Increased Kupffer Cell CYP2E1 Expression Regulate Synergistic Inflammation By Alcohol And Fatty Acid Via NF-κB Pathway

Objective: To investigate the role of cyp2e1 in the pathogenisis of the pro-inflammatory relative signal transduction in synergistic steatohepatitis induced by alcohol and free fatty acid.Methods: 1、Make the cyp2e1 over-expression in Raw 264.7 through lentivirus transduction(named RE1 cell).2、stimulate the inflammation factor by alcohol and free fatty acid to establish the inflammation model in vitro with 4 group:(1)control.(2)alcohol(50mM).(3)free fatty acid(o.5mM,oleic acid:palmitic acid=2:1).(4)alcohol+free fatty acid. 3 、 after alcohol and FFA,The lipid droplet in the Raw 264.7 and the RE1 cell were detected by oid red o staining.4、The expressions of CYP2E1 and TNF-α were examined with RT-qPCR.5、The expressions of NF-κB、MAPK、STAT3 were examined with western blot.6、culture the BRL-3A cell with the supernatant in the inflammation model for 4 hours,then detected the Alanine aminotransferase and glutamic-oxalacetic transaminase in the supernatant.The lipid droplet in the BRL cell were detected by oid red o staining.Result:(1) Make the cyp2e1 over-expression in Raw 264.7 through lentivirus transduction,the expression of CYP2E1 mRNA in RE1 group was 46 times higher than the Raw 264.7 cell(P<0.05) by Real-time PCR,20 times higher at the level of protein(P<0.05) by western blot.(2)Lots of lipid droplet in the group of alcohol and free fatty acid after CYP2E1 over-expressed dected by oil red o staining;(3)TNF-α was significantly higher in the alcohol and/or free fatty acidgroup(P<0.05), especially higher than the control group(P<0.05).(4)the expressions of phosph-STAT3 、 phospho MAPK 、phospho NF-κB in CYP2E1 over-expressed group were significantly increased than the control group(P<0.05).(5)Increased Kupffer cell CYP2E1 expression by alcohol and fatty acid upregulate the production of the inflammatory factor, then injury the hepatocyte,make the Alanine aminotransferase increased when they are co-cultured(P<0.05).Conclusion: Increased Kupffer cell CYP2E1 expression may regulate synergistic inflammation by alcohol and fatty acid via NF-κB pathway.and then promote the inflammation in cells through upregulating the expression of inflammatory factor,such as TNF-α.