关键词: 自噬, 铁死亡, 程序性坏死, 肝损伤, 肝再生

Abstract: Objective To investigate the role of autophagy in acetaminophen(APAP)-induced liver injury and regene-ration in normal mouse hepatocytes(AML-12)and its potential mechanisms. Methods AML-12 cells were cultured in vitro, and Western blotting was employed to detect the protein expression of microtubule-associated protein 1A/1B light chain 3-II(LC3II)at 0, 1, 4, 12, 24, 48, and 72 hours after APAP treatment. This approach enabled identification of the time points corresponding to significant liver injury and liver regeneration following APAP administration. On one hand, the autophagy agonist rapamycin(RAPA)was administered at the point of hepatic injury, which was divided into control group, RAPA group, APAP 4 h group and APAP 4 h + RAPA group. Western blotting was used to detect protein expressions of LC3II, autophagy-related protein 5(ATG5), sequestosome 1(p62), receptor-interacting protein kinase 1(RIP1), receptor-interacting protein kinase 3(RIP3), glutathione peroxidase 4(GPX4), solute carrier family 7 member 11(SLC7A11)and p53; RT-qPCR was used to detect mRNA levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), ferritin heavy chain 1(FTH1), and nuclear receptor coactivator 4(NCOA4); the Fe²⁺ levels were detected using a ferrous ion assay kit. On the other hand, the autophagy inhibitor chloroquine(CQ)was administered at the point of liver regeneration, with the groups divided into control group, CQ group, APAP 24 h group, and APAP 24 h + CQ group. Western blotting was utilized to detect the protein expressions of LC3II, ATG5, p62, proliferating cell nuclear antigen(PCNA), and Cyclin D1; RT-qPCR was employed to detect the mRNA expressions of Ki-67, yes-associated protein 1(YAP1), and wingless-type MMTV integration site family member 2(Wnt2). Results In terms of injury, compared with the control group, the APAP 4 h group exhibited increased expressions of RIP1, RIP3, and p53 proteins, as well as TNF-α, IL-6, NCOA4 mRNA, and Fe²⁺(P<0.05), conversely, expressions of LC3II, SLC7A11, and GPX4 proteins, along with FTH1 mRNA, were downregulated(P<0.05); compared with the APAP 4 h group, the APAP 4 h + RAPA group showed increased expressions of LC3II, ATG5, SLC7A11, and GPX4 proteins, as well as FTH1 mRNA(P<0.05), conversely, p62, RIP1, RIP3, and p53 protein expressions, as well as TNF-α, IL-6, NCOA4 mRNA, and Fe²⁺ levels, were downregulated(P<0.05). In terms of regenerations, compared with the control group, the APAP 24 h group exhibited increased expression of LC3II protein, as well as Ki-67, Wnt2, and YAP1 mRNA(P<0.05), while p62 and PCNA proteins expressions were decreased(P<0.05); compared with the APAP 24 h group, the APAP 24 h + CQ group showed increased expressions of LC3II and p62 proteins(P<0.05), while the expressions of ATG5, PCNA, and Cyclin D1 proteins, as well as Ki-67, Wnt2, and YAP1 mRNA, were decreased(P<0.05). Conclusion Activating autophagy reduces inflammatory responses, programmed necrosis, and ferroptosis, ultimately mitigating APAP-induced liver injury; conversely, inhibiting autophagy may impair the liver regeneration process following APAP-induced liver injury. Autophagy agonists hold promise as novel therapeutic targets for treating APAP-induced liver injury.

Key words: Autophagy, Ferroptosis, Programmed necrosis, Liver injury, Liver regeneration