| Nonalcoholic fatty liver disease(NAFLD)is a chronic liver disease characterized by hepatic lipid accumulation in the presence of non-alcoholic drinking history and other liver diseases,persistent non-alcoholic fatty liver disease is the main cause of cirrhosis and liver cancer.Oxidative stress plays a key role in the lipid peroxidation of nonalcoholic fatty liver disease.Studies have shown that when the etiology(high-fat,high-sugar diet)is removed,the liver’s lipid accumulation can spontaneously reverse.Cytochrome P450 1A1(CYP1A1)is a widely expressed cytochrome P450 enzyme.Aromatic receptors(AHRs)are ligand-activating transduction factors.Polycyclic aromatic hydrocarbons benzopyrene(BaP)is an AHR agonist.AHRs are translocated to the nucleus when the agonist binds to the aromatic receptor Dimerizes with ARNT and then forms a dimer that binds to the promoter DRES / XRES synthesized by the CYP1A1 protein to activate the transcription and translation of the CYP1A1 protein.As a member of the cytochrome P450 enzyme family,CYP1A1 is an important enzyme that regulates the production of reactive oxygen species.Studies have shown that CYP1A1 protein expression in non-alcoholic fatty liver is elevated,but its role in non-alcoholic fatty liver is unclear.In this study,we want to prove that CYP1A1 plays a catalytic role in the lipid peroxidation of non-alcoholic fatty liver and reduces the high expression of CYP1A1 in non-alcoholic fatty liver,which can reduce lipid peroxidation in non-alcoholic fatty liver.In order to further investigate the role of CYP1A1 in non-alcoholic fatty liver,we examined the changes of liver tissues in the non-alcoholic fatty liver during the advanced and the reverse phase.HepG2 cells were selected as experimental subjects to detect the changes and roles of CYP1A1 in the progression and reversal of fatty liver in vitro.The main contents of this paper include the following three parts: 1.The protein expression of CYP1A1 in non-alcoholic fatty liver formation and recovery 36 mice(male,4 weeks old,20-22g)were randomLy divided into control group,non-alcoholic fatty liver model group and non-alcoholic fatty liver reversal group,12 rats in each group.The model mice were given MCD diet for 4 weeks to establish non-alcoholic fatty liver model.The mice in reversal group were fed with MCD diet for 4 weeks,then MCD diet was stopped and re-fed normal diet(negative feed corresponding to MCD feed)for 4 weeks to establish spontaneous reversal model of non-alcoholic fatty liver.Mice were sacrificed at 0 and 4 weeks after normal diet,serum and liver samples were collected for use.The liver pathological changes were observed by H & E staining and oil red staining,and the levels of serum ALT and AST were measured to observe the degree of liver injury.Real-time PCR,Western Blot detection of CYP1A1 protein expression and mRNA level.2.The effect of ilencing CYP1A1 protein expression on lipid peroxidation in non-alcoholic fatty liverHepG2 cells were stimulated by different gradient concentrations(0,0.1,0.2,0.5,1.0,2.0mM;24h)at different time points(0,12,24,48h;0.2mM).The expression of CYP1A1 in HepG2 cells was detected by western blot.The results showed that: 0.1mM,0.2mM,0.5mM oleic acid solution concentration of cell proliferation had no obvious killing effect,so the choice of 0.2mM concentration as the concentration of the model fluid for future experiments.CYP1A1 was upregulated in oleic acid-induced HepG2 cells,with the highest protein expression at 24 h.HepG2 cells were pretreated with CYP1A1-siRNA and then treated with oleic acid(0.2mM)for 24 hours.Real-time PCR and Western-blot were used to detect the mRNA and protein expressions of CYP1A1 and CYP1A2 in HepG2 cells.Oxidative Stress indicators ROS and SOD,and lipid peroxidation indicators(MDA and HNE).3.Effects of CYP1A1 overexpression by BAP on lipid peroxidation in non-alcoholic fatty liver.After 24 h induced by oleic acid,HepG2 cells were removed from the model fluid and replaced with normal DMEM for 48 h.Real-time PCR and Western Blot were used to detect the expression of CYP1A1 in HepG2.The results showed that CYP1A1 was up-regulated in oleic acid-induced HepG2 cells and down-regulated in the reversed group.HepG2 cells were induced by oleic acid for 24 h,then BaP(5 μM)was added to the cells for further cultivation for 24 h.The mRNA and protein expression of CYP1A1 in HepG2 cells were detected by Real-time PCR and Western-blot.The oxidative stress indicators ROS and SOD,and lipid peroxidation(MDA and HNE)were detected by kit.The results showed that CYP1A1 was up-regulated in non-alcoholic fatty liver and decreased in non-alcoholic fatty liver restored spontaneously.The results showed that after the silencing of CYP1A1 protein expression was significantly decreased compared with the model group,mRNA levels than the model group also decreased significantly.Protein silence,lipid peroxidation indicators MDA and HNE decreased significantly,the oxidative stress index ROS decreased compared with the model group,transfection of SOD value than the model group increased significantly.According to the results of CYP1A2 protein and mRNA,we can see that CYP1A2 transfected with no significant difference compared with the model group or the normal group.The results show that: stimulation of CYP1A1 protein expression with agonist,non-alcoholic fatty liver will be increased lipid peroxidation.In this study,in vivo reversal experiments,in vitro cell silencing and overexpression experiments demonstrated that cytochrome P4501A1 enzyme can catalyze the lipid peroxidation of non-alcoholic fatty liver. |
