CD39-mediated ATP-adenosine Signaling Promotes Alcoholic Liver Disease

Backgrounds:Long-term excessive alcohol consumption can lead to a series of liver damage,from steatosis,alcoholic hepatitis to alcoholic liver fibrosis,and finally to acute cirrhosis or liver failure,collectively known as alcoholic liver disease（ALD）.Among them,liver fibrosis has been proved to play a key role in the process of alcoholic liver disease.During the development of ALD,a large amount of ATP is released.Adenosine triphosphate（ATP）is hydrolyzed to adenosine by different enzymes including CD39 and CD73,which regulate the physiological and pathological processes of many diseases.CD39 is involved in a variety of physiological and pathological processes,such as cell proliferation and differentiation,but this change and function has not been confirmed in alcoholic liver disease.Therefore,we investigated whether CD39 can be used as a therapeutic target for alcoholic liver fibrosis and whether CD39-mediated ATP and adenosine signaling pathway can regulate the activation of HSCs.Methods:In this study,a model of alcoholic liver fibrosis induced by carbon tetrachloride（CCl₄）plus ethanol was established in C57BL/6 mice.The CD39 inhibitor sodium polyoxotungstate（POM1）and colchicine were intraperitoneally injected from the fifth to the eighth week,and then the primary hepatic stellate cells of mice were extracted by liver perfusion in situ.In vivo,serum ALT,AST,LN and HA were detected by colorimetric assay.Liver damage was observed by HE staining and Collagen deposition was observed by Masson staining.Expression ofα-SMA in liver tissue was detected by immunohistochemical staining and immunofluorescence staining.Western blot and RT-q PCR were used to detect the expression of CD39,CD73,α-SMA,TGF-β,Collagen I and Collagen III.Rat hepatic stellate cells（HSC-T6）,human HSC and primary mouse HSC were stimulated with 200μM acetaldehyde for 48 h to establish alcoholic liver fibrosis models in vitro;POM1（48 h）and exogenous ATP（24 h）were sequentially adde to HSC-T6 cells stimulate with 200μM acetaldehyde.HSC-T6 cells in the transfection group were transfected with Si-RNA for 6 h with Lipofiter3.0^TM,then Opti-MEM was replaced with fresh DMEM（containing 5%fetal bovine serum）,and HSC-T6 cells were stimulated with 200μM acetaldehyde for 48 h.ATP concentration in the supernatant is measured by an ATP Colorimetric/Fluorometric Assay Kit.The expressions of CD39,CD73,α-SMA,TGF-β,Collagen I,Collagen III,adenosine A2A receptor,adenosine A2B receptor and Smad3 were detected by Western blot and RT-q PCR.The cell cycle of HSC-T6 cells was detected by cell cycle kit;Immunofluorescence staining was used to detect the expression of CD39 andα-SMA.Results:In vivo,the serum ALT,AST,LN and HA of the model group were significantly higher than those of the control group,the liver cell cords were disordered,inflammatory cells were infiltrated in portal and vascular areas,the number of inflammatory cells was increased,accompanied by a large number of fat vacuoles and collagen deposition;The expression of CD39,CD73 and profibrotic factors（α-SMA,TGF-β,Collagen I,Collagen III）were up-regulated.Compared with the model group,CD39 inhibitor could significantly improve the liver injury and the expression of related proteins in a dose-dependent manner,and the effect of high dose was the most obvious.Western blot and RT-q PCR analysis showed that CD39 and CD73 were up-regulated in activated HSC-T6cells;The results of mouse primary hepatic stellate cells and human hepatic stellate cells were consistent with those of HSC-T6.Exogenous ATP and POM1 were sequentially added to HSC-T6 cells stimulated with 200μM acetaldehyde.The results showed that exogenous ATP could increase the expression of CD39 and CD73,and promote cell activation,while blocking or silencing CD39 could prevent acetaldehyde-induced activation of HSC-T6 cells and the expression ofα-SMA,TGF-β,Collagen I and Collagen III.Furthermore,blocking or silencing CD39 inhibits the activation of adenosine A2A receptor,adenosine A2B receptor and TGF-β/Smad3 pathway.Conclusion:（1）The expression of CD39 and CD73 in mice and HSCs alcoholic liver fibrosis model is significantly higher than that in normal group;（2）In the alcoholic liver fibrosis model of HSC-T6 cells,ATP can promote the occurrence of liver fibrosis by activating CD39;（3）Pharmacological blockade of CD39 by POM1 can alleviate alcoholic liver fibrosis in vivo;（4）Silencing CD39 may attenuate HSC-T6-induced cell activation by acetaldehyde.（5）Inhibition/silencing of CD39 attenuates adenosine receptor A2A,A2B and TGF-β/Smad3 signaling pathways.