CD73/NT5E-mediated Ubiquitination Of AURKA Regulates Alcohol-Related Liver Fibrosis Via Modulating Hepatic Stellate Cell Senescence

Backgrounds:Alcohol-related liver disease(ALD)is the most common chronic liver disease in the world,but there is still no effective treatment to prevent the progress of alcohol-related liver fibrosis(ALF).Purine signaling pathway(including extracellular nucleotides,extracellular nucleotidases and specific receptors acting on nucleotides and their derivatives)has been proved to play an important role in a variety of diseases in recent years,including the regulatory process of liver diseases,such as liver inflammation and chronic liver diseases characterized by steatosis,fibrosis,and malignant tumors.Among them,CD39(ectonucleoside triphosphate diphosphohydrolase-1,ENTPD1)and CD73(ecto-5’-nucleotidase,NT5E),as key regulatory molecules of purine signaling pathway,are very important for regulating the duration,content,and composition of"purine energy halo" around immune cells,and their expression and activity will change dynamically with the different pathophysiological environment.Therefore,it is of great significance to explore their dynamic changes in different stages of alcohol-related liver disease.The previous experiment of the research group has proved that inhibition of CD39/CD73 can reduce alcohol-related liver fibrosis,but which one plays a more important role in it has aroused our interest.Therefore,we compared the role of CD39 and CD73 in HSC proliferation,activation,and apoptosis,and further explored the specific mechanism of CD73’s role in alcohol-related liver fibrosis.Objective:(1)To observe the dynamic changes of CD39-CD73-adenosine signaling pathway in the progression of alcohol-related liver disease and compare the roles of CD39 and CD73 in the proliferation,activation,and apoptosis of HSCs;(2)To explore the specific mechanism of CD73 in alcohol-related liver fibrosis.Methods:C57BL/6 male mice and CD73 knockout mice(8-12 weeks old)were randomly divided into four groups:(ⅰ)WT;(ⅱ)WT,EtOH-fed+CCl4;(ⅲ)KO;(ⅳ)KO,EtOH-fed.WT,EtOH-fed+CCl4 group and KO,EtOH-fed+CCl4 group were continuously fed a liquid diet containing 5%ethanol for 8 weeks and were gavaged with high concentration alcohol twice a week(5 g/kg).In the last two weeks,CCl4 was injected intraperitoneally twice a week(10%in olive oil,1 ml/kg).The liver tissue and blood of these mice were collected for further analysis after the first,second,fourth,sixth and eighth weeks of alcohol infusion.And the primary hepatic stellate cells were isolated by in situ liver perfusion separation.The contents of ALT and AST in serum were detected by serum biochemical analyzer in vivo.HE staining was used to reflect the degree of liver injury.Masson staining and Sirius red staining were used to reflect the collagen deposition.Immunohistochemical staining,Western blot and RT-qPCR were used to detect the expression of COLlal and a-SMA comprehensively reflects the degree of fibrosis in mice.Immunofluorescence double staining of CD39/CD73 and α-SMA/CK19/F4/80 was used to localize CD39 and CD73.In vitro,200 μM acetaldehyde stimulates HSC-T6 cells and 600 μM acetaldehyde stimulates LX-2 cells to establish the model.CD39 and CD73 were silenced and overexpressed by CD39-siRNA/CD73-siRNA and pEX3-CD39/pEX3CD73,respectively,and the proliferation and apoptosis of HSC-T6 cells were detected by Western blot and flow cytometry.Proteomic sequencing and STRING database were used to screen signal pathways related to CD73 changes and proteins that might interact with CD73.p53-siRNA and pEX3-p53 were used to silence and overexpress p53.MLN8237 inhibits the expression of aurora kinase A(AURKA).β-galactosidase staining and the protein expression of p53,p21 and p16 was used to reflect the degree of cell senescence.Molecular docking technology and immunoprecipitation were used to detect the interaction between CD73 and AURKA.Chloroquine(CQ,50 μM)、N-[N-(N-AcetylL-leucyl)L-leucyl]-L-norleucine(ALLN,5 μM)and MG-132(1 μM)were used to inhibit autophagy-lysosome pathway,calpain system and ubiquitin-proteasome system respectively to explore the specific mechanism of CD73 increasing the stability of AURKA.Results:In vivo experimental results showed that compared with the Pair-fed group,the EtOH-fed+CCl4 group had excessive inflammatory cell infiltration,liver cell necrosis and collagen deposition,and the expression of CD39,CD73 and adenosine were increased in the EtOH-fed+CCl4 group.In addition,we observed that the expression of CD39 and CD73 in the EtOH-fed+CCl4 group increased significantly from the second week.In vitro,the expression of CD39 and CD73 was also increased in HSC-T6 cells stimulated by acetaldehyde.Compared with silencing CD39 alone and silencing CD39 and CD73 together,silencing CD73 alone can better reduce the expression of fibrosis factor(COL1al and a-SMA),and the protein levels of c-Myc and CyclinD1.At the same time,the proportion of HSC-T6 cells apoptosis was increased.We detected the expression of CD73/NT5E in normal and fibrotic human liver,and the results were consistent with in vivo and in vitro experiments.At the same time,the deletion of CD73/NT5E has a protective effect on the mouse model of alcoholic liver fibrosis.In addition,KEGG pathway analysis showed that the overexpression of CD73/NT5E was related to p53 signal pathway,which could regulate cell senescence.The STRING database was used to predict the proteins interacting with p53.The overlap between proteomic analysis and STRING database is aurora kinase A(AURKA),a cell cycle regulating kinase.The direct interaction between CD73/NT5E and AURKA was confirmed by immunoprecipitation analysis and molecular docking.We found that the overexpression of CD73/NT5E inhibited the ubiquitination of AURKA,while the p53 signal was downregulated.CD73/NT5E regulates alcohol-related liver fibrosis and activation and senescence of hepatic stellate cells by combining with AURKA.These findings suggest that CD73/NT5E is a potential therapeutic target for alcohol-related liver fibrosis.Conclusion:In this study,we demonstrated that CD73/NT5E has a more significant regulatory effect on the activation,proliferation,and apoptosis of hepatic stellate cells(HSC)than CD39/ENTPD1.Moreover,CD73/NT5E can inhibit its ubiquitination and enhance its stability by directly combining with AURKA,thus regulating alcohol-related liver fibrosis and activation and senescence of hepatic stellate cells.