Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston, SC, United States of America; Departments of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC, United States of America.
Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston, SC, United States of America; Departments of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, Charleston, SC, United States of America; Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, United States of America.
Toxicol Appl Pharmacol. 2023 Nov 15;479:116722. doi: 10.1016/j.taap.2023.116722. Epub 2023 Oct 15.
Acetaminophen (APAP) overdose disrupts hepatocellular lysosomes, which release ferrous iron (Fe) that translocates into mitochondria putatively via the mitochondrial calcium uniporter (MCU) to induce oxidative/nitrative stress, the mitochondrial permeability transition (MPT), and hepatotoxicity. To investigate how MCU deficiency affects mitochondrial Fe uptake and hepatotoxicity after APAP overdose, global MCU knockout (KO), hepatocyte specific (hs) MCU KO, and wildtype (WT) mice were treated with an overdose of APAP both in vivo and in vitro. Compared to strain-specific WT mice, serum ALT decreased by 88 and 56%, respectively, in global and hsMCU KO mice at 24 h after APAP (300 mg/kg). Hepatic necrosis also decreased by 84 and 56%. By contrast, when MCU was knocked out in Kupffer cells, ALT release and necrosis were unchanged after overdose APAP. Intravital multiphoton microscopy confirmed loss of viability and mitochondrial depolarization in pericentral hepatocytes of WT mice, which was decreased in MCU KO mice. CYP2E1 expression, hepatic APAP-protein adduct formation, and JNK activation revealed that APAP metabolism was equivalent between WT and MCU KO mice. In cultured hepatocytes after APAP, loss of cell viability decreased in hsMCU KO compared to WT hepatocytes. Using fructose plus glycine to prevent cell killing, mitochondrial Fe increased progressively after APAP, as revealed with mitoferrofluor (MFF), a mitochondrial Fe indicator. By contrast in hsMCU KO hepatocytes, mitochondrial Fe uptake after APAP was suppressed. Rhod-2 measurements showed that Ca did not increase in mitochondria after APAP in either WT or KO hepatocytes. In conclusion, MCU mediates uptake of Fe into mitochondria after APAP and plays a central role in mitochondrial depolarization and cell death during APAP-induced hepatotoxicity.
对乙酰氨基酚（APAP）过量会破坏肝细胞溶酶体，溶酶体释放二价铁（Fe），通过线粒体钙单向转运体（MCU）易位进入线粒体，从而诱导氧化/硝化应激、线粒体通透性转换（MPT）和肝毒性。为了研究 MCU 缺乏如何影响 APAP 过量后线粒体铁摄取和肝毒性，我们在体内和体外使用全局 MCU 敲除（KO）、肝细胞特异性（hs）MCU KO 和野生型（WT）小鼠进行 APAP 过量处理。与特定于品系的 WT 小鼠相比，在 APAP（300mg/kg）后 24 小时，全局和 hsMCU KO 小鼠的血清 ALT 分别降低了 88%和 56%。肝坏死也分别减少了 84%和 56%。相比之下，当 Kupffer 细胞中的 MCU 被敲除时，APAP 过量后 ALT 释放和坏死没有变化。活体多光子显微镜证实 WT 小鼠中央区肝细胞的活力丧失和线粒体去极化，而在 MCU KO 小鼠中则减少。CYP2E1 表达、肝 APAP-蛋白加合物形成和 JNK 激活表明 WT 和 MCU KO 小鼠的 APAP 代谢相当。在 APAP 后培养的肝细胞中，hsMCU KO 肝细胞的细胞活力丧失比 WT 肝细胞减少。使用果糖加甘氨酸防止细胞杀伤，在用线粒体铁指示剂 mitoferrofluor（MFF）处理后，APAP 后线粒体铁逐渐增加。相比之下，在 hsMCU KO 肝细胞中，APAP 后线粒体铁摄取受到抑制。Rhod-2 测量显示，WT 或 KO 肝细胞中的线粒体在 APAP 后 Ca 没有增加。总之，MCU 介导 APAP 后线粒体中铁的摄取，并在 APAP 诱导的肝毒性中发挥核心作用，导致线粒体去极化和细胞死亡。
