| Background:Alcoholic liver disease(ALD)is a liver disease caused by long-term heavy drinking.Initially it usually presents as fatty liver,which can then develop into alcoholic hepatitis,alcoholic liver fibrosis and cirrhosis.Its molecular and pathological mechanisms are unknown,and there is no definite drug for ALD in clinic at present.In recent years,purinergic signaling has been found to be involved in the pathophysiological regulation of many diseases,including liver diseases.Under external stimulation,such as ethanol,cells will release intracellular high concentration of nucleotides(such as ATP)out of them,which serves as a "danger signal" to prompt the body to start immune defense.When cellular stress or damage occurs,ATP is released extracellular and immediately hydrolyzed to AMP(Adenosine Monophosphate)by the extracellular enzyme CD39(ecto-nucleoside triphosphate diphosphohydrolase,NTPDase1),while extracellular CD73(Ecto-5’-Nucleotidase,NT5E)hydrolyzes AMP to adenosine.Adenosine deaminase(ADA)hydrolyzes and inactivates adenosine,so the extracellular enzyme CD39-CD73-ADA cascade co-regulates adenosine concentration.CD39 and CD73 are also expressed in Hepatic stellate cells(HSCs),but their role in ALD inflammation and fibrosis has not been systematically reported so far.Our previous study found that the Adenosine Receptors(ARs)of HSCs activated by acetaldehyde(Ace)can promote activation and proliferation of HSCs through c AMP-PKA-CREB signaling pathway.In addition,ARs antagonists and Caffeine can reduce activation and proliferation of HSCs in chronic alcoholic hepatic fibrosis,further confirming that Adenosine signaling pathway regulates ALD fibrosis.Research purpose: In this study,mouse models and cellular models of acute alcoholic liver injury and chronic alcoholic hepatic fibrosis were established respectively according to previous literatures published by our research team.To investigate the role of ATP-CD39-CD73-Adenosine signaling pathway in regulating HSCs’ inflammatory factors and fibrosis factors secretion in the processes of acute alcoholic liver injury and chronic alcoholic liver fibrosis.Methods:(1)Mouse models of acute alcoholic liver injury and chronic alcoholic liver fibrosis were established respectively by referring to the methods in previous articles of our research team.HSCs were extracted and purified from mouse liver.Cellular models of acute alcoholic liver injury and chronic alcoholic liver fibrosis were established by referring to the methods in the previous articles of our research team.(2)Mean body weight and liver index were calculated.Biochemical and fibrosis indexes of animal serum were detected by automatic biochemical analyzer.Histopathology was performed by Hematoxylin-Eosin(HE)staining,Masson’s trichrome staining,Van Gieson(VG)staining and Sirius red staining.Immunohistochemical method was used to analyze protein expression ofα-SMA in liver tissue.Protein expressions of CD39 and CD73 in liver tissue were detected by double immunofluorescent staining.HE staining was performed on the liver tissues of the high,medium and low dose groups of POM-1(Sodium Polyoxotungstate).The purpose of these experiments was to elucidate that CD39 and CD73 may play important roles in animal models of acute alcoholic liver injury and chronic alcoholic liver fibrosis.(3)Gene expressions of CD39,CD73,inflammatory cytokines and fibrogenic cytokines were detected by RTq PCR.Protein expressions of CD39,CD73,inflammatory cytokines and fibrogenic cytokines were detected by Western blotting.ATP concentration was determined by bioluminescence and adenosine content was determined by HPLC.The purpose of these experiments was to elucidate that CD39 and CD73 may play important roles in cellular models of acute alcoholic liver injury and chronic alcoholic liver fibrosis.(4)To elucidate the role of ATP-CD39-CD73 adenosine signaling pathway in cellular models of acute alcoholic liver injury and chronic alcoholic liver fibrosis,cellular models of acute alcoholic liver injury and chronic alcoholic liver fibrosis were established respectively.The effects of agonists and antagonists of CD39 and CD73 on protein expressions of inflammatory cytokines and fibrogenic cytokines were detected,and the content changes of ATP and adenosine in HSCs culture supernatant were detected at the same time.(5)CD39 and CD73 genes were silenced by CD39 si RNA and CD73 small interfering RNA/silencing RNA(si RNA),respectively,protein expressions of CD39,CD73,inflammatory cytokines and fibrogenic cytokines were detected by Western blotting.The purpose of these experiments was to elucidate the role of CD39 and CD73 silencing in cellular models of acute alcoholic liver injury and chronic alcoholic liver fibrosis.The data was analyzed by Statistical Product and Service Solutions(SPSS)17.0 software and images were drawn by Graphpad Prism 8.0 software.Results:(1)We found that results of average body weights,liver indexes,serum biochemical parameters,serum fibrosis indexes,liver histopathology,immunohistochemistry and double immunofluorescent staining had signifcant changes in two model groups compared with two control groups.Results of HE staining showed that high dose group of POM-1 could alleviate acute alcoholic liver injury and chronic alcoholic liver fibrosis.Results of double immunofluorescent staining indicated that protein expressions of CD39 and CD73 in mouse liver tissue was increased in model groups of acute alcoholic liver injury and chronic alcoholic liver fibrosis.These results suggested that animal models of acute alcoholic liver injury and chronic alcoholic liver fibrosis were successfully established.(2)In vitro,protein expressions of CD39,CD73,inflammatory cytokines and fibrogenic cytokines were significantly increased in model group,suggesting that CD39 and CD73 might play regulatory roles in acute alcoholic liver injury and chronic alcoholic liver fibrosis.(3)Furthermore,in cell experiments,CD39 antagonist ARL67156 and CD73 antagonist APCP can significantly decrease protein expressions of CD39 and CD73 respectively,and both of them can decrease protein expressions of inflammatory cytokines and fibrogenic cytokines.Meanwhile,concentrations of ATP and adenosine in cell culture supernatant also changed correspondingly.It is suggested that ATP-CD39-CD73-adenosine signaling pathway plays a regulatory role in cellular models of acute alcoholic liver injury and chronic alcoholic liver fibrosis.(4)Besides,CD39 si RNA and CD73 si RNA can decrease protein expressions of CD39 and CD73 in cellular models of acute alcoholic liver injury and chronic alcoholic liver fibrosis,respectively.And both of them can decrease protein expressions of inflammatory cytokines and fibrogenic cytokines.Meanwhile,concentrations of ATP and adenosine in cell culture supernatant also changed correspondingly.It is suggested that ATP-CD39-CD73-adenosine signaling pathway plays a regulatory role in cellular models of acute alcoholic liver injury and chronic alcoholic liver fibrosis.Conclusion:(1)ATP-CD39-CD73 signaling pathway plays an important role in acute alcoholic liver injury and chronic alcoholic liver fibrosis.(2)Inhibition or silencing of CD39 can alleviate acute alcoholic liver injury and chronic alcoholic liver fibrosis.(3)Inhibition or silencing of CD73 can alleviate acute alcoholic liver injury and chronic alcoholic liver fibrosis. |
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