Department of Laboratory Center, Affiliated People's Hospital of Jiangsu University, Zhenjiang, China.
Department of General Surgery, Affiliated People's Hospital of Jiangsu University, Zhenjiang, China.
Free Radic Biol Med. 2020 Nov 20;160:871-886. doi: 10.1016/j.freeradbiomed.2020.09.015. Epub 2020 Sep 16.
Activation of nucleotide-binding domain leucine-rich repeat containing family pyrin domain containing 3 (NLRP3) inflammasome in Kupffer cells (KCs) contributes significantly to hepatic ischemia/reperfusion (I/R) injury, while the mechanism of how NLRP3 inflammasome is regulated remains less well defined. Recent evidence has showed that mitophagy acts as a central player for maintaining mitochondrial homeostatis through elimination of damaged mitochondria, leading to the prevention of hyperinflammation triggered by NLRP3 activation. In this study, we aimed at investigating the potential role of PTEN-induced kinase 1 (PINK1)-mediated mitophagy in hepatic I/R injury. C57BL/6 mice subjected to partial warm hepatic I/R or primary KCs exposed to anoxia/reoxygenation (A/R) was used as in vivo or in vitro model, respectively. Mitophagy was measured by protein levels of PINK1, Parkin, LC3B-II, TOMM20 and p62. NLRP3, caspase-1 and IL-1β at mRNA and/or protein levels were used as indicators of inflammasome activation. Our results demonstrated remarkable hepatic inflammation and NLRP3 inflammasome activation during hepatic I/R, along with increased PINK1-mediated mitophagy. Notably, overexpression of PINK1 in vivo attenuated hepatic I/R injury, ROS production, NLRP3 activation and hepatic inflammation. In parallel, A/R challenge in vitro also triggered NLRP3 activation in KCs accompanied by increase in mitophagy. Enhanced mitophagy mediated by PINK1 overexpression further inhibited NLRP3 activation and reversed the KC-mediated inflammatory injury to hepatocytes. Kinase-dead mutation of PINK1 completely abolished the above protective effects by PINK1. Blocking of mitophagy/autophagy by silencing of PINK1/Parkin, ATG5, NDP52 or OPTN showed the totally opposite effects, respectively. Treatment with different autophagic inhibitors also consistently reversed the PINK1-mediated effects, suggesting that an intact PINK1-mediated mitophagy signaling was crucial for ablation of NLRP3 signaling in the presence of A/R. Together, these results support a critical role of PINK1-mediated mitophagy in mitochondrial quality control for KC activation and function in hepatic I/R.
核苷酸结合域富含亮氨酸重复蛋白家族成员 N 端亮氨酸重复结构域包含蛋白 3（NLRP3）炎性小体在库普弗细胞（KCs）中的激活对肝脏缺血再灌注（I/R）损伤有重要贡献，而 NLRP3 炎性小体的调节机制仍不太明确。最近的证据表明，线粒体自噬通过消除受损的线粒体作为维持线粒体稳态的核心机制，从而防止 NLRP3 激活引发的过度炎症。在这项研究中，我们旨在研究 PTEN 诱导的激酶 1（PINK1）介导的线粒体自噬在肝脏 I/R 损伤中的潜在作用。使用 C57BL/6 小鼠进行部分温热性肝 I/R 或原代 KCs 进行缺氧/复氧（A/R）作为体内或体外模型。通过 PINK1、Parkin、LC3B-II、TOMM20 和 p62 的蛋白水平来衡量线粒体自噬。NLRP3、caspase-1 和 IL-1β 的 mRNA 和/或蛋白水平被用作炎性小体激活的指标。我们的结果表明，在肝脏 I/R 期间，肝脏炎症和 NLRP3 炎性小体激活明显增加，同时 PINK1 介导的线粒体自噬增加。值得注意的是，体内过表达 PINK1 减轻了肝脏 I/R 损伤、ROS 产生、NLRP3 激活和肝脏炎症。同时，体外 A/R 刺激也触发了 KCs 中的 NLRP3 激活，同时增加了线粒体自噬。PINK1 过表达增强的线粒体自噬进一步抑制 NLRP3 激活，并逆转了 KC 介导的对肝细胞的炎症损伤。PINK1 的激酶缺失突变完全消除了 PINK1 的上述保护作用。通过沉默 PINK1/Parkin、ATG5、NDP52 或 OPTN 阻断线粒体自噬/自噬，分别显示出完全相反的效果。用不同的自噬抑制剂处理也一致逆转了 PINK1 介导的作用，表明在 A/R 存在的情况下，完整的 PINK1 介导的线粒体自噬信号对于 NLRP3 信号的消融至关重要。综上所述，这些结果支持 PINK1 介导的线粒体自噬在 KC 激活和肝脏 I/R 中的功能中对线粒体质量控制的关键作用。
