CD73 Regulates Hepatic Stellate Cells Activation And Proliferation Through Wnt/β-catenin Signaling Pathway

Backgrounds:Alcoholic liver fibrosis（ALF）is a kind of proliferative pathological process caused by long-term excessive drinking in the transformation process of alcoholic hepatitis and alcoholic cirrhosis.It is characterized by the huge accumulation of extracellular matrix（ECM）in the liver.Hepatic stellate cells（HSCs）activation can lead to ECM synthesis and up regulation ofα-smooth muscle actin（α-SMA）expression,thus aggravating liver fibrosis.Therefore,inhibiting the activation and proliferation of HSCs and promoting the apoptosis of activated HSCs become the key to prevent and treat ALF.As the key molecule of purine signaling pathway,CD73 has been demonstrated in liver inflammation and hepatocellular carcinoma.However,the function and mechanism of CD73 in alcoholic liver fibrosis are still unclear.This study focused on the function and possible mechanism of CD73 regulating HSCs activation,proliferation and apoptosis in ALF.Objective:（1）To investigate the role of CD73 in alcoholic liver fibrosis;（2）to explore whether CD73 regulates alcoholic liver fibrosis by activating the activation and proliferation of hepatic stellate cells.Methods:Male 6-8 weeks old C57 BL/6J mice（21-24g）in vivo were used to establish alcoholic liver fibrosis model by continuous alcohol liquid feed for 8 weeks,followed by intraperitoneal injection of CCl₄for 4 weeks.The mice were randomly divided into 6groups with 12 mice in each group:control feeding group（Vehicle）,alcohol plus CCl4feeding group（Et OH+CCl₄）,three groups of APCP low,medium and high treatment groups（1,3 and 9 mg/kg）and Colchicine group（0.2 mg/kg）.Simultaneously,the control feeding group was supplied with liquid control fodder for 8 weeks.At the same time of modeling,from the fifth week,the mice in each administration group were intraperitoneally offered with the targeted medicine,while the mice in the control feeding group and Et OH+CCl₄group were injected with the equal dose of normal saline.After modeling,primary HSCs were isolated by in situ perfusion and CD73expression was detected.Sirius red staining and Masson staining were used to observe the deposition of liver fiber.Western blot,RT-q PCR and immunofluorescence double staining were used to detect the localization and expression of CD73 and adenosine test kit was used to detect the concentration of adenosine in serum.In vitro experimental operation,HSC-T6 cells were activated by acetaldehyde.The expressions of CD73,α-SMA and Col1α1 were examined by Western blot and RT-q PCR.HSC-T6 cells were transfected with p EX2-CD73 and CD73-si RNA separately.Flow cytometry and Western blot were used to examine whether CD73 could affect the activation,proliferation and apoptosis of HSC-T6 cells.Results:（1）In vivo,compared with the control group,the expression of AL T and AST in the alcohol plus CCl₄group were increased,and there were obvious fibrous tissue hyperplasia and collagen deposition in the liver;the expression of CD73 and fibrosis indexes（α-SMA and Col1α1）were up-regulated.Compared with alcohol plus CCl₄group,APCP group significantly reduced liver fibrosis in a dose-dependent manner.（2）In vitro,CD73 was up-regulated in acetaldehyde activated HSC-T6 cells.Silencing CD73 could inhibit the activation and proliferation of HSC-T6 cells and promote the apoptosis of activated HSC-T6 cells,showing significant down-regulation of fibrosis indexes,decrease of cell viability,cell cycle arrest and up-regulation of apoptosis related proteins.Overexpression of CD73 had the adverse impact.Silencing CD73 also significantly inhibited the expression of Wnt/β-catenin pathway related proteins,while overexpression of CD73 reversed.Conclusion:（1）Inhibiting the expression of CD73 can inhibit the activation and proliferation of HSCs and promote the apoptosis of activated HSCs,thus reducing alcoholic liver fibrosis.（2）CD73 might modulate the activation and propagation of hepatic stellate cells via Wnt/β-catenin pathway.