| Objective: To investigate the mechanism of cytochrome P4502E1(CYP2E1)gene regulating alcohol-induced inflammatory cytokines expression in rat adrenal pheochromocytoma(PC12)cells and insulin resistance in the central nervous system.Methods: 1? The model of CYP2E1 gene knockout neural cell was constructed by using lentivirus technique in rat adrenal pheochromocytoma cells(PC12)(CYP2E1KD),and using scramble PC12 cells(CYP2E1WT)as control.2? CYP2E1 WT group,CYP2E1 mRNA expression was detected by real-time quantitative PCR during time-course and dose-response alcohol-induced experiments;After the alcohol dose was decided,CYP2E1 protein expression was confirmed by immunofluorescence test.3 ? The expression of protein kinase B(AKT)which is an key protein in insulin signaling pathway were detected by Western blot(WB)in the PC12 cells,after alcohol stimulation,both two groups PC12 cells were divided into four groups respectively:(1)Blank control group(C group);(2)blank control + insulin group(100 nM,C + Al);(4)alcohol + insulin group(the same dose with the former,A + I).The effect of alcohol on insulin signaling pathway were detected by WB.4?Establish a model of alcohol-induced inflammation,both two groups PC12 cells were distributed into two groups:(1)blank control group(C)and(2)alcohol group(Al),After alcohol stimulation for 24 hours,the changes of IKK? / NF-?B were detected by western blot and the mRNA expressions of CYP2E1,TNF-?,IL-1??IL-6 and iNOS were detected by q-PCR;Final ROS was detected by ROS assay kit.Result: 1.CYP2E1 knockdown PC12 neural cell line was successfully constructed.Both the mRNA expression of CYP2E1 were significantly reduced by 18-fold(P<0.05)in CYP2E1 group compared with the control group,also protein expression of CYP2E1 were decreased significantly.2?The model of alcohol-induced CYP2E1 was successfully established.CYP2E1 mRNA were induced under the concentration of 100 mM for 24 hours,during time-course and dose-response alcohol-induce experiment(P<0.05),increased of protein expression also confirmed by immunofluorescence test;3?Lost of CYP2E1 can alleviate the role of alcohol-inhibited insulin signaling pathway(PI3K / AKT): alcohol can inhibited phosphorylation of AKT(p-AKT)after insulin stimulation;p-AKT473 were highly expressed in both PC12 cells(compare to unstimulated groups,P<0.05),and there was no significant difference between CYP2E1 KD and CYP2E1 WT cells.After alcohol treatment,AKT phosphorylate was significantly inhibited(compare to unstimulated groups,P<0.05),there are no effect of alcohol on the insulin signaling pathway in CYP2E1 KD cells.Similarly,P-AMPK(phosphorylated adenylate activated protein kinase)expression was significantly lower in CYP2E1 WT cells than in CYP2E1 KD cells(compare to unstimulated groups,P<0.05).4?The alcohol not only induced CYP2E1 mRNA expression,but also activated NF-?B signaling pathway,and increased level of inflammatory cytokines(TNF-?,IL-6,IL-1? and iNOS mRNA)in CYP2E1 WT cells.However the effect of alcohol-induced inflammatory response was inhibited in CYP2E1 KD cells.5?Alcohol results in the increase of oxidative stress with an increased production of reactive oxygen species(ROS)in CYP2E1 WT cells compare to CYP2E1 KD cells.Conclusion: Alcohol can increase the expression of CYP2E1 mRNA and protein in central nervous system(PC12)cells and caused insulin resistance through inhibits the phosphorylation of AKT and AMPK which are key proteins in the insulin signal pathways.Our data demonstrated that alcohol induced oxidative stress and the activated of IKK? / NF-?B signaling pathway,resulting in the increase of phosphorylated NF-?B and release of inflammatory cytokines.However,the lost of CYP2E1 can inhibite the effect of alcohol on generation of ROS and alleviate insulin resistance.Hence,there is a need for further study on mechanism. |
PDF Full Text Request