Perfluoroalkyl acid exposure induces protective mitochondrial and endoplasmic reticulum autophagy in lung cells
Perfluoroalkyl acid exposure induces protective mitochondrial and endoplasmic reticulum autophagy in lung cells
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全氟烷基酸暴露诱导肺细胞中保护性线粒体和内质网自噬
DOI：
10.1007/s00204-018-2266-0
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发表时间：
2018-07
影响因子：
6.1
通讯作者：
Yan Xin;B. Wan;Yu Yang;Xuejing Cui;Yi-Chun Xie;Liang-Hong Guo
中科院分区：
文献类型：
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作者：
Yan Xin;B. Wan;Yu Yang;Xuejing Cui;Yi-Chun Xie;Liang-Hong Guo
全氟烷基酸的广泛应用引起了人们对其对人体健康的副作用的极大关注。PFAAs已被证明主要在肝脏中积累并引起肝毒性。然而，PFAAs也可以通过空气中的颗粒沉积在肺组织中，并引起严重的肺毒性。但其潜在机制在很大程度上仍是未知的。自噬是一种类似于坏死和凋亡的程序性细胞死亡，可能与PFAAs的肺毒性有关。本研究以肺癌细胞A549为模型，研究了三种不同碳链长度的PFAAs对细胞自噬的影响。通过对非细胞毒浓度（200µM）和细胞毒浓度（350µM）下细胞LC3-I/II比值的Western blot分析，我们发现细胞中自噬体呈浓度依赖性增加，并通过细胞超薄切片TEM检查和活细胞自噬体荧光成像进一步证实了这一点。p62的丰度随着PFAAs浓度的增加而增加，表明自噬通量被阻断。此外，我们在形态学上发现线粒体自噬（mitophagy）和内质网自噬（ER-phagy）是自噬的主要类型，表明线粒体和内质网自噬受到破坏。ROS的过度生成、线粒体膜电位的超极化以及内质网应激相关蛋白ATF4和p-IRE1的上调证实了这些细胞器损伤。进一步信号通路分析表明，PFAAs激活MAPK通路，抑制PI3K/Akt通路，其效价顺序为PFDA > PFNA > PFOA。抗氧化（NAC）处理不能使细胞免于死亡，表明氧化应激不是细胞毒性的原因。Atg5 siRNA和氯喹抑制PFAAs自噬甚至增加了PFAAs的毒性，提示PFAAs自噬是细胞器损伤的继发性效应，在细胞死亡过程中起保护作用。
Wide application of perfluoroalkyl acids (PFAAs) has raised great concerns on their side-effects on human health. PFAAs have been shown to accumulate mainly in the liver and cause hepatotoxicity. However, PFAAs can also deposit in lung tissues through air-borne particles and cause serious pulmonary toxicity. But the underlying mechanisms are still largely unknown. Autophagy is a type of programmed cell death parallel to necrosis and apoptosis, and may be involved in the lung toxicity of PFAAs. In this study, lung cancer cells, A549, were employed as the model to investigate the effects of three PFAAs with different carbon chain lengths on cell autophagy. Through Western blot analysis on LC3-I/II ratio of cells exposed to non-cytotoxic concentration (200 µM) and cytotoxic concentration (350 µM), we found concentration-dependent increase of autophagosomes in cells, which was further confirmed by TEM examination on ultra-thin section of cells and fluorescence imaging on autophagosomes in live cells. The abundance of p62 increased with the PFAAs concentration indicating the blockage of autophagy flux. Furthermore, we identified the mitochondrial autophagy (mitophagy) and endoplasmic reticulum autophagy (ER-phagy) morphologically as the major types of autophagy, suggesting the disruption on mitochondria and ERs. These organelle damages were confirmed by the overgeneration of ROS, hyperpolarization of mitochondrial membrane potential, as well as the up-regulation of ER-stress-related proteins, ATF4 and p-IRE1. Further analysis on the signaling pathways showed that PFAAs activated the MAPK pathways and inhibited the PI3K/Akt pathway, with potencies following the order of PFDA > PFNA > PFOA. Anti-oxidant (NAC) treatment did not rescue cells from death, indicating that oxidative stress is not the reason of cytotoxicity. Inhibition of autophagy by Atg5 siRNA and chloroquine even increased the toxicity of PFAAs, suggesting that PFAAs-autophagy was induced as the secondary effects of organelle damages and played a protective role during cell death.