Asiatic Acid Regulates The Mechanism Of Liver Fibrosis Via Lipin1/TLR4 Signaling Pathway

Objective:Asiatic acid(ASA)is one of the five ring three terpenoids of Chinese traditional Asiatic Herba.Some studies have found that it has a better protective effect on liver.This study used thioacetamide induced liver fibrosis in mice and the activation of hepatic stellate cells,macrophages,explore the asiatic acid through Lipin 1/TLR4-IRAK4 signaling pathway in liver fibrosis effect and mechanism of regulation.Methods:The mice liver fibrosis model was induced by intraperitoneal injection of TAA for 28 days,two times a week for first weeks,next three weeks:three times a week.Daily gavage treatment different doses of asiatic acid(5mg/kg,10mg/kg,20mg/kg)were given to mice for all days.The serum and liver were collected at-80 C.Serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured by serum assay kit.Adopt Masson,H&E and Sirius red,immunohistochemical staining of 4 methods,pathological changes of the liver were observed.The protein and mRNA expression levels of alpha-SMA,Collagen-I,MMP-13,TIMP-1,TLR4 and Lipin-1 were detected.Used logarithmic mouse hepatic stellate cells(HS),TGF-beta(10 ng/ml)stimulation 1 h,and then were incubatedwith different asiatic acid concentrations(1.56,3.125,6.25,12.5,25 ?mol/L)for 6 h,cells were collected and analyzed by Western blot.The total proteins were extracted.The protein expression levels of TLR4,Lipin-1 and P2X7R were measured.Results:In vivo experiment,asiatic acid inhibited the activity of ALT and AST in serum compared with thioacetamide group.Asiatic acid inhibited thioacetamide induced the expression of-SMA and Collagen-I and at the same time adjusted the ratio of MMP-13/TIMP-1.Asiatic acid significantly decreased the expression of TLR4/IRAK4 and Lipin-1.In vitro,asiatic acid in concentration range(1.65-?25 mol/L)had no cytotoxicity on mouse hepatic stellate cells;Acanthopanic acid can obviously inhibit theexpression of TGF-beta-stimulated alpha-SMA,in a dose dependent manner,while acanthoic acid dosage is 6.25-25?M TLR4 expression decreased significantly.LPS is a lipid polysaccharide,which stimulates mouse macrophages(Raw264.7)expression inflammatory factors and induces activation mononuclear macrophages(THP-1).Compared with the single with LPS stimulation Raw264.7 and THP-1 cells,in different concentrations(1.56,3.125,6.25,12.5,25?M)after incubation of asiatic acid,asiatic acid can significantly inhibit Lipin-1 activation in a dose-dependent manner,when the concentration is 25 ?M,TLR4 and P2X7R also have the same effect.Activated hepatic stellate cells with different concentrations(1.56,3.125,6.25,12.5,25 mu M)of asiatic acid,at concentrations 3.125,6.25,12.5 and 25 ?M can significantly reduce the protein level of alpha-SMA,and 25 M M asiatic acid can significantly reduce the protein level of Collagen-I,while asiatic acid at 6.25,12.5 and 25 M can significantly reduce the protein level of TIMP-1.Asiatic Acid with a dose of 6.25,12.5 and 25 inhibited the expression of Lipin-1 and then inhibited the expression of TLR4 in a dose-dependent manner.Conclusion:Asiatic acid may inhibit the expression of Lipin-1 by inhibiting TLR4 and P2X7R,and thus improve the liver fibrosis induced by thioacetamide.And by inhibiting NLRP-3 inhibition of activation of hepatic stellate cells.LPS is combined with r Toll like receptor 4(Toll like receptor 4,TLR4)to form a complex,triggering activation of mononuclear macrophages and inducing the occurrence hepatitis.Previous studies suggest that LPS plays the role of activated cells mainly by initiating transmembrane signal transduction,and the internalization of LPS is related to the degradation of metabolism.In this study,it was found that LPS could enter the inner body through the internalization of TLR4,and then the activation of macrophages.Therefore,the aim of this study is to explore the internalization pathway of LPS-TLR4 complex and its role in LPS activation of macrophages and its possible mechanism.LPS activates the expression of lipid metabolic factor Lipin-1 in activating Raw264.7 and THP-1 cells,and then the TLR4 signaling pathway was activated.Therefore,asiasalic acid effectively inhibits the expression of Lipin-1 and P2X7R and then inhibits the expression of TLR4/IRAK4.