Nrf2-ARE信号通路遗传变异在抗结核药致肝损害发生中的作用及相关机制研究

Anti-tuberculosis induced liver injury (ATLI) is a serious adverse drug reaction during anti-tuberculosis treatment. Though the pathogenic mechanism of ATLI is still largely obscure, it is suggested that toxic intermediates, rather than direct toxicities of the parent drugs, are responsible for most idiosyncratic drug reactions. Studies have shown that toxic intermediates can cause oxidative stress, and could be further detoxified by phase Ⅱ metabolizing enzymes and antioxidant enzymes which could be induced to express by Nuclear factor erythroid 2-related Factor 2 (Nrf2)-Antioxidant Response Element (Nrf2-ARE) signaling pathway. So the genetic variation of Nrf2-ARE signaling pathway and downstream target gene expression levels may affect the clearance of toxic intermediates and even occurrence of ATLI. In the present study, new diagnosed tuberculosis patients were recruited and followed up. Periodic liver function tests and detection of oxidative stress indicators would be conducted. Potential functional SNPs in genes of Nrf2-ARE signaling pathway would be screened and detected, and some significant SNPs' biological function would be studied. Allele frequency and oxidative stress indicators would be analyzed among ATLI group, liver adaptation group, and normal liver function group. The relationship of genotype-phenotype, different genotype, different levels of oxidative stress, environment factors and their interactions would be evaluated in the risk of liver injury. All these would probably illustrate the role of genetic variation of Nrf2-ARE signaling pathway in the risk of liver injury, and show a potential mechanism of ATLI. The present study would contribute to early identification, prevention, clinical classification, prognosis of ATLI, and would provide a scientific basis to develop individualized treatment to tuberculosis.

抗结核药引起的肝损害是一种严重不良反应，其机制仍不清楚，可能与代谢产生的毒性中间产物有关。毒性中间产物会引起机体氧化应激，通过启动核因子E2相关因子2(Nrf2)-抗氧化反应元件(ARE)信号通路(Nrf2-ARE信号通路)，增加下游Ⅱ相代谢酶和抗氧化酶表达来清除。因此，Nrf2-ARE信号通路遗传变异及其所调控下游靶基因表达水平就会影响毒性中间产物清除，继而导致肝损害发生。本研究以抗结核治疗病人为随访队列，定期监测肝功能和检测氧化应激指标，筛选并检测Nrf2-ARE信号通路相关基因潜在功能SNPs和生物学功能研究。通过比较肝损害组、肝适应组、肝功能正常组等位基因频率、氧化应激指标差异以及基因型-表型关系，评价不同基因型、氧化应激水平及交互作用对肝损害发生风险的影响，阐明Nrf2-ARE信号通路遗传变异在肝损害发生中的作用及可能的机制，为早期识别和预防肝损害及制定个体化治疗提供依据。

抗结核药致肝损害（ATLI）是结核病治疗中的严重不良反应，但其机制仍不清楚，可能与代谢的毒性中间产物有关。而毒性中间产物一般会引起机体氧化应激，通过启动核因子E2相关因子2(Nrf2)-抗氧化反应元件(ARE)信号通路(Nrf2-ARE信号通路)增加代谢酶和抗氧化酶表达而清除。因此，推测Nrf2-ARE信号通路遗传变异可能影响肝损害发生。本项目基于前瞻性队列研究设计，选择新登记结核病人进行门诊随访收集其肝功能信息，判断ATLI的发生，动态分析病人氧化应激指标与ATLI的关系。同时，基于随访队列开展1:2匹配巢式病例对照研究，筛选并检测Nrf2-ARE信号通路相关基因多态性位点，分析基因型、单倍型、交互作用等与ATLI的关系，同时对于阳性位点进行生物学功能研究。截至2017年10月31日，抗结核治疗随访队列共有3294名患者，完成治疗2412人，判断为ATLI有336人，肝酶正常者1318人，ATLI发生率为13.9%。ATLI组在治疗15天和30天时仅有SOD显著低于肝酶正常组，其他指标未见明显变化。1:2匹配巢式病例对照研究包括313例ATLI病例和626例肝酶正常者。基于Nrf2-ARE信号通路11个基因50个位点的检测结果，条件Logistic回归分析显示Bach1、XPO1、MnSOD、HMOX1、UGT1A1基因位点与ATLI有关联，但经过Bonferroni校正后均无统计学联系，分层分析结果经校正后也无统计学联系。各个基因的单倍型在两组的分布也无统计学差异，交互作用显示KEAP1-rs1048290位点与保肝药存在相乘交互作用，会增加ATLI发病风险，但Bonferroni校正后无统计学意义（P>0.05）。采用中国结核病防治规划抗结核病药品不良反应研究（ADACS）的89个ATLI病例和356个对照（1:4匹配病例对照研究）进行外部验证，结果也确证了本次阴性结果。基于本次的现场随访和实验室检测，未能发现Nrf2-ARE信号通路相关基因多态性与ATLI存在联系，也基本排除了氧化应激反应通路中的遗传变异在肝损害发生中的作用，这对于进一步认识ATLI发生机制和探寻新的研究思路和方向有一定帮助。