Atractylenolide Ⅰ Regulates Hepatic Stellate Cell Autophagy And Its Mechanism Of Action On Liver Fibrosis

Research background and purpose: Liver fibrosis refers to the excessive deposition of diffuse extracellular matrix(ECM),a process that leads from chronic liver disease to cirrhosis.Activation of hepatic stellate cells(HSCs)is considered to be a key step in the development of liver fibrosis,and HSCs are the most important cellular source of ECM.Autophagy is an essential metabolic pathway that enables cells to sustain homeostasis by degrading their own cytoplasmic components.However,in the process of fibrosis,autophagy is one of the reasons for the activation of HSCs and can provide energy for the activation of HSCs.Atractylenolide I(ATL-1)is a sesquiterpene obtained from the rhizomes of atractylodes,which has various biological activities such as neuroprotective,anti-allergic,anti-inflammatory,and anticancer.It has been shown to have hepatoprotective effects,but whether it regulates autophagy and its exact molecular mechanisms are still unclear.The aim of this study was to reveal that ATL-1 regulates autophagy and ameliorates liver fibrosis by its specific molecular mechanism.To this end,we used LX-2 cells and C57BL/6 mice for in vitro and in vivo studies to provide reliable experimental data and theoretical support for ATL-1 as a clinical antihepatic fibrosis drug.Materials and Methods: The cell model was stimulated by human hepatic stellate cells line LX-2 and platelet-derived growth factor(PDGF)-BB,with the intervention of ATL-1.CCK-8,clonal formation to detect cell proliferation;Oil Red O has been used to measure the number of cellular lipid droplets.Immunoblotting,immunofluorescence,and RT-q PCR were performed to detect the expression of α-SMA and COL1A1 proteins,as well as autophagy-related molecules P62,Becn1,ATG5,LC3-I,and LC3-II;immunoblotting was used to detect the expression of phosphorylated and non-phosphorylated proteins of PI3 K,Akt,and m TOR.Electron microscopy is used to examine the autophagosomes.Autophagy flux was detected by the autophagy double-label lentivirus m RFP-GFPLC3.The mouse liver fibrosis model was set up by intraperitoneal injection of CCl4 and ATL-1 injected intraperitoneally.6 weeks later,serum concentrations of Alanine aminotransferase(ALT)and Aspartate aminotransferase(AST)were measured in each group of mice,and the liver was observed by general observation.HE,Sirius Red,and Masson staining were used to observe the microstructure and collagen changes in the liver,and the expression of α-SMA and COL1A1 in the liver was detected by Western blot and immunohistochemistry.Result: 1.ATL-1 significantly inhibited the proliferation and activation of LX-2 cells.PDGFBB increased the expression of α-SMA and COL1A1,increased the number of clone formations,and decreased the number of lipid droplets in LX-2 cells,whereas ATL-1 promoted the accumulation of lipid droplets,reduced the expression of α-SMA and COL1A1,and the number of clone formations in LX-2 cells.2.ATL-1 inhibited autophagy in hepatic stellate cells.ATL-1 was able to result in increased expression of p62,reduced expression of ATG5,Beclin-1,and LC3-II proteins,and reduced number of LC3 fluorescent spots as well as autophagic vesicles in LX-2 cells.3.ATL-1 regulates the PI3K/Akt/m TOR signaling pathway involved in cellular autophagy and liver fibrosis.The expression of phosphorylated PI3 K,Akt,and m TOR proteins increased in ATL-1-treated cells,while the expression levels of the native proteins remained unchanged;after treatment with Rapamycin,a targeted inhibitor of m TOR(also an agonist of autophagy),the expression of p-m TOR and P62 decreased,while the α-SMA,COL1A1,Beclin-1,LC3-II expression appeared to rise again.4.ATL-1 can improve CCl4-induced liver fibrosis in mice.CCl4 increased collagen fibers in the liver of mice,while ATL-1 treated mice reduced collagen fiber deposition,α-SMA and COL1A1 protein expression,and serum AST and ALT concentrations.Conclusion: The experiments showed that ATL-1 could inhibit the activation and proliferation of hepatic stellate cells,inhibit the autophagy of LX-2 cells,activate the PI3K-Akt-mTOR pathway,and ameliorate CCl4-induced liver fibrosis in mice.This further recognizes the drug activity of ATL-1 and provides a new approach for the clinical treatment of liver fibrosis.