基于斑马鱼模型研究炎症状态下异烟肼经内质网应激-自噬通路诱发肝毒性的作用机制

Isoniazide (INH) is an important first-line drug that is used to treat tuberculosis. However, hepatotoxicity is the major adverse effect of INH and the mechanism of INH-induced hepatotoxicity is still unknown. Patients with tuberculosis are often associated with inflammation, which may improve the sensitivity of drug toxicity. We have determined that inflammatory state could aggravate INH-induced hepatotoxicity via mild inflammation transgenic zebrafish model. On this basis of experimental results, we employed in vivo imaging, Real-time PCR and Western Blotting technology to research the function of autophagy and endoplasmic reticulum stress which exposed INH on zebrafish model under condition of mild inflammation. In order to research the hepatotoxicity of INH on inflammatory zebrafish, and further clarify the the mechanism of INH induced hepatotoxicity by endoplasmic reticulum stress-autophagy pathways under mild inflammatory state, tool drugs (autophagy inducer / inhibitor and endoplasmic reticulum stress inhibitor) and gene editing techniques (Morpholino, CRISPR/Cas9) were used to investigate the expression of autophagy, endoplasmic reticulum stress as well as the key factor in autophagy upstream pathways (IRE1, ATF6, PERK and DAPK). We also inspect the protective effect of antioxidants for INH-mediated hepatotoxicity under mild inflammatory state. Our research will provide beneficial guide for the clinical medication safety of INH, and will offer reliable reference for hepatoxic evaluation of similar anti-tubercular drugs.

异烟肼是一线抗结核药，机体伴发炎症的结核病患者在用药时，肝脏不良反应发生率高，严重困扰临床治疗，但炎症状态下异烟肼的肝毒性作用机制尚不明确。我们利用转基因斑马鱼构建了基于炎症状态斑马鱼的药物肝毒性评价模型，确定了炎症状态下异烟肼的肝毒性作用增强。在此基础上，采用活体成像、Real-time PCR、Western Blotting等技术，研究异烟肼对炎症状态斑马鱼的自噬和内质网应激作用。应用工具药物（自噬诱导剂、抑制剂和内质网应激抑制剂）和基因编辑技术（Morpholino基因敲降、CRISPR/Cas9基因敲除），干扰自噬、内质网应激以及自噬上游通路中关键因子（IRE1、ATF6、PERK、DAPK）表达，考察异烟肼的肝毒性变化，阐明炎症状态下异烟肼经内质网应激-自噬通路诱发肝毒性的分子调控机制，为异烟肼的临床安全用药提供指导，并为类似的抗结核药物肝毒性评价提供研究思路。

结核病患者在用药时可能因机体伴发炎症，对异烟肼肝毒性的敏感性增强而出现肝损伤。但炎症状态下异烟肼造成肝毒性的机制尚不明确。本项目利用低剂量内毒素（LPS）预处理和创伤性损伤（断尾）的方法，建立了基于炎症状态斑马鱼的药物肝毒性评价模型。实验发现异烟肼导致炎症状态斑马鱼的肝毒性加剧，炎症反应参与了异烟肼引起的肝细胞损伤过程。异烟肼导致炎症状态斑马鱼肝细胞内部分细胞器溶解，轮廓不清楚，出现自噬体。LPS和异烟肼联用组斑马鱼机体自噬相关因子Beclin1、LC3、Atg3和Atg12的mRNA表达水平比异烟肼单用组显著上调。提示异烟肼可能导致炎症状态斑马鱼发生自噬，导致细胞损伤。自噬抑制剂（3-MA）对异烟肼导致的斑马鱼肝毒性有一定的减缓作用，自噬激动剂（雷帕霉素）显著加重了异烟肼对炎症状态斑马鱼的肝毒性。异烟肼导致炎症状态机体ROS含量显著增加，斑马鱼肝细胞内质网发生扩张、肿胀、断裂现象，内质网应激标志性分子 GRP78、GRP94、IRE1、XBP1s、ATF6、PERK的mRNA表达水平比正常状态斑马鱼的表达水平显著增加，说明内质网应激参与了炎症状态导致异烟肼肝毒性增强的过程。内质网应激抑制剂（4-PBA）缓解了异烟肼导致的炎症状态斑马鱼肝毒性。利用Morpholino敲降内质网应激活化的UPR和Ca2+信号通路中的关键因子IRE1、ATF6、PERK和DAPK。结果发现ATF6敲降可减轻异烟肼导致的炎症状态斑马鱼肝毒性，揭示异烟肼可能通过激活ATF6信号途径，诱导内质网应激发生，激活细胞自噬，导致肝脏损伤。通过本项目的实施，明确了异烟肼对炎症状态斑马鱼的肝毒性作用，进一步明确了内质网应激和自噬通路参与了异烟肼对炎症状态斑马鱼的肝毒性作用。项目的开展为临床上异烟肼的安全用药、寻找降低异烟肼肝毒性的治疗策略和方案提供科学参考依据，为类似药物的肝毒性评价建立技术平台。