The Protection Role And Mechanism Research Of CD38 Gene Deficiency On Alcohol Induced Oxidative Stress Injury On Mouse Primaryhepatocytes

Objective:Alcoholic liver diseases(ALD)was defined as a metabolism disease which induced by sustained and large volume alcohol consumpion.Its main Pathological changes include fat accumulation,fibrosis and liver cirrhosis.According to statistics,at least 60% of liver disease-related deaths are caused by excessive drinking.At Present,oxidative stress is considered to be the main cause of the occurrence and development of alcoholic liver disease,and anti-oxidant stress is considered to be the most direct and effective treatment strategy for alcoholic liver disease.Our Previous studies showed that mouse embryonic fibroblasts from CD38 knockout mice are resistant to hydrogen Peroxide and hypoxia-reoxygenation induced oxidative stress injury.Moreover,CD38 interference can improve oxidative stress injury induced by cardiac lipid overload.However,whether CD38 can affect the role of alcohol in liver injury through oxidative stress has not been reported.This work was aimed to study the role and mechanism of CD38 gene in alcohol-induced oxidative stress hepatocyte injury,thereby Provied new targets and research ideas for the Prevention and treatment of alcoholic liver disease.Methods:1.Extraction of Primary hepatocytes and Preparation of alcohol-induced hepatocyte injury model: 1)Isolation and culture of Primary hepatocytes from C57BL/6 mice aged 6-8 weeks by in vivo Perfusion;2)The Primary hepatocytes cultured in vitro were treated with gradient concentration alcohol for 10 hours.The reactive oxygen species(ROS)was measured by fluorescence microscope after labeled with DCFH-DA fluorescent probe.3)The primary hepatocytes cultured in vitro were treated with 1% alcohol and ROS was detected at different time points.4)Q-PCR and Western Blot were used to detect the expression of CD38 after alcohol treatment.2.Effect of CD38 knockout on alcohol-induced oxidative stress injury in Primary hepatocytes: The Primary hepatocytes of wild type and CD38 knockout mice were isolated,and treated with 1% alcohol for 10 hous to induce oxidative stress injury.1)Q-PCR and Western Blot was used to detect knockout efficiency of CD38;2)Alcohol induced hepatocyte ROS detect by flowcytometre;3)MDA was measured by microplate reader of multiwavelength;4)Mitochondrial membrane potential was detected by flourence microscope.3.Mechanism study: 1)Western blot analysis was performed to detect the protein expression of oxidative stress relative SOD2 ? NOX2 and its upstream transcripional activator FOXO3;2)Total SOD activity was measured by WST analysis;3)Q-PCR was used to analyze ALDH2 mRNA expression in CD38knockout?wild type and CD 38 over expression hepatocyte.Results:1.After 10 hours of alcohol concentration gradient treatment,the ROS level of primary hepatocytes was increased in a concentration-dependent manner,and the expression of CD38 mRNA was decreased in a concentration-dependent manner.When Primary hepatocytes were treated with 1% alcohol,ROS accumulated in a time-dependent manner and reached its maximum in about 10 hours.2.CD38 gene knockout could significantly reduce the contents of ROS and MDA,and improve the decline of mitochondrial membrane potential after 1% alcohol treated for 10 hours.3.CD38 knockout inhibited the expression but promoted the protein activity of SOD2 activity,and did not affect the expression of FOXO3.After treated with alcohol,the expression of NOX2 was decreased in CD38 deficiency cells.4.ALDH2 mRNA expression was increased in CD38 knockout hepatocytes,but decreased in CD38 over expression cells.Conclution:CD38 gene deficiency protect hepatocytes from alcohol induced injury,the mechanism may be to relative with promoting SOD activity and ALDH2 expression in hepatocytes to alleviate alcohol-induced oxidative stress.