Drug-induced liver injury is the major cause of acute liver failure. However, the underlying mechanisms seem to be multifaceted and remain poorly understood, resulting in few effective therapies. Here, we report a novel mechanism that contributes to acetaminophen-induced hepatotoxicity through the induction of ferroptosis, a distinctive form of programmed cell death. We subsequently identified therapies protective against acetaminophen-induced liver damage and found that (+)-clausenamide ((+)-CLA), an active alkaloid isolated from the leaves of Clausena lansium (Lour.) Skeels, inhibited acetaminophen-induced hepatocyte ferroptosis both in vivo and in vitro. Consistently, (+)-CLA significantly alleviated acetaminophen-induced or erastin-induced hepatic pathological damages, hepatic dysfunctions and excessive production of lipid peroxidation both in cultured hepatic cell lines and mouse liver. Furthermore, treatment with (+)-CLA reduced the mRNA level of prostaglandin endoperoxide synthase 2 while it increased the protein level of glutathione peroxidase 4 in hepatocytes and mouse liver, confirming that the inhibition of ferroptosis contributes to the protective effect of (+)-CLA on drug-induced liver damage. We further revealed that (+)-CLA specifically reacted with the Cys-151 residue of Keap1, which blocked Nrf2 ubiquitylation and resulted in an increased Nrf2 stability, thereby leading to the activation of the Keap1–Nrf2 pathway to prevent drug-induced hepatocyte ferroptosis. Our studies illustrate the innovative mechanisms of acetaminophen-induced liver damage and present a novel intervention strategy to treat drug overdose by using (+)-CLA.

药物性肝损伤是急性肝功能衰竭的主要原因。然而，潜在的机制似乎是多方面的，并且仍然知之甚少，导致几乎没有有效的治疗方法。在这里，我们报告了一种通过诱导铁死亡（一种独特的程序性细胞死亡形式）导致对乙酰氨基酚引起的肝毒性的新机制。我们随后确定了防止对乙酰氨基酚引起的肝损伤的治疗方法，并发现 (+)-clausenamide ((+)-CLA)，一种从Clausena lansium的叶子中分离出来的活性生物碱(Lour.) Skeels 在体内和体外均抑制对乙酰氨基酚诱导的肝细胞铁死亡。一致地，(+)-CLA 在培养的肝细胞系和小鼠肝脏中显着减轻了对乙酰氨基酚诱导或埃拉汀诱导的肝脏病理损伤、肝功能障碍和脂质过氧化的过度产生。此外，用 (+)-CLA 处理降低了前列腺素内过氧化物合酶 2 的 mRNA 水平，同时增加了肝细胞和小鼠肝脏中谷胱甘肽过氧化物酶 4 的蛋白质水平，证实了铁死亡的抑制有助于 (+)- CLA 对药物性肝损伤的影响。我们进一步发现 (+)-CLA 与 Keap1 的 Cys-151 残基发生特异性反应，从而阻断 Nrf2 泛素化并导致 Nrf2 稳定性增加，从而导致 Keap1-Nrf2 通路的激活，以防止药物诱导的肝细胞铁死亡。我们的研究阐明了对乙酰氨基酚引起的肝损伤的创新机制，并提出了一种使用 (+)-CLA 治疗药物过量的新干预策略。