CD73 Aggravated Alcohol-related Liver Fibrosis By Regulating Hepatic Stellate Cells Autophagy And Its Mechanism

Alcohol-related liver fibrosis（ALF）is an important pathological process of alcohol-related liver diseases,the main characteristics is extracellular matrix excessively accumulate in liver.The activation of HSCs give play to vital function in development of liver fibrillation,some studies have shown that inhibition of the activation of them can effectively reduce the synthesis and deposition of ECM,thereby reducing ALF.CD73 is a membrane-bound glycoprotein that can dephosphorylate AMP to adenosine,adenosine and its receptor can affect the development of liver inflammation,fibrosis and carcinoma,CD73 also bring into play a certain action as adenosine,and it also participates in cell autophagy.Some studies have found that increased the autophagy of HSCs can promote its activation.In this paper,we used CD73 knockout mice and rat hepatic stellate cells（HSC-T6）as research subjects,and found that knockout of CD73 could attenuate ALF and downregulate liver autophagy levels in mice,and the autophagy inhibitor 3-MA could reduce the activation of HSC-T6 cells by acetaldehyde.In addition,the results showed that CD73 regulates autophagy of HSC-T6 cells,which in turn promotes their activation.This study illustrated the role and mechanism of CD73 in ALF and the activation of HSCs,which provides a new direction for the treatment of alcohol-related liver fibrosis.Objective:The purpose of this study is to explore the role of CD73 in Et OH+CCl₄induced alcohol-related liver fibrosis and acetaldehyde-stimulated autophagy of HSC-T6cells and its possible molecular mechanism.Methods:1.Changes of CD73 and autophagy level in mice alcohol-related liver fibrosis and acetaldehyde-activated HSC-T6 cells.In vivo,C57BL/6 mice that are 8-10 weeks old randomly divided into Control group and Et OH+CCl₄ group.Mice in Et OH+CCl₄ group were fed with Lieber-Decarli ethanol liquid diet for 8 weeks,gavaged with alcohol（twice a week）,and intraperitoneally injected with CCl₄ in last two weeks to establish ALF model.On the last day of modeling,mice in Et OH+CCl₄ group were given alcohol by gavage.9 h later,weighed the weight of the mice,took blood from the eyes,collected and weighed liver tissues.The lesions of liver were evaluated via serum ALT,AST and hematoxylin eosin（HE）staining.The condition of collagen deposition in liver can be examined by Masson Trichrome staining.Western blot and qRT-PCR were used to detect the expression of CD73,and myofibroblast markersα-SMA,TGF-β1 and Collagen1.In vitro,the effect of acetaldehyde at different concentrations on the viability of HSC-T6 was detected by CCK-8 kits,and the appropriate concentration and time of acetaldehyde to activate HSC-T6 cells were determined by Western blot and qRT-PCR.In addition,using Western blot,qRT-PCR and IF to detect the changes of CD73,α-SMA,TGF-β1 and Collgen1 as also as the changes of autophagy-related indicators LC3,Beclin1,and ATG5 in HSC-T6 cells.2.Autophagy inhibitor 3-MA can reduce the activation of HSCHSC-T6 cells were pretreated with autophagy inhibitor 3-MA for 6 h,then using acetaldehyde stimulate cells for 48 h.Western blot,qRT-PCR and IF were used to detect the changes of LC3,Beclin1,ATG5 and fibrosis related indicatorsα-SMA and Collagen1in cells,and the fluorescence intensity of LC3 and ATG5 in cells was detected with IF.3.CD73 aggravates alcohol-related liver fibrosis by promoting HSCs autophagyA model of alcohol-related liver fibrosis was established in wild-type（WT）and CD73 knockout(CD73^-/-)C57BL/6 mice（8-10 weeks old,male）.All mice were randomly divided into four groups,as follows:wild-type control group[WT（control）],wild-type Et OH+CCl₄ group[WT（Et OH+CCl₄）],CD73 knockout control group[CD73^-/-（control）],CD73 knockout Et OH+CCl₄ group[CD73^-/-（Et OH+CCl₄）].The liver damage was evaluated via serum ALT and AST in mice,hematoxylin eosin（HE）staining after CD73knockout.The condition of collagen deposition in liver can be examined by Sirius Red staining after CD73 knockout.Western blot and qRT-PCR tested the expression ofα-SMA,Collagen1,IHC determined autophagy-related indicators.In vitro,silence or overexpression of CD73 in HSC-T6 cells,Western blot,qRT-PCR and IF detected the expression ofα-SMA and Collagen1,autophagy level,and the protein levels of P-AMPK,P-AKT and P-mTOR in cells.Result:1.In Et OH+CCl₄ induced mice,the liver index increased,contents of serum biochemical indicators ALT and AST significantly increased,inflammatory cells infiltrated in the liver and collagen deposition all increased contrasted to control group.The expression of CD73,α-SMA and Collagen1 all increased in acetaldehyde activated HSC-T6 cells,and autophagy-related indicators also increased in acetaldehyde-induced HSC-T6 cells.2.Compared with the Acetaldehyde group,3-MA（2 m M）reduced the expression of Beclin1 and ATG5 protein,decreased the fluorescence intensity of LC3 and ATG5 in cells,and could significantly reduce the expression ofα-SMA,TGF-β1 and Collagen1.3.Compared with[WT（Et OH+CCl₄）],the expression of SMA and Collagen1 in mice liver tissues of[CD73^-/-（Et OH+CCl₄）]group significantly decreased,and knockout of CD73 alleviated infiltration of inflammatory cells and liver injury,reduced liver collagen deposition and improved liver fibrosis,in addition,CD73 knockout also decreased the levels of autophagy in liver tissues.In vitro,silencing CD73 could significantly reduce the expression ofα-SMA,TGF-β1,Collagen1 protein and m RNA,decreased the ratio of LC3Ⅱ/LC3Ⅰ,weaken LC3 and ATG5 fluorescence intensity in cells,and also reduced ATG5 and Beclin1 protein,but increased the level of p62 protein.In addition,silencing CD73 down-regulated P-AMPK,but up-regulated P-AKT and P-mTOR protein.Overexpression of CD73 resulted in the opposite result of silencing CD73.Conclusion:（1）CD73 knockout can downregulate HSCs autophagy,reduce alcohol-related liver fibrosis,（2）CD73 may regulate the autophagy of hepatic stellate cells through AMPK/AKT/mTOR pathway,thus affecting the activation of HSCs.