| Objective: Hepatic satellate cells(HSC) activation is general regardedas the key event in the development of liver fibrosis.Then the activatedhepatic satellate cells secreted a large number of extracellularmatrix,resulting in the occurrence of liver fibrosis.Wnt signalingpathway,including the canonical Wnt pathway and the noncanoncial Wntpathway,is widely involved in cell differentiation， apoptisis and immunemechanisms in human and animals.There were evidences thatβ-cateninsignaling pathway and Wnt/JNK signaling pathway are involved in thedevelopment and progression of liver fibrosis,but considerable differenceamong their researches.This study focued on the effect of the Wnt/β-cateninsignaling pathway inhibitior XAV939and the Chinese traditional medicineGanfukang on the activation of hepatic satellate cells.Chinese traditionalmedicine Ganfukang has been useed several times by our laboratoty andXAV939is a selective Wnt/β-catenin-mediated transcription inhibitor.Wecompared the roles of Wnt/β-catenin signaling pathway and Wnt/JNKpathway in liver fibrosis and tried to purse a new target to the treat thedisease.Methods:Rat HSC-T6cell line was cultured in Dulbecco,s modifiedeagle medium(DMEM) containing10%fetal bovine serum(FBS) at37℃in5%plus CO2.The cells were plated35mm plates and culutred for about1-2weeks.Then the cells were pretreated with serum-free DMEM for24hours when they coverd80%-90%of the bottom of the plates.The cells for MTT assay were separated into:nomoral serumgroup(DMEM with10%nomoral rats serum only),XAV9391umol/L group(DMEM with10%nomoral rats serum and XAV9391umol/L),XAV9392umol/L group(DMEM with10%nomoral rats serumand XAV9392umol/L),XAV9394umol/L group(DMEM with10%nomoralrats serum and XAV9394umol/L),XAV9398umol/L group(DMEM with10%nomoral rats serum and XAV9398umol/L),GFK group（DMEM with10%serum of medium-does GFK treated rats）.All the groups were culturedin96well plate at37℃in5%CO2gas mixture for48hours.The cells for RT-PCR and Western blot assay were separatedinto:nomoral serum group(treated with DMEM contains10%nomoral ratsserum only),XAV9391umol/L group(DMEM with10%nomoral rats serumand XAV9391umol/L),XAV9392umol/L group(DMEM with10%nomoralrats serum and XAV9392umol/L),GFK group（DMEM with10%serum ofmedium-does GFK treated rats).All the cells were cultured in35mm platesat37℃in5%CO2gas mixture for48hours.The mRNA expression ofcollagen I， a-SMA,β-catenin， Wnt1， Frizzled1， Frizzled2， MKK4，MKK7and JNK were analyzed by RT-PCR and the protein expression ofβ-catenin and p-JNK were tested by Western-blot.Results:1.MTT assay showed that:compared with the normal serumgroup,the OD value of the other five groups decreasedremarkably(P<0.05).But the differences among XAV9392umol/L,XAV9394umol/L group and XAV9398umol/L group were not statisticalsignificant.2.RT-PCR assay showed that:compared with the normalgroup,the mRNA expression of collagen I,a-SMA,β-catenin，Wnt1，Frizzled1， Frizzled2， MKK4， MKK7and JNK1was signsficantly declined in theGFK group(P<0.05).The mRNA expression of collagenI,a-SMA,β-catenin，Wnt1，Frizzled1and signsificantly decreased in theXAV9391umol/L and XAV9392umol/L compare to the normal serumgroup(P<0.05),at the same time,the mRNA expression between XAV9392umol/L and the GFK group had no signsificant change， while Frizzled2，MKK4， MKK7and JNK1expression had no signsificant change.3.Western-blot analysis for β-catenin and P-jnk proteinexpression,compared to the normal serum group,the protein expression of β-catenin and P-jnk decreasd evidently in the GFK group（P<005).Theprotein expression of β-catenin in XAV9391umol/L and XAV9392umol/Ldownregulated as the GFK group while the protein expression of p-jnk didnot.Conclusion:1.Wnt/β-catenin signaling pathway maybe play a moreimprtant role in the process of liver fibrosis than Wnt/JNK did.2.Chinesetraditional medicine GFK blocked both Wnt/β-catenin and Wnt/JNKsignaling pathway,and the therapeutic effect was highly dependent on theinhibition of Wnt/β-catenin signaling pathway. |
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