CYP4A11 Is Involved In The Development Of Non-alcoholic Fatty Liver Through ROS-induced Lipid Peroxidation And Inflammation

Nonalcoholic fatty liver disease is a disorder of fatty acid metabolism in hepatocytes,and the development of simple steatosis has a tendency toward nonalcoholic steatohepatitis and liver fibrosis.Oxidative stress is considered to be a major inducer of NAFLD development to NASH.With the study of the pathogenesis of NAFLD,mitochondria,microsomes and peroxisomes play an important role in the oxidative metabolism of lipids in hepatocytes in the pathogenesis of NAFLD.Cytochrome P450 enzyme in NAFLD metabolizes extra fatty acids and causes the production of reactive oxygen species in the liver cells.The involvement of CYP4 A family enzymes in the development of NAFLD is associated with the induction of ROS production.The literature indicates that in mice with a high-fat diet or medicience diets,the expression level of CYP4A14 in liver tissue is significantly increased with the production of ROS,indicating that CYP4 A is involved in the progression of NAFLD and may contribute to the progression of the disease.This study will investigate the role of the CYP4 A,particularly the human-specific CYP4A11,in the pathogenesis of NAFLD in vivo and in vitro.Objective Through reviewing the literature and analyzing the literature,this study will verify the effect of CYP4A11 enzyme(CYP4A)on the development of NAFLD through fatty acid metabolism.Method In this paper,in vivo and in vitro experiments will be conducted to investigate the expression changes of CYP4A11 enzyme(CYP4A)during the progression of NAFLD.The in vivo experiment was based on the high fat diet induced C57BL/6 mouse NAFLD model.Human hepatoma cells(Hep G2 cells)were induced by free fatty acids(FFA)in vitro as an in vitro cell model for studying NAFLD.In addition,relevant indicators for serum testing of clinical NAFLD patients were collected.1.Changes of CYP4 A expression level over time in the high-fat diet rat model A total of fifty C57BL/6 mice aged 8 weeks and weighing 20 ± 2 g were selected.According to the literatures,mice in the model group were given self-made high-fat emulsions for intragastric administration,which were divided into groups of 4 weeks,6 weeks,8 weeks and 10 weeks.Mice in each group were sacrificed at the corresponding time to collect serum and liver;a portion of the liver tissue was placed in 4% paraformaldehyde for HE staining and the other portion was stored at-80 °C refrigerator.2.Effect of FFA-induced Hep G2 cells on CYP4A11 expression Hep G2 cells were incubated with FFA solution for 24 hours using CYP4A11 expression inducer(clofibrate)and inhibitor(HET0016),respectively,to investigate the changes of lipid deposition and oxidation in Hep G2 cells during high expression and inhibition of CYP4A11.The expression level of CYP4A11 in cells was analyzed by Western blot.3.Effects of overexpression of CYP4A11 and silencing of CYP4A11 on inflammatory cytokines and NF-?B signaling pathways We transfected the plasmid carrying the CYP4A11 gene into Hep G2 cells while silencing the expression of CYP4A11 using si RNA.The expression levels of inflammatory factors TNF-?,IL-6,IL-1? m RNA were detected by RT-PCR and phosphorylated p65 were detected by westerning blot.4.Correlation between LPO level and CYP4A11 expression in patients with NAFLD The contents of LPO and CYP4A11 in plasma were detected by ELISA,and further linear correlation analysis was performed on the values of LPO and CYP4A11 in the Excel table.Results In vivo,the levels of ALT,AST,MDA,TG and liver ROS in C57BL/6 mice after high-fat emulsion administration were highest in the 10-week group.At the same time,the expression level of CYP4 A and 20-HETE were also the most obvious increase in the 10-week group.In vitro,the expression of CYP4A11 in cells increased after FFA treatment of Hep G2 cells.In further studies,the expression of CYP4A11 protein was significantly increased or decreased on the basis of FFA induction after treatment with Hep G2 cells with the inducer of CYP4A11(clofibrate)or inhibitor(HET0016).The results of oil red staining showed that the degree of lipid accumulation in Hep G2 cells was more serious than that in the model group after using clofibrate.The detection of intracellular TG content showed that clofibrate increased intracellular TG synthesis.Incubation of Hep G2 cells with HET0016 significantly attenuated the degree of cells oil red staining and decreased intracellular TG synthesis.The fluorescence intensity of ROS was the strongest in the clofibrate group.The expression of MDA in the cells was significantly higher than that in the model group.The activity of SOD in the cells was significantly lower than that in the model group,suggesting that CYP4A11 can enhance oxidative stress and increase peroxidation products and weaken antioxidant capacity.The use of HET0016 reduced FFA-induced ROS,decreased intracellular MDA expression,and increased intracellular SOD activity,suggesting that CYP4A11 is involved in the regulation of oxidative stress.When CYP4A11 gene was overexpressed,the m RNA expression levels of TNF-?,IL-6 and IL-1? were significantly higher than those in the model group,while the m RNA expression of TNF-?,IL-6 and IL-1? was lower than the model group when CYP4A11 gene was silenced.This indicates that the CYP4A11 gene affects the secretion of pro-inflammatory factors.Plasma analysis of patients with NAFLD showed increased expression of CYP4A11 and LPO in patients with NAFLD,and linear regression analysis showed that the expression levels of the two were highly correlated.Conclusion CYP4A11 is involved in the oxidative metabolism of excess fatty acids and promotes the production of ROS.It further promotes lipid peroxidation through oxidative stress and promotes the secretion of inflammatory factors through ROS/NF-?B pathway,and participates in the progression of NAFLD.