Mechanism Of 1,25（OH）₂D₃ Inhibit Hepatic Stellate Cell Activation By Regulating MiRNA Expression

Background and Objective:Liver fibrosis is a common pathological process in which various liver diseases develop to cirrhosis and even hepatocellular carcinoma.The current study shows that,liver fibrosis is a highly dynamic process,effective interventions could delay or even reverse the occurrence and development of liver fibrosis.Therefore,looking for effective drugs or therapeutic targets to reverse the process is particularly important.1,25（OH）₂D₃ is the active form of vitamin D in the body,our team in previous experiments found that it has the effect of inhibiting proliferation and promoting apoptosis of rat hepatic stellate cells,and has the role of anti-liver fibrosis.However,the specific mechanism is not yet clear.Mi RNA is a class of non-coding RNA which is 18-25 nt in length,participating the regulation of other protein-coding gene expression.Studies found that many mi RNAs involved in the activation and proliferation of HSC,promoting or inhibiting the occurrence and development of liver fibrosis.The purpose of this study was to explore whether 1,25（OH）₂D₃ could inhibit hepatic stellate cell activation by regulating the expression of mi RNA or regulating mi RNA-mediated signaling pathways,and then exert anti-hepatic fibrosis.Methods:By subcutaneous injection of CCL4 and 10 pmmol / L TGF-β1 stimulate hepatic stellate cells to construct in vitro and in vivo models of hepatic fibrosis.Transfection of mi RNA mimics / inhibit to overexpression / inhibit of mi RNA in hepatic stellate cells.And then using 1,25（OH）₂D₃ respectively act on hepatic stellate cells and SD rats.q PCR was used to screen differentially expressed of mi RNA and verify if the transfection was successful and each group of differential expression of mi RNA expression in hepatic stellate cells.Detectioning the proliferation of hepatic stellate cells by CCK8.Flow cytometry was used to detect apoptosis.Detecting the SD rat serum ALT 、 AST and HE staining methods to detect the degree of liver tissue fibrosis.Using bioinformatics technology to analyze mi RNA target gene.Western blot method to verify the target gene expression.Results1.Compared with the primary hepatic stellate cells,the expressions of miR-146a,mi R-30 c,mi R-193,mi R-101,and mi R-200 a in activated hepatic stellate cells were abnormal,but miR-146a was statistically significant.2.CCK8 results showed that compared with miR-146a mimics NC group,the proliferation rate of hepatic stellate cells in miR-146a mimics transfection group was significantly decreased,and compared with miR-146a inhibit NC group,the proliferation rate in miR-146a inhibit group was significantly increased,while compared with DMSO control group,1,25（OH）₂D₃ group the hepatic stellate cells proliferation rate was significantly reduced（P<0.05）.Flow cytometry results showed that compared with miR-146a mimics NC group,the rat hepatic stellate cell apoptosis rate was significantly increased in miR-146a mimics transfected group;compared with miR-146a inhibit NC group,the cell apoptosis rate was significantly reduced in miR-146a inhibit group,while compared with DMSO group,the 1,25（OH）₂D₃ group of hepatic stellate cell apoptosis rate was significantly increased,（P<0.05）,the difference was statistically significant.3.The expression of ALT and AST in the serum of the model group and the drug control group was significantly higher than that of the normal group.The expression of ALT and AST in the drug treatment group was significantly lower than that in the drug control group（P<0.05）.The difference was statistically significant.4.HE staining showed that compared with the normal group,liver fibrosis in the model group and the drug control group was significant,while compared with the drug control group,the degree of hepatic fibrosis in the drug treatment group was significantly reduced.5.q PCR result showed that the expression of miR-146a in model group and drug control group was significantly lower than the normal group,while in drug treatment group the expression of miR-146a was significantly higher than the drug control group,the difference was statistically significant.6.Biotechnological analysis showed that DLL1 is a potential target gene of miR-146a,and the expression level of DLL1 in the model group and the drug control group was significantly higher than that in the normal group（P<0.05）,while the expression level of DLL1 in the drug treatment group was significantly lower than that in the drug control group（P<0.05）,the difference was statistically significant;Conclusions:1.The expression of miR-146a in the TGF-β1 stimulated HSC and SD rat liver fibrosis models is down-regulated.2.1,25（OH）₂D₃ could inhibit the activation of HSC and the formation of hepatic fibrosis.3.The mechanism of 1,25（OH）₂D₃ inhibit hepatic stellate cell activation may be related to up-regulation of miR-146a expression.4.DLL1 may be one of the targets gene of miR-146a anti-liver fibrosis5.1,25（OH）₂D₃ may be by regulating the expression of miR-146a and thus inhibiting the Notch signaling pathway to play the role of anti-hepatic fibrosis.