High Expression Of CYP2E1 Can Influence The Inflammation Of Hepatic Macrophages By Alcohol Synergistic With Free Fatty Acid

Objective:To explore the influence of CYP2E1 to the inflammation of hepatic macrophages by alcohol synergistic with free fatty acid.Methods:1.Created the CYP2E1 overexpression cell through lentvirus transducing the RAW264.7 cell and got Kupffer cells from the CYP2E1knock-down mouse liver by perfusing in vivo and gradient centrifugation.Real-Time PCR and Western-blot were used to test the expression of CYP2E1 mRNA and protein in them.2.Stimulated the three cells to create the alcohol and free fatty acid(FFA)synergistic inflammation model with alcohol and FFA.Divided three cells into four groups:(1)control group,(2)alcohol group,(3)FFA group,(4)alcohol + FFA group.When stimulating the cells,the alcohol and FFA were used with 50 mM and 0.5mM.3.After stimulated of alcohol and FFA,the steatosis of three cells was examined by oil red O staining.4.The expression of mRNA of CYP2E1,TNF-α,iNOS and Arg1 were examined by Real-Time PCR.Results: The CYP2E1 overexpression cell could be created through lentvirus transducing the RAW264.7 cell.Kupffer cells,however,were not isolated successfully.The expression of CYP2E1 mRNA and protein in overexpression cells were significantly higher than the RAW264.7 cell(p<0.01).Stimulated by alcohol and FFA,two cells suggested that :(1)Alcohol and FFA alone or together both could make two cells steatosis in difference degree and the OD values of them were significantly higher than the controlgroup(p<0.05);(2)CYP2E1 could make the macrophage more sensitive to FFA and the transcriptional level of CYP2E1 was up-regulated under the stimulation of FFA(p<0.01).What’s more,alcohol and FFA had synergistic effect(p<0.01).(3)The mRNA expression of TNF-α in all of the experiment groups of the overexpression cells were significantly higher than the control groups of RAW264.7 cell(p<0.05)and the mRNA expression of TNF-αin all of the experiment groups were significantly higher than the control groups of two cells(p < 0.01).(4)Alcohol and FFA could increase the expression of iNOS significantly(p<0.05,p<0.01)and have the synergistic effect.The mRNA expression of Arg1,however,was down-regulated significantly(p<0.01).The value of iNOS/Arg1 was significantly increased when stimulated by alcohol and FFA(p<0.01).Conclusion: Alcohol and FFA can make the expression and activity of M1/M2 macrophages polarization out of balance with the influence of CYP2E1.The oxidative stress and nitrosative stress are more severe.M1 macrophage polarization increases and pro-inflammation is more strong.