| Objective: To explore the molecular role of CYP2E1 in the regulation of macrophages in synergistic steatohepatitis by alcohol and free fatty acid through metabolic reprogramming.Methods:1.M1-polarized cell model,M2-polarized cell model and synergistic steatohepatitis by alcohol and free fatty acid cell model were established with CYP2E1 overexpressed cells and CYP2E1 no-load cells.The CYP2E1 overexpressed cells(OE)were divided into 6 groups given the stimulation of alcohol,oleic acid,alcohol with oleic acid,LPS+IFN-?,IL-4 and blank control,the same as CYP2E1 no-load cells(NC).2.3h after the establishment of the cell model,the expressing level of CYP2E1 gene,typical gene of polarization and inflammatory cytokines,was detected by RT-qPCR.The level of reactive oxygen species(ROS)was measured by laser confocal microscope and flow cytometry.The level of NO was evaluated through NO kit.3.24 h after the establishment of the cell model,the expressing level of key nodal enzyme genes in glucose metabolic pathway was detected by RT-qPCR.The content of glucose metabolites were measured by gas chromatography-mass spectrometer(GC-MS).Result:1.M1-polarized cell model,M2-polarized cell model and synergistic steatohepatitis by alcohol and free fatty acid cell model were all successfully established.And the expressing level of typical inflammatory cytokines are significantly higher than that in the blank control group(p<0.05).And the level of ROS are significantly increased(p<0.05),too.2.In the glucose metabolism pathway,alcohol promoted an up-regulated expression of pgd in OE cells(p<0.05),and Irg1?ASS1 in NC cells(p<0.05).Oleic acid promoted the up-regulated expression of IDH1?IDH2?GPX1?Irg1?Gsr?Got1/2?ASS1 in OE cells(p<0.05),and Irg1?Gsr?ASS1 in NC cells(p<0.05),while the down-regulation of IDH1?NOX2 in NC cells(p<0.05).Alcohol with oleic acid promoted the down-regulation of IDH1?IDH2(p<0.05)while the up-regulated expression of Irg1?Gsr?Got1/2?ASS1 in OE cells.Besides,Alcohol with oleic acid promoted the up-regulated expression of Irg1?Gsr?ASS1 in NC cells(p<0.05).3.The data of GC-MS detection in glycometabolism showed that oleic acid and alcohol with oleic acid promoted the down-regulation of 3-phosphoglyceric acid(p<0.05)in NC cells,while such effect was not observed in OE cells.Oleic acid significantly down-regulated pyruvate levels in NC cells(p<0.01),but not obviously in OE cells.The levels of succinic acid in both OE and NC cells were down-regulated by alcohol,oleic acid and alcohol with oleic acid(p<0.05).Alcohol increased the levels of fumaric acid and malic acid in NC cells(p<0.05),but not in OE cells.Compared with NC and OE cells,CYP2E1 gene promoted down-regulation of 3-phosphoglyceric acid(p<0.05)and up-regulation of pyruvate,citric acid,alpha-ketoglutaric acid,fumaric acid,malic acid and lactic acid(p<0.05).Conclusion:1.Alcohol and free fatty acids have independent interactive promoting effects with CYP2E1 gene,and they can synergistically increase the expression of CYP2E1.2.Both alcohol and free fatty acids can independently stimulate the secretion of pro-inflammatory cytokines TNF-?,IL-1?,IL-6,HIF-1? by macrophages.By the influence of CYP2E1 gene,the ability of macrophages to secrete these pro-inflammatory cytokines was further enhanced.3.CYP2E1 gene,involved in the production of ROS,plays an important role in inducing oxidative stress and lipid peroxidation,and may participate in nitriding stress at the same time.4.Under the intervention of free fatty acids,CYP2E1 gene may change the metabolic pathway by regulating macrophages' polarization to m1-type,resulting in metabolic reprogramming.5.Macrophages may rapidly promote the polarization of macrophages toward m1-type by enhancing the metabolism of anaerobic glycolysis,pentose phosphate pathway and aspartate-arginine bypass,under the positive regulation of CYP2E1 gene and the synergistic effect of alcohol and free fatty acids. |
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