Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, MS 1018, Kansas City, KS, 66160, USA.
Arch Toxicol. 2019 Jan;93(1):163-178. doi: 10.1007/s00204-018-2331-8. Epub 2018 Oct 15.
We previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300 mg/kg APAP followed by 20 mg/kg MT 1.5 h or 3 h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24 h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) 2 h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.
我们之前报道过，线粒体靶向超氧化物歧化酶模拟物 Mito-tempo（MT）延迟治疗可通过抑制过氧亚硝酸盐形成来保护对乙酰氨基酚（APAP）肝毒性的早期阶段。然而，这种保护是否持续到毒性的晚期阶段尚不清楚。为了研究晚期保护，用 300mg/kgAPAP 处理 C57Bl/6J 小鼠，然后在 1.5 或 3 小时后用 20mg/kgMT 处理。我们发现，两种 MT 处理均在 12 和 24 小时时保护免受 APAP 肝毒性的晚期影响。令人惊讶的是，MT 处理的小鼠表现出凋亡的肝细胞明显增加，而仅在用 APAP 处理的小鼠中观察到坏死表型。此外，MT 处理的小鼠肝脏中的半胱天冬酶-3 活性和切割明显增加。用活性半胱天冬酶-3 免疫染色显示，阳性染色的肝细胞仅存在于中央小叶区域。在用 pan-caspase 抑制剂 ZVD-fmk（10mg/kg）在 APAP 后 2 小时处理后，这种半胱天冬酶激活被中和，并为 APAP 肝毒性提供了额外的保护。用当前对 APAP 中毒的标准治疗 N-乙酰半胱氨酸治疗虽然提供了保护，但不会诱导这种凋亡表型。从机制上讲，MT 治疗抑制了 APAP 诱导的 RIP3 激酶表达，而 RIP3 缺陷型小鼠的肝细胞中半胱天冬酶激活和凋亡形态类似于 MT 治疗。这些数据表明，虽然坏死是 APAP 肝毒性后细胞死亡的主要原因，但抗氧化剂 MT 的治疗可能会使一些细胞的细胞死亡模式转变为继发性凋亡。线粒体氧化应激和 RIP3 激酶表达的调节在这种转变中起着关键作用。
