SIRT1及炎性细胞因子在不同抗结核药物致肝损伤中的作用

Role of SIRT1 and Inflammatory Factors in Liver Injury Induced by Different Anti-Tuberculosis Drugs

目的探讨沉默信息调节因子1 (SIRT1)及相关炎性细胞因子在异烟肼(INH)、利福平(RFP)、吡嗪酰胺(PZA)致人肝细胞损伤过程中的作用。方法传代培养人正常肝细胞HL-7702细胞,分为3组:INH组、RFP组、PZA组。各组再分为以下几个亚组:空白对照组、药物组、药物+SIRT1激动剂组、激动剂对照组、药物+SIRT1抑制剂组和抑制剂对照组。采用赖氏法检测每组细胞培养液中ALT、AST含量;实时PCR法检测肝细胞中SIRT1、NF-κB p65、IL-6、TNF-αmRNA表达;Western blotting检测SIRT1、NF-κB p65蛋白表达;ELISA法检测IL-6、TNF-a蛋白表达。结果与其空白对照组比较,INH、RFP、PZA组SIRT1 mRNA和蛋白表达均降低(均P <0.05);INH组NF-κB p65、IL-6及TNF-αmRNA与蛋白表达水平均增加(均P <0.05)。INH与SIRT1激动剂联用可抑制NF-κB p65、IL-6及TNF-αmRNA与蛋白表达(均P <0.05);与SIRT1抑制剂联用则使NF-κB p65、IL-6及TNF-αm RNA与蛋白表达升高(均P <0.05);与SIRT1激动剂对照组和SIRT1抑制剂对照组比较,RFP、PZA组炎性细胞因子表达水平均无明显变化(均P> 0.05)。结论 SIRT1参与INH致人肝损伤的机制是通过作用于NF-κB p65信号通路进而影响炎性细胞因子表达来实现的,推测SIRT1通过调控过氧化物酶增殖物激活受体(PPAR)信号通路参与RFP、PZA致人肝细胞损伤的过程。

Objective To investigate the role of silent mating type information regulation 2 homolog-1(SIRT1) and related inflammatory factors in the process of human hepatocyte injury induced by isoniazid(INH),rifampicin(RFP),and pyrazinamide(PZA). Methods Human normal hepatocytes HL-7702 were subcultured and treated with INH,RFP or PZA to induce hepatocyte injury. The treated hepatocytes were divided into blank control,drug,drug + SIRT1 agonist,agonist control,drug + SIRT1 inhibitor,and inhibitor control groups. The levels of ALT and AST in each group were detected by Rye’s method;the expressions of SIRT1,NF-κB p65,IL-6,and TNF-α mRNA in hepatocytes were detected by RT-PCR;the expressions of SIRT1 and NF-κB p65 protein were detected by Western blotting;and the expressions of IL-6 and TNF-α protein were detected by ELISA. Results The expressions of SIRT1 mRNA and protein in INH-,RFP-,and PZA-treated hepatocytes were lower compared to their expressions in the blank control group(P < 0.05). The mRNA and protein expression levels of NF-κB p65,IL-6,and TNF-α were increased in INH-treated hepatocytes(P < 0.05). The combination treatment of INH and SIRT1 agonist inhibited the expressions of NF-κB p65,IL-6,and TNF-α mRNA and protein(all P < 0.05),while the combination treatment of INH and SIRT1 inhibitor increased the expressions of NF-κB p65,IL-6,and TNF-α mRNA and protein(all P < 0.05). Compared to the treatment with SIRT1 agonists and inhibitors,no significant changes were observed in the expressions of inflammatory cytokines in the RFP-and PZA-treated hepatocytes(P > 0.05). Conclusion Our study suggests that SIRT1 is involved in INH-induced liver injury because it specifically affects the expressions of NF-κB p65 signaling pathway components and inflammatory factors. SIRT1 is also involved in the process of RFP-and PZA-induced hepatocyte injury as it regulates the peroxidase proliferator activated receptor(PPAR) signaling pathway.