Effect Of GFK And XAV939on Wnt/RhoA/ROCK Signaling Pathway

Objective: Liver Fibrosis has been recognized as one of the inevitable processesthrough the lesions of mostchronic liver disease, and it can further result in cirrhosis andeven Hepatocellular Carcinoma (HCC). HCC is one of the most high-rate carcin oma inChina. Therefore, Chinese clinical medicine has been committed to studying HCCcontrol and HCC intervention. It has been acknowledged through the current medicalfield that the process of Liver Fibrosis growing to cirrhosis is the key to whether thedisease is reversible because Liver Fibrosis is reversible but cirrhosis is not. Therefore,the treatment and intervention of Liver Fibrosis should be significant to cirrhosisprevention and treatment. The core of Liver Fibrosis process is the activation of HepaticSatellate Cells (HSC). A lot of research has proved that Wnt pathway should beclassified in to CanoncialWnt pathway and NoncanoncialWnt pathway due to whetherWnt protein will produce endogenous integrated β-cateninsignals through theprogression of Liver Fibrosis. A lot of research has been conducted to Canoncial Wntsignaling pathway (Wnt/β-catenin signaling pathway) during the past few years, but fewhas been done to Noncanoncial Wnt pathway since it is more complicatedand containsthree signaling pathway, one of which is RhoA/ROCK signaling pathway that is relatedto various diseases including cell con traction, migration, prolif eration, apoptosis, andgene experssion. It has been proved that small molecule XAV939takes a positive effectin intervening canonical Wnt signaling pathway. However, such researches related tohepatic cell are few. By applying Chinese traditional medicine Ganfukang and smallmolecule inhibitor XAV939to HSC-T6, this thesis studies the effects of Ganfukang and XAV939on Liver Fibrosis level and RhoA/ROCK signaling pathway in noncanonicalWnt sigaling pathway, and further explores the connection between canon ical Wntpathway and noncanonical Wnt signaling pathway.Methods:HSC-T6cells was cultured in Dulbecco,s modified eagle medium(DMEM)containing10%fetal bovine serum, on temperature of37℃and5%CO2incubator,subculture.Wait for growth, pairs of cells with DMEM medium for culture, the cultureconcentration do not contain fetal bovine serum, to cultivate a day of hunger.Cells in six orifice were randomly divided into: normal serum group (DMEMmedium containing10%normal rat serum);XAV9391umol/L group (DMEM mediumcontaining10%normal rat serum and join XAV9391umol/L);XAV9392umol/L(DMEM medium containing10%normal rat serum and join XAV9392umol/L);GanFuKang treatment group (containing10%GanFuKang treatment group ratsserum DMEM medium).Put the six orifice plate in the incubator, at the temperature of37℃, CO2content is5%, training for two days.Reverse transcription-polymerase chainreaction (reverse transcriptase polymerase chain reaction, RT-PCR) method fordetermination of lcollagenI, collagenIII, a-SMA, FAK vinculin and RhoA, ROCKImRNA expression in cells;Immune protein imprinting method (western blot) detectionmeasure ROCK1protein expression in cells.Results:1. GFK and XAV939drug serogroup influences on the targets of extracellularmatrix (ECM)：GFK serogroup compared with normal serogroup collagen I, collagen Ⅲ,a-SMA expression at the genetic level has obvious decline（P<0.05），XAV9391umol/L,2umol/L serum group compared with normal serum collagen I, collagen Ⅲ, a-SMA expression at the genetic level has obvious decline（P<0.05），And XAV9392umol/Lserogroup effect is stronger than1umol/L group.2.GFK and XAV939drugs effects on cytoskeleton change: GFK serogroupcompared with normal serogroup FAK,vinculin mRNA level express has obviousdecline（P<0.05），XAV9391umol/L,2umol/L serum group compared with normal serogroup FAK,vinculin expression at the genetic level has obvious decline（P<0.05），And XAV9392umol/L serogroup effect is stronger than1umol/L group.3.GFK and XAV939drug serum of RhoA/ROCK signaling pathway.3.1RT-PCR results show XAV939serogroup Rho RhoA/ROCK signalingpathways, ROCK1mRNA expression compared with normal serum group, nosignificant cha nges occurred,GFK serum drug group of RhoA mRNA expression wassignificantly lower (P <0.05).3.2Western-blot shows XAV939serogroup Rho/ROCK signaling pathway ofROCK1protein expression compared with normal serum group, no significant changesoccurred,GFK serum drug group ROCK1protein experssion sign ificantly lower (P <0.05).It consistent with the results of RT-PCR.Conclusions:1.GFK and XAV939obvious inhibitory HSC that was activated。2.GFK and XAV939obvious inhibitory noncanoncial Wnt/RhoA/ROCK signalingpathway。3.XAV939to noncanoncia Wnt RhoA/ROCK signaling pathway effect is notobvious。...