Regulatory effect of PINK1/Parkin axis on mitophagy in isoniazide-induced hepatocellular injury.

Abstract

Patients with tuberculosis, who are treated with long-term high-dose combined use of anti-tuberculosis drugs, can cause many adverse reactions such as liver damage, but the mechanism is still unclear. Although phosphatase and tensin homolog induced kinase 1 (PINK1)/Parkin axis might participate in the process of liver damage through regulating mitochondrial autophagy and oxidative stress in liver cells. However, the association between the mitochondrial autophagy regulated by the PINK1/Parkin axis and the liver injury induced by anti-tuberculosis drugs is unknown. This study aims to explore the mechanism of PINK1/Parkin signal axis in regulating hepatocellular injury induced by anti-tuberculosis drugs through mitochondrial autophagy, and to provide new therapeutic targets for the patients with liver damage caused by anti-tuberculosis drugs.Mouse hepatocytes AML-12 were treated with isoniazide (INH) to induce liver injury. The mRNA and protein levels of PINK1, Parkin and autophagy associated factors were detected by real-time PCR and Western blotting in the normal AML-12 cells (control group), the AML-12 cells treated by INH (model group) and the AML-12 cells treated with INH and salidroside together (intervention group). Meantime, ELISA kit was used to detect the level of reactive oxygen species (ROS) in AML-12 cells, and the cell morphology and injury was observed by HE staining and transmission electron microscope (TEM).Compared with the control group, the mRNA (all P<0.01) and protein levels (all P<0.05) of PINK1, Parkin and microtubule associated protein 1 light chain 3 (LC3) were significantly decreased, the ubiquitination level was significantly decreased (P<0.05), and the ROS level was increased (P<0.05), and the hepatocellular cell showed obvious damage in the model group. Compared with the model group, the expression of PINK1, Parkin and LC3 were significantly increased in the intervention group (all P<0.05), as well as the ubiquitination level was also increased (P<0.05). The ROS level mediated by mitochondria was decreased (P<0.05). The results of HE staining and TEM showed that the cell status was improved and the damage of hepatocytes was significantly attenuated.In this study, the mechanism of mitochondrial autophagy mediated by PINK1/Parkin signal axis may be related to INH-induced injury in liver cells and it can regulate the damage state in vitro, indicating that Parkin is a potential molecular target for the prediction, diagnosis and treatment for liver injury induced by anti-tuberculosis drugs.目的: 结核病患者长期大剂量联合使用抗结核药物会引起肝损伤等不良反应，但引起损伤的机制尚不明确。尽管同源性磷酸酶及张力蛋白诱导激酶1(phosphatase and tensin homolog induced kinase 1，PINK1)/Parkin信号轴可能通过调控线粒体自噬和肝细胞内氧化应激水平而参与肝损伤过程，但目前尚不清楚药物致肝损伤与该信号轴调控的线粒体自噬机制间的相关性。本研究旨在探究PINK1/Parkin信号轴是否可通过调控线粒体自噬改善抗结核药物用药过程中出现的肝细胞损伤，从而为临床结核病患者使用抗结核药物治疗过程导致的肝损伤提供潜在的药物治疗靶点。方法: 使用异烟肼(isoniazide，INH)处理小鼠肝实质细胞AML-12以模拟肝损伤状态，分别采用real-time PCR和蛋白质印迹法检测正常AML-12细胞(对照组)、INH刺激的肝损伤模型(模型组)及INH和PINK1激动剂红景天甙共同刺激下的AML-12细胞(干预组)中PINK1、Parkin以及自噬相关分子的表达水平。利用ELISA试剂盒检测肝细胞内活性氧(reactive oxygen species，ROS)水平，采用HE染色及透射电镜技术观察细胞的肝细胞形态结构。结果: 与对照组比较，模型组中PINK1、Parkin和微管相关蛋白1轻链3(microtubule associated protein 1 light chain 3，LC3)的mRNA表达水平(P<0.01)以及蛋白质水平(P<0.05)均显著下降，同时泛素化蛋白水平显著下调(P<0.05)，ROS水平升高(P<0.05)，细胞损伤明显。与模型组相比，干预组中使用红景天甙后PINK1、Parkin和自噬相关分子LC3的mRNA及蛋白质水平均显著升高(均P<0.05)，泛素化蛋白水平亦显著升高(P<0.05)，同时线粒体介导的ROS水平下降(P<0.05)，肝细胞损伤状态明显减轻。结论: 本研究利用体外细胞实验证明了PINK1/Parkin信号轴介导的线粒体自噬机制可能与异烟肼所致肝细胞的损伤相关，并能调控损伤状态，提示Parkin是一个潜在的药物致肝损伤预测及诊疗的分子靶点。.