Hepatocyte Nrf2 Regulates The Role Of CYP2E1 Protein Expression In Liver Tumor Induced By Diethylnitrosamine

Objective: The liver is the main organ for metabolism of endogenous chemicals and exogenous poisons.The most common primary tumor in the liver is liver cancer,which ranks 6th in terms of cancer incidence and 3rd in terms of cancer mortality.Many animal models have been developed to elucidate the pathogenesis of liver cancer and study the effects of potential therapies.Chemically-induced animal liver tumors are the most commonly used animal models with many morphological,histological and biochemical characteristics of human liver cancer.Nuclear factor erythroid2-related factor 2(Nrf2)is a transcription factor that regulates defense against toxicity and oxidative damage by expressing genes involved in oxidative stress response and detoxification.Nrf2 activates and regulates cells to resist chemical carcinogens and inflammation,and coordinates cells to adapt to various pressures to maintain homeostbalance and inhibit the occurrence of tumors.Meanwhile,its overactivation promotes the development of liver cancer by protecting cancer cells from excessive oxidative stress,chemotherapy drugs and radiation.Therefore,this paper summarizes the function of Nrf2 in hepatocytes and describes its role in chemical-induced liver tumors.Methods: 1.Hepatocellular specific Nrf2 knockout(Nrf2(L)-KO)mice with C57BL/6J genetic background were used as the research object.The DEN induced liver tumor model was established by intraderitoneal injection of DEN(25 mg/kg BW)or 0.9%Na Cl solution at the age of 2 weeks.The diet,water intake and body weight of mice were monitored.The occurrence of liver tumor was evaluated by liver gross image,Doppler ultrasound,tumor load and tumor number.H&E staining was used to detect pathological changes of liver.Immunohistochemical staining was used to detect liver cancer markers AFP,PCNA and abnormal proliferation of liver cells.2.Male C57BL/6J hepatocellular specific Nrf2 knockout(Nrf2(L)-KO)mice(8-12 weeks of age)were injected with DEN(100 mg/kg BW)or 0.9% Na Cl solution,and collected at 3,6,12 and 24 h,respectively.Acute liver injury model caused by DEN was established(n =6).DNA damage was detected by immunohistochemical staining of γ-H2 AX and O6-Etd G.The contents of ALT and AST were detected by Elisa.m RNA levels of Tnf-α,Il-6,Il-1β,Nrf2,Hmox-1,Gsr,Gclm and Cyp2e1 were detected by RT-q PCR.Western blot analysis was used to detect the expression levels of NRF2 and CYP2E1.The content of acetaldehyde was determined by gas chromatography.Results: 1.Nrf2 deficiency in hepatocyte had no significant effect on diet,water intake and body weight of DEN treated mice.After DEN treatment,liver organ coefficient increased,and Nrf2 deficiency in hepatocyte inhibited DEN induced liver organ coefficient increased(p < 0.05).2.DEN treatment can induce liver tumors in mice,and liver tumor volume,tumor load and tumor index can all show that hepatocyte specific Nrf2 deletion reduces DEN-induced liver tumors(p < 0.05)..H&E staining showed that hepatocyte specific Nrf2 knockout mice attenuated the liver structural damage caused by DEN exposure.3.The results of γ-H2 AX staining showed that DNA damage began to occur at 3 h after exposure to DEN,and the damage worsened over time,while hepatocyte-specific Nrf2 deletion alleviated the liver DNA damage caused by acute DEN exposure.3.After DEN treatment,inflammation was found in the liver at 3 h,and the structure of the liver was changed at 12 h,resulting in bridging necrosis,which was aggravated over time.Hepatocyte specific Nrf2 deletion inhibited the liver damage effect caused by DEN.ALT and AST,as indicators of liver injury,were not significantly increased after acute DEN exposure,and hepatocyte specific Nrf2 deletion had no effect on them.Meanwhile,m RNA levels of inflammatory cytokines in mice were detected,and it was found that after acute DEN exposure,m RNA levels of Tnf-α increased at 3h,Il-1β and Il-6 increased at 6 h,and gradually decreased at 12 h.Hepatocyte specific Nrf2 deletion inhibited DEN-induced inflammation(p < 0.05).4.DNA damage began to occur at 3 h after exposure to DEN,and increased with time,while hepatocyte specific Nrf2 deletion mitigated liver DNA damage caused by acute DEN exposure.5.A significant increase in Nrf2 protein and m RNA levels was observed after DEN treatment(p < 0.05).We also detected the m RNA levels of downstream Nrf2 genes Hmox-1,Gsr,and Gclm,and found that Hmox-1 increased at 6 and 12 hours.Hepatocyte specific Nrf2 deletion inhibited the increase of Hmox-1(p < 0.05).6.After DEN treatment,at 24 hours,acetaldehyde levels in urine of Nrf2(L)-KO mice were significantly lower than those of Nrf2-KI mice.Meanwhile,Nrf2-specific deletion of hepatocytes decreased the number of O6-Etd G positive staining cells.7.The expression of Cyp2e1 m RNA was decreased(p < 0.05),while hepatocellular specific Nrf2 deletion had no effect on Cyp2e1 m RNA.But inhibited the expression level of CYP2E1 protein(p < 0.05).Conclusion: Nrf2 deletion in hepatocytes inhibits the activation of DEN metabolism mediated by CYP2E1 protein,thereby attenuating DEN-induced DNA damage in hepatocytes and liver tumor effects.