ligand regulates the progression of hepatic fibrosis through the suppression of HSC acti-vation [11]，but there is no confirmed evidence of the uptake of bile acid by HSC and bile acids were ignored in the updated study of HSC．Instead ，vari-ous receptors and cond mesngers localized near plasma membrane have been considered as the target molecules of bile acids in HSC [7]．手机串号查询
Activation of HSCs is the most important steps for HSCs during liver fibrogenesis．Generally ，activa-
tion of HSCs is accompanied by incread migration and proliferation [2，12－14]．It is reported that HSCs are resistant to bile acid-induced cell apoptosis and can survive even in the prence of bile acids．Further-more ，the fact that bile acid facilitates the prolifera-tion of HSCs indicates that bile acid may facilitate the liver fibrogenesis．Our data also demonstrated that 50μmol ／L GCDCA ，a toxic concentration for hepa tocytes ，enhanced the proliferation of HSCs ，implying the possible involvement of bile acid in hepatic fibro-genesis．MAPK signaling pathway is known to play a key role in cell survival and proliferation ，and bile acids can induce the phosphorylation of MAPKs ，
especially p38and JNK [15，16]．Therefore ，bile acid could play a role in sustaining the proliferation of the activated HSCs ，thus preventing their apoptotic death through regulating the MAPKs signaling．To test this hypothesis we determined the MAPKs phosphorylation at the same time．We found that HSC proliferation is paralleled by the phosphorylation of JNK and p38，indicating that besides EGFR signaling ，MAPKs signa-ling takes part in the proliferation of HSCs too．
Cell migration is a key aspect of many physiolo-gical and pathological process [17，18]．In respon to various stimuli ，the activated HSC migrates to the injured area and releas ECM [19－21]．It is generally
accepted that the driving force for cell movement is provided by the dynamic reorganization of the cyto-skeleton，directing protrusion at the front of the cell and retraction at the rear．Meanwhile，the complicat-ed net-work driving the cell to move also depends on the cooperation of intermediate filaments，adhesion molecules，and accessory proteins[22，23]．In our experi-ment，wound-healing was utilized to asss the effect of bile acid on cellular motility and the results demon-strated that GCDCA stimulated the movement of HSCs and facilitated the wound closure compared to control cells．Meanwhile，the enhanced movement is accom-panied with the phosphorylation of M
APKs．The characters induced by GCDCA are independent of proliferative effect of bile acids under our condition．Yang et al[20]mentioned that the cell migration induced by PDGF-BB was associated with incread prolifera-tion，whereas TGF-β1／EGF-induced migration was proliferation independent．Therefore，it is possible that the bile acids facilitate the HSC activation via proliferation dependent and independent way．Our da-ta showed that bile acids enhance the HSC prolifera-tion only in the ca of the prence of10％FCS．This suggests that some factors in the FCS are neces-sary for the proliferative effects of GCDCA．In turn，the factors in the FCS are not necessary for migratory effect of GCDCA．Moreover，compared with control group，a greater number of HSC expod to GCDCA prented a phenotype with lamellipodia，a sheet-like protrusive structure found at the leading edge，con-sisting of a cross-linked meshwork of actin filaments （data not shown）．The prence of lamellipodia indi-cates the collaboration of multiple molecular and cy-tokines in liver fibrogenesis，though further investiga-tion is necessary for elucidating the mechanisms．Various evidences have confirmed that MAP ki-na families，especially p38and JNK take important part in cell proliferation and cell migration．In the prent study，elevated phosphorylation of p38and JNK were paralleled with cell proliferation and cell migration after bile acid treatment，indicating the in-volvement of the two kinas in the cellular motility and proliferation induced by bile acid．In addition，in a bile duct ligation model，the express
ion of TGF-β，especially of TGF-β1was only found to increa dra-matically in HSCs among the multiple liver cells[24]．Moreover，the p38as well as JNK have been identi-fied as the molecules under the control of TGF-βsig-naling pathway．Since TGF-βis the most potent pro-fibrogetic cytokine inducing HSC activation，the idea that bile acids induce TGF-βsignaling is worthwhile to discuss．The expression of TGF-βin the prence of bile acids is now under investigation in our labora-tory．The“real”target of bile acid should be further identified in order to understand the pathological mechanism of cholestatic fibrosis，and this may lead to the further development of new therapeutic concept of the dias．
In conclusion，bile acid enhanced proliferation and cellular motility of HSCs．P38／JNK signaling is involved in the effects．Bile acid may aid the deve-lopment of liver fibrosis through enhancing the proliferation and cellular motility of HSCs．
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（Received November2，2009）营城
