Department of Toxicology, School of Public Health, Anhui Medical University, Hefei City, Anhui Province, 230032, China; Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, 66160, USA.
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS, 66160, USA.
Redox Biol. 2019 Apr;22:101148. doi: 10.1016/j.redox.2019.101148. Epub 2019 Feb 20.
Mitochondria damage plays a critical role in acetaminophen (APAP)-induced necrosis and liver injury. Cells can adapt and protect themselves by removing damaged mitochondria via mitophagy. PINK1-Parkin pathway is one of the major pathways that regulate mitophagy but its role in APAP-induced liver injury is still elusive. We investigated the role of PINK1-Parkin pathway in hepatocyte mitophagy in APAP-induced liver injury in mice. Wild-type (WT), PINK1 knockout (KO), Parkin KO, and PINK1 and Parkin double KO (DKO) mice were treated with APAP for different time points. Liver injury was determined by measuring serum alanine aminotransferase (ALT) activity, H&E staining as well as TUNEL staining of liver tissues. Tandem fluorescent-tagged inner mitochondrial membrane protein Cox8 (Cox8-GFP-mCherry) can be used to monitor mitophagy based on different pH stability of GFP and mCherry fluorescent proteins. We overexpressed Cox8-GFP-mCherry in mouse livers via tail vein injection of an adenovirus Cox8-GFP-mCherry. Mitophagy was assessed by confocal microscopy for Cox8-GFP-mCherry puncta, electron microscopy (EM) analysis for mitophagosomes and western blot analysis for mitochondrial proteins. Parkin KO and PINK1 KO mice improved the survival after treatment with APAP although the serum levels of ALT were not significantly different among PINK1 KO, Parkin KO and WT mice. We only found mild defects of mitophagy in PINK1 KO or Parkin KO mice after APAP, and improved survival in PINK1 KO and Parkin KO mice could be due to other functions of PINK1 and Parkin independent of mitophagy. In contrast, APAP-induced mitophagy was significantly impaired in PINK1-Parkin DKO mice. PINK1-Parkin DKO mice had further elevated serum levels of ALT and increased mortality after APAP administration. In conclusion, our results demonstrated that PINK1-Parkin signaling pathway plays a critical role in APAP-induced mitophagy and liver injury.
线粒体损伤在乙酰氨基酚（APAP）诱导的坏死和肝损伤中起着关键作用。细胞可以通过自噬作用清除受损的线粒体来适应和保护自己。PINK1-Parkin 途径是调节自噬的主要途径之一，但它在 APAP 诱导的肝损伤中的作用仍不清楚。我们研究了 PINK1-Parkin 途径在 APAP 诱导的肝损伤中肝细胞自噬中的作用。野生型（WT）、PINK1 敲除（KO）、Parkin KO、PINK1 和 Parkin 双敲除（DKO）小鼠分别用 APAP 处理不同时间点。通过测量血清丙氨酸氨基转移酶（ALT）活性、肝组织 H&E 染色和 TUNEL 染色来确定肝损伤。串联荧光标记的线粒体内膜蛋白 Cox8（Cox8-GFP-mCherry）可用于监测基于 GFP 和 mCherry 荧光蛋白不同 pH 稳定性的自噬。我们通过尾静脉注射腺病毒 Cox8-GFP-mCherry 在小鼠肝脏中转染 Cox8-GFP-mCherry。通过共聚焦显微镜评估 Cox8-GFP-mCherry 斑点、电镜（EM）分析自噬体和 Western blot 分析线粒体蛋白来评估自噬。Parkin KO 和 PINK1 KO 小鼠在用 APAP 治疗后提高了生存率，尽管 PINK1 KO、Parkin KO 和 WT 小鼠之间血清 ALT 水平没有显著差异。我们只发现 PINK1 KO 或 Parkin KO 小鼠在用 APAP 后自噬的轻微缺陷，而 PINK1 KO 和 Parkin KO 小鼠的生存改善可能是由于 PINK1 和 Parkin 的其他功能与自噬无关。相比之下，PINK1-Parkin DKO 小鼠的 APAP 诱导的自噬明显受损。PINK1-Parkin DKO 小鼠在用 APAP 给药后血清 ALT 水平进一步升高，死亡率增加。总之，我们的结果表明，PINK1-Parkin 信号通路在 APAP 诱导的自噬和肝损伤中起着关键作用。
