Zhou Qian, Zhang Han, Wu Qin, Shi Jingshan, Zhou Shaoyu
Joint International Research Laboratory of Ethnomedicine, Key Laboratory of Basic Pharmacology of Ministry of Education, Zunyi Medical UniversityGuizhou, China.
Int J Biochem Mol Biol. 2017 Jun 15;8(2):13-22. eCollection 2017.
Environmental exposure to paraquat has been reported to be associated with Parkinson's disease (PD). In experimental animal models paraquat reproduces features of PD, however, the exact mechanism of PD-induced neurotoxicity has not been fully established. This study was designed to investigate paraquat-mediated interference with mitochondrial function and autophagy, and determine the impact of the modulation of autophagy flux on paraquat-induced cell toxicity. Rat adrenal pheochromocytoma PC12 cells were treated with paraquat for 24 h to establish a cellular mode of PD induced neurotoxicity. Pre-incubation of PC12 cells with an antioxidant N-acetyl-L-cysteine (NAC) or autophagy modulators rapamycin and chloroquine was conducted to determine the effect of modulation of oxidative status and autophagy flux on paraquat-elicited cytotoxicity. Mitochondrial functions and dynamics were analyzed by measuring oxygen consumption in a high-resolution oxygraph and imaging with a fluorescent mitochondrial dye (MitoTracker). Reactive oxygen species was determined by flow cytometry using fluorescent probe DCFH-DA. Autophagic flux was determined by Western blot analysis of autophagy marker LC3-II as well as p62 expression. It was found that treatment of cells with paraquat caused a concentration-dependent loss of cell viability that was accompanied by a decrease in cell respiration and reduction of polarized mitochondria, which was prevented by pretreatment of cells with NAC. Analysis of autophagy showed that NAC inhibited basic autophagy flux of PC12 cells, as evidenced by a decrease in LC3-II level and an increase in p62 expression. However, this modulation of autophagy by NAC may not be implicated into its cellular protective mechanism over paraquat cytotoxicity as inhibition of autophagy by chloroquine significantly enhanced paraquat induced cytotoxicity. Furthermore, the autophagy inducer rapamycin dramatically decreased paraquat induced cellular toxicity in PC12 cells. The present study demonstrates that basal autophagy plays a protective role in paraquat-induced cell toxicity. Antioxidant NAC confers protective role in paraquat toxicity mainly through maintaining mitochondrial dynamics and function, other than a modulation of autophagy flux.
据报道，环境接触百草枯与帕金森病（PD）有关。在实验动物模型中，百草枯可重现PD的特征，然而，PD诱导神经毒性的确切机制尚未完全明确。本研究旨在探讨百草枯介导的对线粒体功能和自噬的干扰，并确定自噬通量调节对百草枯诱导的细胞毒性的影响。用百草枯处理大鼠肾上腺嗜铬细胞瘤PC12细胞24小时，以建立PD诱导神经毒性的细胞模型。用抗氧化剂N-乙酰-L-半胱氨酸（NAC）或自噬调节剂雷帕霉素和氯喹对PC12细胞进行预孵育，以确定氧化状态和自噬通量调节对百草枯诱导的细胞毒性的影响。通过在高分辨率氧电极中测量氧气消耗并使用荧光线粒体染料（MitoTracker）成像来分析线粒体功能和动力学。使用荧光探针DCFH-DA通过流式细胞术测定活性氧。通过对自噬标记物LC3-II以及p62表达的蛋白质印迹分析来确定自噬通量。发现用百草枯处理细胞导致细胞活力呈浓度依赖性丧失，同时伴有细胞呼吸减少和极化线粒体减少，而用NAC预处理细胞可防止这种情况。自噬分析表明，NAC抑制PC12细胞的基础自噬通量，这通过LC3-II水平降低和p62表达增加得以证明。然而，NAC对自噬的这种调节可能与其对百草枯细胞毒性的细胞保护机制无关，因为氯喹抑制自噬可显著增强百草枯诱导的细胞毒性。此外，自噬诱导剂雷帕霉素显著降低了百草枯诱导的PC12细胞的细胞毒性。本研究表明基础自噬在百草枯诱导的细胞毒性中起保护作用。抗氧化剂NAC在百草枯毒性中发挥保护作用主要是通过维持线粒体动力学和功能，而不是通过调节自噬通量。
