Regulation Of CD39 Expression In ATP-P2Y2-Mediated Alcoholic Liver Steatosis And Inflammation

Backgrounds:Alcoholic liver disease（ALD）is an abnormal liver cell structure and dysfunction caused by long-term excessive drinking.Inflammation plays an important role in the progression of alcoholic liver disease.As an important signal transducer,ATP plays an important role not only in cellular energy metabolism and also serves as a“danger signal”in the pathophysiology of chronic liver diseases including ALD.In the extracellular compartment,ATP acts as a signaling molecule primarily by activating the purinergic P2 receptor.Irachetavellve et al.found that in addition to the release of cytokines/inflammatory factors in ALD,a large amount of ATP is generated.P2Y2R,a subtype of P2Y receptor family,has a unique structure and function,which is activated by the ligand UTP,triggering Ca²⁺influx,which mediates a series of cell signal transduction pathways.It has been reported that P2Y2R expression in bone marrow-derive cells is essential for neutrophil liver infiltration and subsequent injury.Extracellular ATP is primarily hydrolyzed to ADP and AMP by the action of the CD39,and AMP is primarily converted to adenosine by CD73.Recent studies have shown that CD39 alleviates liver damage caused by sepsis.Interestingly,Beldi G et al.have reported that the failure of extracellular nucleotides in VEGF receptor-2/KDR transactivation of CD39-deficient endothelial cells is associated with P2Y2 receptor desensitization.Therefore,we investigated whether ATP-P2Y2R and CD39 can be used as therapeutic targets as well as the mutual regulation of CD39 and P2Y2 in alcoholic steatohepatitis.Objective:（1）To explore the role of ATP-P2Y2R and CD39 in alcoholic steatohepatitis;（2）To explore the mutual regulation between ATP-P2Y2R and CD39.Methods:Male C57BL/6 mice（18-22 g）in vivo for 6-8 weeks were used to establish an alcoholic steatohepatitis model by continuous alcohol feeding followed by acute alcohol gavage.The mice were randomly divided into pair-fed group（n=8）,Et OH-fed group（n=16）,and mice treated with suramin at doses of 5,10,and 20 mg/kg（n=16）.First fed with Lieber-De Carli alcoholic liquid feed containing 5%ethanol for 10 days.Meanwhile,from day 4 to day 10,mice were injected intraperitoneally with Suramin once daily,whereas the pair-fed and the Et OH-fed group were injected with an identical volume of physiological saline.On day 11,mice were gavaged with single-binge alcohol（5 g/kg body weight）or isocaloric maltose-dextrin（9 g/kg body weight）.After 9h,the mice were sacrificed under anesthesia.RAW264.7 cells were stimulated with100m M ethanol in vitro for 24 hours to establish an alcoholic hepatitis model.Cells were maintained in DMEM supplemented with 10%（v/v）heat-inactivated fatal bovine serum（FBS）and incubated at 37°C with 5%CO2.RAW264.7 cells were pre-treated with 100m M Et OH for 12 h before treatment with ATP,UTP,or suramin for 24 h.RAW264.7 cells were cultured in the indicated wells of the plate for 12 h and transfected with Si RNA using Lipofectamine TM2000 according to the manufacturer’s protocol.After 6 h,Opti-MEM was replaced by DMEM（10%FBS）,and 100m M Et OH was added for 36h.In vivo,serum ALT,AST,TC and TG were detected by colorimetric assay.Liver damage was observed by HE staining and lipid accumulation was observed by oil red O staining.In vitro,cell viability was detected by MTT.Western blot,RT-q PCR and ELISA were used to detect the expression of CD39,P2Y2R,IL-6,IL-1βand TNF-αin liver as well as ATP concentration measured by an ATP Colorimetric/Fluorometric Assay Kit both in vivo and vitro.Results:In vivo,compared with Pair-fed group,alcohol feeding significantly increased serum ALT,AST,T-CHO and TG levels,leading to liver fat accumulation and disorganized liver cord.The expression of ATP,CD39,P2Y2R,and the pro-inflammatory factors including IL-6,IL-1βand TNF-αwere all up-regulated.In comparision with the Et OH-fed group,the P2Y2R inhibitor group significantly improved the liver injury in a dose-dependent manner,and the effect was most obvious in the high-dose group.Interestingly,the expression of CD39 also decreased with the decrease in P2Y2R.In vitro,compared with the control group,alcohol group increased ATP,CD39,P2Y2R levels,promoting secretion of IL-6,IL-1βand TNF-α.Compared with the ethanol group,the addition of exogenous ATP increased the expression of P2Y2R,IL-6,IL-1βand TNF-α.The suramin group and P2Y2R-Si RNA group down-regulated the expression of CD39 while down-regulating pro-inflammatory factors（IL-6,IL-1βand TNF-α）.And the UTP group were the opposite,which was consistent with the results of previous in vivo experiments.In the CD39-Si RNA group,the expressions of ATP,P2Y2R,IL-6,IL-1βand TNF-αall increased with the decrease of CD39.Conclusion:（1）Extracellular ATP acting via P2Y2R contributes to Et OH-primed inflammation.（2）P2Y2R blockade alleviates alcoholic liver steatosis and inflammation.（3）CD39 has a protective effect on Et OH-primed inflammation.（4）CD39 and ATP-P2Y2 regulate each other in alcoholic liver steatosis and inflammation.