BACKGROUND: Chronic alcohol consumption induces CYP2E1, which is involved in the development of alcoholic hepatitis (AH). However, the mechanisms underlying the induction of CYP2E1 by alcohol remain unclear. Therefore, we herein investigated the induction of drug-metabolizing enzymes, particularly CYP2E1, by hydrogen peroxide (H2O2), the concentration of which is elevated under inflammatory conditions.
OBJECTIVE: The mechanisms underlying the induction of CYP2E1 by H2O2 were examined with a focus on Keap1, a target factor of H2O2.
METHODS: We assessed changes in the expression of drug-metabolizing enzymes in the human hepatoma cell line, Hep3B, following treatment with H2O2, and evaluated changes in the expression of the NFkB-related factor RelA(p65) after the knockdown of Keap1, a regulator of Nrf2 expression by reactive oxygen species. We also performed a promoter analysis using the upstream region of the CYP2E1 gene. We herein used the GSE89632 series for non-alcoholic hepatitis (NASH) and the GSE28619 series for AH.
RESULTS: The induction of CYP2E1 by H2O2 was significantly stronger than that of other drugmetabolizing enzymes. On the other hand, the knockdown of Keap1, a target of H2O2, markedly increased RelA(p65), an NFkB factor. Furthermore, the overexpression of RelA(p65) strongly induced the expression of CYP2E1. Four candidate p65-binding sequences were identified upstream of the CYP2E1 gene, and promoter activity assays showed that the third sequence was responsive to the overexpression of RelA(p65). We used the GSE89632 series for NASH and the GSE28619 series for AH in the present study. The expression of CYP2E1 mRNA in the liver was significantly lower in AH patients than in HC patients, but was similar in HC patients and NASH patients.
CONCLUSIONS: We herein demonstrated that the expression of CYP2E1 was induced by H2O2. The overexpression of RelA(p65) also induced CYP2E1 mRNA expression, whereas H2O2 did not after the knockdown of RelA. These results suggest that H2O2 acts on Keap1 to upregulate RelA (p65) in the NFkB system. One of the mechanisms underlying the induction of CYP2E1 was dependent on the H2O2-Keap1-RelA axis. The results of the database analysis revealed that the expression of CYP2E1 in the liver was significantly lower in AHH patients than in NASH patients, suggesting that CYP2E1 is not the main cause of AH; however, CYP2E1 may exacerbate the pathogenesis of AH.
背景:长期饮酒会诱导CYP2E1,这与酒精性肝炎(AH)的发展有关。然而,酒精诱导CYP2E1的潜在机制尚不清楚.因此,我们在这里研究了药物代谢酶的诱导,特别是CYP2E1,通过过氧化氢(H2O2),其浓度在炎症条件下升高。
目的:研究了H2O2诱导CYP2E1的潜在机制,重点研究了H2O2的靶因子Keap1。
方法:我们评估了人肝癌细胞系中药物代谢酶表达的变化,Hep3B,在用H2O2处理后,并评估了NFkB相关因子RelA(p65)的表达变化,Keap1是活性氧的Nrf2表达调节剂。我们还使用CYP2E1基因的上游区域进行了启动子分析。我们在此使用GSE89632系列用于非酒精性肝炎(NASH)和GSE28619系列用于AH。
结果:H2O2对CYP2E1的诱导作用明显强于其他药物代谢酶。另一方面,H2O2靶标Keap1的击倒显著增加RelA(p65),NFkB因子。此外,RelA(p65)的过表达强烈诱导CYP2E1的表达。在CYP2E1基因的上游鉴定了四个候选p65结合序列,和启动子活性测定显示第三序列对RelA(p65)的过表达有反应。在本研究中,我们将GSE89632系列用于NASH,将GSE28619系列用于AH。AH患者肝脏中CYP2E1mRNA的表达明显低于HC患者,但在HC患者和NASH患者中相似。
结论:我们在此证明CYP2E1的表达是由H2O2诱导的。RelA(p65)的过表达也诱导CYP2E1mRNA表达,而H2O2在RelA敲低后没有。这些结果表明H2O2作用于Keap1以上调NFkB系统中的RelA(p65)。CYP2E1诱导的机制之一取决于H2O2-Keap1-RelA轴。数据库分析结果显示,AHH患者肝脏中CYP2E1的表达明显低于NASH患者,表明CYP2E1不是AH的主要原因;然而,CYP2E1可能会加剧AH的发病机制。