Study Of The Mechanism Of Baicalin Via Up-regulating MiR-3595 Targeting The Expression Of ACSL4 Inhibits Hepatic Fibrosis In Rats

Object:This study aims to explore the role of miRNAs and their target genes in the process of regulating the function of activated hepatic stellate cells by baicalin,so as to clarify the mechanism of baicalin against hepatic fibrosis and provide scientific basis for the clinical application of baicalin.Methods:Part 1:1.The effect of baicalin on the morphology of activated hepatic stellate cells was observed by using microscope;2.The expression level of a-SMA in activated hepatic stellate cells was detected by using qPCR and western blot assay;3.The effects of baicalin on migration,invasion and proliferation ability of activated hepatic stellate cells were detected by using transwell assay and CCK-8 Kit.;4.Cell-cycle distribution and apoptosis level of activated hepatic stellate cells after the cells treated with baicalin were detected by using flow cytometry;5.The effect of baicalin on the expression spectrum of miRNAs in activated hepatic stellate cells was analyzed based on miRNA chip array.Part 2:1.The expression level of miR-3595 in activated hepatic stellate cells after treated with baicalin was detected by using qPCR;2.MiR-3595 inhibitor was synthesized and transfected into activated hepatic stellate cells,to detect the effects of miR-3595 on migration,invasion,proliferation,cell-cycle distribution,apoptosis level and hepatic fibrosis-related protein expression in the cells after treated with baicalin;3.The target gene(ACSL4)of miR-3595 was predicted based on the online database Targetscan,and the direct targeting relationship between miR-3595 and ACSL4 was verified by double luciferase reporter gene assay;4.ACSL4-expressing plasmid was constructed and transfected into activated hepatic stellate cells,to detect the effects of ACSL4 on migration,invasion,proliferation,cell-cycle distribution,apoptosis level and hepatic fibrosis-related protein expression in the cells after treated with baicalin.Part 3:1.Rat model of hepatic fibrosis was established by using dimethyl-nitrosamine(DMN).2.The protein expression levels of a-SMA,Desmin and ACSL4 were detected by using western blot assay;3.The gene expression levels of miR-3595 and TGF-β1 were detected by using qPCR;4.The effects of baicalin on hepatic fibrosis in rats were detected by using HE staining and Masson staining;5.The effects of baicalin on serum biochemical indices were detected by using automatic biochemical analyzer or ELISA Kit;6.The effects of baicalin on the contents of MDA,Hyp,SOD and GSH-Px in rat liver were detected by using ELISA Kit.Results:Part 1:1.Baicalin could affect the morphology of activated hepatic stellate cells;2.Baicalin could inhibit the expression of a-SMA in activated hepatic stellate cells,presenting a concentration-dependent effect;3.Baicalin could inhibit the migration,invasion and proliferation ability of activated hepatic stellate cells,presenting a concentration-dependent effect;4.Baicalin could induce G1 arrest and apoptosis of activated hepatic stellate cells;5.MiRNA chip array revealed that the affection of baicalin on the function of activated hepatic stellate cells might be related to the up-regulation of miR-3595.Part 2:1.Baicalin could significantly up-regulate the expression of miR-3595 in activated hepatic stellate cells;2.The down-regulation of miR-3595 could reverse the inhibited migration,invasion,proliferation,cell-cycle distribution,apoptosis level and hepatic fibrosis-related protein expression of activated hepatic stellate cells after treated with baicalin;3.Double luciferase reporter gene assay showed that miR-3595 could directly target the 3’UTR region of the ACSL4 gene;4.The overexpression of ACSL4 could reverse the inhibited migration,invasion,proliferation,cell-cycle distribution,apoptosis level and hepatic fibrosis-related protein expression of activated hepatic stellate cells after treated with baicalin.Part 3:1.Baicalin could down-regulate the expression levels of a-SMA,Desmin and ACSL4 proteins in rat liver tissue;2.Baicalin could up-regulate the expression of miR-3595 and down-regulate the gene expression of TGF-β1 in rat liver tissue;3.Baicalin could reduce the degree of hepatic fibrosis in rats;4.Baicalin could improve serum biochemical indices in rats with hepatic fibrosis;5.Baicalin could decrease in MDA and Hyp contents in liver tissue of rats with hepatic fibrosis,and increase in SOD and GSH-Px contents.Conclusion:This study suggests that baicalin can inhibit the occurrence and development of hepatic fibrosis in rats by up-regulating miR-3595 and targeting the expression of ACSL4.