Effect Of Curcumol On Rho-ROCK Signaling Pathway Of Hepatic Stellate Cells In Mice With Hepatic Fibrosis

Objective:The present study was designed to investigate the effects of curcumol on Rho-ROCK signaling pathway in hepatic stellate cells in mice with hepatic fibrosis,and to explore the underlying molecular mechanism of its action.Hopefully,our study could provide scientific basis for anti-fibrosis of curcumol,and lay theoretical foundation for further development and research of genuine medicinal materials in Guangxi,curcuma in anti-hepatic fibrosis.Methods:In-depth analysis of hepatic fibrosis and curcuma was performed using bioinformatics.KM mice were divided into three groups namely blank group,model group,and curcumol group.Carbon tetrachloride was used to establish hepatic fibrosis model,followed by gavage of curcumol.Liver structural changes were observed by HE and Masson staining,and four indexes for liver fibrosis as well as liver function were detected using an automated analyzer.In addition,mouse hepatic stellate cells were cultured and then grouped.MTT colorimetric assay was used to determine the proliferation rate of cells in each group,to determine the high,medium and low concentrations of curcumol by eight concentration gradients.Cells were then divided into six groups: blank control group,model group,positive control group,low-concentration curcumol group,medium-concentration curcumol group and high-concentration curcumol group.Real-time quantitative PCR(RT-q PCR)was used to detect the mRNA expression of Rho A and ROCK2,and Western blot was utilized for the protein expression of Rho A and ROCK2.Results: 1.Results of bioinformatics analysis: Bioinformatics analysis indicated that the important target genes of hepatic fibrosis were closely associated with the Rho-ROCK signaling pathway.And the genes enriched in the pathway of pharmacological action of curcumol were closely related to Rho-ROCK signaling pathway.2.Construction of hepatic fibrosis model: In the blank group,hepatocyt es were radially arranged around the portal area,without obvious pathol ogical changes,and almost no collagen fiber formation.In the model gr oup,hepatocytes were irregularly arranged,with vacuole-like changes,c ollagen fiber deposition,widened fiber spacing,and obvious pseudolobul i formed by the fibrous tissue surrounding the hepatic lobules.In the c urcumol group,collagen deposition was less,with alleviated hepatic lesi ons compared to the model group.3.Four indexes of hepatic fibrosis and liver function indexes: The four indexes of hepatic fibrosis and liver function indexes were significantly higher in the model group and the curcumol group than those in the control group,which were significantly higher in the model group compared to the curcumol group(P<0.05).4.MTT assay: Eight concentration gradients of curcumol were set.As a result,curcumol inhibited cell proliferation in all groups except the blank group.Three doses of curcumol at 1.5626ug/ml,3.1252ug/ml and 6.2504ug/ml were selected as the low,medium and high concentration groups of curcumol,due to the relatively high cell proliferation rate of these three groups.In the eight groups,with the increased concentration of curcumol,the cell proliferation rate was significantly decreased,indicating the dose-dependent effect of curcumol on cell proliferation(P<0.05).5.RT-qPCR: The mRNA expression of RhoA and ROCK2 was significantly higher in in the model group than that in the blank control group(P<0.05).The m RNA expression of Rho A and ROCK2 was significantly higher in the positive control group than the that in model group(P<0.05).Moreover,the m RNA expression of Rho A and ROCK2 was significantly lower in the curcumol groups with different concentrations in a dose-dependent pattern,compared to that in the model group(P< 0.05).6.Western blot: The protein expression of Rho A and ROCK2 was significantly higher in the model group than that in the blank control group(P<0.05).Similarly,the protein expression of Rho A and ROCK2 was significantly higher in the positive control group than that in the model group(P<0.05).The protein expression of Rho A and ROCK2 was significantly lower in the in the curcumol group compared to that in the model group(P<0.05).Conclusion: Rho-ROCK signaling pathway is closely associated with hepatic fibrosis and curcuma.Curcumol exerts a certain anti-hepatic fibrosis effect in KM mouse model.In addition,curcumol inhibits Rho-ROCK signaling pathway in hepatic stellate cells,which is dose-dependent within a certain dose range.