The Role Of Hepatic Cytochrome P4501A1, 2E1, 3A1 In The Pathogenesis Of NAFL In Rats

Objective: To investigate the role of hepatic cytochrome P4501A1, 2E1, 3A1 in the pathogenesis of NAFL and the mechanism of NAFL in rats to provide theoretical basis for early prevention and treatment of NAFL.Methods: Sixty-four male Wister rats were divided randomly into the basal feed group(Control ) and high fat feed group (F). Each group was subdivided into 4 groups at different time points, i.e., 2,4,8,12 weeks respectively, with 8 rats in each subgroups. The following changes in each group were observed and determined dynamically: ①Animal body masses and liver wet weights; ②Histopathology of liver using HE-staining under a light-microscope and ultra-structures of hepatic tissue under a electron microscope; ③ Contents of triglyceride(TG)and malondialdehyde(MDA) in hepatic tissue and concentrations of TG and free fatty acid(FFA) in serum; ④Activities of CYP1A1 (7-ethoxyl isoxazole O-deethylase,EROD), CYP2E1 (aniline hydroxylase, ANH) and CYP3Al(erythromycin N-demethylase,ERD) in rat hepatic tissue using enzymatic methods; ⑤Distribution of CYP4501A1,2E1,3A1 in hepatic tissue using immunohistochemical methods; ⑥ mRNA and protein levels of CYP4501A1, 2E1, 3A1 in rat liver using RT-PCR and Western blotting.Results: 1. Changes in rat body masses and liver wet weights: The body masses and liver wet weights of group F were significantly higher than those of group C at 12 weeks (P<0.01); 2.HE staining revealed fatty infiltration of the liver at 2 weeks, mild fatty liver at 4 weeks and moderate to severe fatty liver at 8-12 weeks in group F. Under the electron microscope, there were a number of deposits of round lipid droplets in the cytoplasm of liver cells, the organelles and nuclei were pushed to the side of the cells and the cytoplasm seemed puff and dropsy at 12 weeks in group F. 3. The contents of liver TG, MDA in group F were significantly higher than that of group C at 2 weeks (P<0.05), which were increased as the time elapsed and reached a peak at 12 weeks (P<0.01); The changes of serum FFA, TG were similar to that of liver TG and MDA; 4. The activities of CYP1A1 (7-ethoxylisoxazole O-deethylase, EROD), CYP2E1 (aniline hydroxylase, ANH) and CYP3Al(erythromycin N-demethylase, ERD) in rat hepatic tissue were enhanced after 2 weeks in group F (P<0.05), which were increased as the time elapsed and reached a peak at 12 weeks (PO.01); 5. Immunohistochemical staining revealed that there were yellow CYP450IAl-positive particles diffusely distributed in the cytoplasm of liver cells in group C. At 2 weeks, the staining was heavier in group F compared with group C. At 8 and 12 weeks the staining became even heavier and the positive cell numbers increased, heavily stained at the sites of obvious fatty degeneration. Similar staining was found of CYP4502E1 and 3A1; 6. RT-PCR revealed that the expression of CYP4501A1 mRNA in Group F was significantly increased compared to group C at 2 weeks (P<0.05). As the time elapsed, the expression was significantly higher than normal at 12 weeks (P<0.01). Similar results were found of the expression of CYP4502E1 and 3A1 mRNA. 7. Western blotting demonstrated that the expression of CYP4501A1 protein in Group F was significantly increased at 2 weeks compared to group C (PO.05). As the time elapsed, the increase became obvious at 12 weeks (P<0.01). Similar results were found of the expression of CYP4502E1 and 3A1 proteins.Conclusions: With the development of NAFL in rats, the expression of hepatic cytochromes P4501A1, 2E1, 3A1 was upregulated and the activities of related enzymes were increased, which were involved in the fatty acid metabolism and lipid peroxidation,and then, resulting in the pathgenesis of NAFL in rats.