自噬在APAP肝损伤及肝再生过程中的调控作用

Role of autophagy in the regulation of APAP liver injury and liver regeneration process

摘要

目的 探究自噬在对乙酰氨基酚(acetaminophen, APAP)诱导的小鼠正常肝细胞(AML-12)肝损伤和肝再生中的作用以及其可能机制。 方法 体外培养AML-12细胞,采用Western blotting法检测APAP作用于该细胞0、1、4、12、24、48、72h微管相关蛋白 1A/1B 轻链 3-II(microtubule-associated protein 1A/1B-light chain 3-II, LC3II)蛋白表达,筛选出APAP作用后出现明显肝损伤和肝再生的时间点。一方面使用自噬激动剂雷帕霉素(rapamycin, RAPA)作用于肝损伤点,分为对照组、RAPA组、APAP 4 h组和APAP 4 h + RAPA 组,采用Western blotting法检测各组细胞LC3II、自噬相关蛋白 5(autophagy related 5, ATG5)、螯合体1(sequestosome 1, p62)、受体相互作用蛋白激酶 1(receptor-interacting protein kinase 1, RIP1)、受体相互作用蛋白激酶 3(receptor-interacting protein kinase 3, RIP3)、谷胱甘肽过氧化物酶 4(glutathione peroxidase 4, GPX4)、溶质载体家族 7 成员 11(solute carrier family 7 member 11, SLC7A11)、p53蛋白水平; 采用RT-qPCR法检测各组细胞肿瘤坏死因子-α(tumor necrosis factor-alpha, TNF-α)、白细胞介素-6(interleukin-6, IL-6)、铁蛋白重链 1(ferritin heavy chain 1, FTH1)、核受体辅激活因子 4(nuclear receptor coactivator 4, NCOA4)mRNA水平;采用亚铁离子试剂盒检测Fe²⁺水平。另一方面使用自噬抑制剂氯喹(chloroquine, CQ)作用于肝再生点,分为对照组、CQ组、APAP 24 h组和APAP 24 h + CQ组,采用Western blotting法检测各组细胞LC3II、ATG5、p62、增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、细胞周期蛋白 D1(Cyclin D1)蛋白水平;采用RT-qPCR法检测各组细胞Ki-67、yes 相关蛋白 1(yes-associated protein 1, YAP1)、无翅型MMTV整合位点家族成员 2(wingless- type MMTV integration site family member 2, Wnt2)mRNA水平。 结果 损伤方面,与对照组相比,APAP 4 h组RIP1、RIP3、p53蛋白水平和TNF-α、IL-6、NCOA4 mRNA、Fe²⁺水平上调(P<0.05),LC3II、SLC7A11、GPX4蛋白水平和FTH1 mRNA水平下调(P<0.05);与APAP 4 h组相比,APAP 4 h + RAPA组LC3II、ATG5、SLC7A11、GPX4蛋白水平和FTH1 mRNA水平上调(P<0.05),p62、RIP1、RIP3、p53蛋白水平和TNF-α、IL-6、NCOA4 mRNA、Fe²⁺水平下调(P<0.05)。再生方面,与对照组相比,APAP 24 h组LC3II蛋白水平和Ki-67、Wnt2、YAP1 mRNA水平上调(P<0.05),p62、PCNA蛋白水平下调(P<0.05);与APAP 24 h组相比,APAP 24 h + CQ组LC3II、p62蛋白水平上调(P<0.05),ATG5、PCNA、Cyclin D1蛋白水平和Ki-67、Wnt2、YAP1 mRNA水平下调(P<0.05)。 结论 激活自噬可降低炎症反应、程序性坏死和铁死亡,最终减轻APAP肝损伤;而抑制自噬可能减轻APAP肝损伤后的肝再生过程。自噬激动剂有望成为治疗APAP肝损伤的新靶点。

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.

关键词

自噬 / 铁死亡 / 程序性坏死 / 肝损伤 / 肝再生

Key words

Autophagy / Ferroptosis / Programmed necrosis / Liver injury / Liver regeneration

参考文献

[1] Kumachev A, Wu PE. Drug-induced liver injury[J]. Can Med Assoc J, 2021, 193(9): E310. doi:10.1503/cmaj.202026

[2] Jaeschke H, Ramachandran A. Acetaminophen hepatoto-xicity: paradigm for understanding mechanisms of durg-induced liver injury[J]. Annu Rev Pathol, 2024, 19: 453-478. doi: 10.1146/annurev-pathmechdis-051122-094016

[3] Verzella D, Pescatore A, Capece D, et al. Life, death, and autophagy in cancer: NF-κB turns up everywhere[J]. Cell Death Dis, 2020, 11(3): 210. doi: 10.1038/s41419-020-2399-y

[4] Frietze KK, Brown AM, Das D, et al. Lipotoxicity reduces DDX58/Rig-1 expression and activity leading to impaired autophagy and cell death[J]. Autophagy, 2022, 18(1): 142-160.

[5] Erfan OS, Sonpol HMA, Abd El-kader M. Protective effect of rapamycin against acrylamide-induced hepatotoxicity: the associations between autophagy, apoptosis, and necroptosis[J]. Anat Rec(Hoboken), 2021, 304(9): 1984-1998.

[6] Liu J, Huang C, Liu J, et al. Nrf2 and its dependent autophagy activation cooperatively counteract ferroptosis to alleviate acute liver injury[J]. Pharmacol Res, 2023, 187: 106563. doi: 10.1016/j.phrs.2022.106563

[7] Zou G, Park JI. Wnt signaling in liver regeneration, di-sease, and cancer[J]. Clin Mol Hepatol, 2023, 29(1): 33-50.

[8] Fan S, Gao Y, Qu A, et al. YAP-TEAD mediates PPAR α-induced hepatomegaly and liver regeneration in mice[J]. Hepatology, 2022, 75(1): 74-88.

[9] 郁福乾, 宋莎莎, 路燕, 等. IL-6在对乙酰氨基酚肝损伤后再生中的作用[J]. 安徽医科大学学报, 2022, 57(6): 866-871.

[10] YU Fuqian, SONG Shasha, LU Yan, et al. Resarch of IL-6 in liver regeneration after APAP-induced acute liver injury[J]. Acta Universitatis Medicinalis Anhui, 2022, 57(6): 866-871.

[11] Yuan M, Sun Z, Manthari RK, et al. Arsenic-induced autophagy regulates apoptosis in AML-12 cells[J]. Toxicol In Vitro, 2021, 72: 105074. doi: 10.1016/j.tiv.2020.105074

[12] 费凡. A20通过自噬调控NLRP3炎性小体影响奶牛子宫内膜上皮细胞炎性反应的机制研究[D]. 扬州: 扬州大学, 2025.

[13] 王安全. CADM1调控骨关节炎软骨细胞合成和分解代谢的分子机制研究[D]. 合肥: 安徽医科大学, 2022.

[14] 李南燕. 苯并芘(BaP)通过ANT1-PARKIN通路影响孕早期卵巢线粒体自噬[D]. 重庆: 重庆医科大学, 2021.

[15] Björnsson HK, Björnsson ES. Drug-induced liver injury: pathogenesis, epidemiology, clinical features, and practical management[J]. Eur J Intern Med, 2022, 97: 26-31. doi: 10.1016/j.ejim.2021.10.035

[16] 李晓敏, 李俊, 徐艺珈, 等. OTUB1基因通过激活SLC7A11信号通路减轻对乙酰氨基酚诱导的急性肝损伤[J]. 江苏大学学报(医学版), 2025, 35(3): 217-229.

[17] LI Xiaomin, LI Jun, XU Yijia, et al. OTUB1 mitigates acetaminophen-induced acute liver injury by activating the SLC7A11 signaling pathway[J]. Journal of Jiangsu University(Medicine Edition), 2025, 35(3): 217-229.

[18] 徐红梅, 宋育林. TFEB-自噬途径在利福平所致肝损伤中的作用及其机制[J]. 安徽医科大学学报, 2025, 60(10): 1827-1832.

[19] XU Hongmei, SONG Yulin. Role of TFEB-autophagy pathway in rifampicin-induced liver injury and its mechanism[J]. Acta Universitatis Medicinalis Anhui, 2025, 60(10): 1827-1832.

[20] Meng NN, Xia LZ, Gong YQ, et al. Autophagy serves as a protective effect against inflammatory injury of oxidative stress in ARPE-19 cell[J]. Int J Ophthalmol, 2025, 18(1): 28-38.

[21] Wang T, Zhao J, Li QY, et al. Poria cocos-derived exosome-like nanovesicles alleviate metabolic dysfunction-associated fatty liver disease by promoting mitophagy and inhibiting NLRP3 inflammasome activation[J]. Int J Mol Sci, 2025, 26(5): 2253. doi: 10.3390/ijms26052253

[22] 沈海涛, 乔亚琴, 董萍, 等. Toll样受体4调控的程序性坏死和铁死亡对对乙酰氨基酚肝损伤的影响[J]. 山东大学学报(医学版), 2024, 62(4): 1-8.

[23] SHEN Haitao, QIAO Yaqin, DONG Ping, et al. Effects of programmed necrosis and ferroptosis regulated by toll-like receptor 4 on acetaminophen-induced liver injury[J]. Journal of Shandong University(Health Sciences), 2024, 62(4): 1-8.

[24] Wang H, Feng X, He H, et al. Crosstalk between autophagy and other forms of programmed cell death[J]. Eur J Pharmacol, 2025, 995: 177414. doi: 10.1016/j.ejphar.2025.177414

[25] Liu N, Liu S, Zhang X, et al. Zinc finger domain of p62/SQSTM1 is involved in the necroptosis of human cisplatin-resistant ovarian cancer cells treated with sulfasalazine[J]. Oncol Lett, 2024, 28(5): 529. doi: 10.3892/ol.2024.14662

[26] Chen Y, Liu M, Wei H, et al. Alcohol induces hepatocytes necroptosis through the LC3/RIPK1/RIPK3 pathway[J]. Food Chem Toxicol, 2023, 182: 114124. doi: 10.1016/j.fct.2023.114124

[27] Chen F, Cai X, Kang R, et al. Autophagy-dependent ferroptosis in cancer[J]. Antioxid Redox Signal, 2023, 39(1-3): 79-101.

[28] Wang H, Guo M, Wei H, et al. Targeting p53 pathways: mechanisms, structures, and advances in therapy[J]. Signal Transduct Target Ther, 2023, 8(1): 92. doi: 10.1038/s41392-023-01347-1

[29] Lin Q, Li S, Jin H, et al. Mitophagy alleviates cisplatin-induced renal tubular epithelial cell ferroptosis through ROS/HO-1/GPX4 axis[J]. Int J Biol Sci, 2023, 19(4): 1192-1210.

[30] Zhang H, Li H, Liu S, et al. Ferroptosis mediated by ferritinophagy is involved in liver injury caused by sepsis[J]. Sci Rep, 2025. doi: 10.1038/s41598-025-29557-3

[31] 张鹏, 陈昆, 宋登辉, 等. 麦冬皂苷介导Wnt/β-catenin通路对大鼠肝部分切除后肝再生的影响[J]. 中华实用诊断与治疗杂志, 2024, 38(6): 552-556.

[32] ZHANG Peng, CHEN Kun, SONG Denghui, et al. Effect of ophiopogonin on liver regeneration in rats after partial hepatectomy via regulating Wnt/β-catenin signaling pathway[J]. Journal of Chinese Practical Diagnosis and Therapy, 2024, 38(6): 552-556.

[33] 郑鑫, 纵加强, 王佳新, 等. 基于Wnt/β-catenin信号通路探讨羟氯喹对烟雾吸入致肺纤维化大鼠的作用机制[J]. 解放军医学院学报, 2023, 44(12): 1391-1398.

[34] ZHENG Xin, ZONG Jiaqiang, WANG Jiaxin, et al. Mechanism of hydroxychloroquine's effect on pulmonary fibrosis after smoke inhalation in rats via Wnt/β-catenin signaling pathway[J]. Academic Journal of Chinese PLA Medical School, 2023, 44(12): 1391-1398.

[35] Ye S, Zhang Y, Wang X, et al. Autophagy positively regulates Wnt signaling in mice with diabetic retinopathy[J]. Exp Ther Med, 2021, 22(4): 1164. doi: 10.3892/etm.2021.10598

[36] Astone M, Tesoriero C, Schiavone M, et al. Wnt/β-Catenin signaling regulates Yap/Taz activity during embryonic development in zebrafish[J]. Int J Mol Sci, 2024, 25(18). doi: 10.3390/ijms251810005

[37] Zhao Y, Zhang F, Zhang X, et al. Transcriptomic ana-lysis of hepatocytes reveals the association between ubi-quitin-specific peptidase 1 and yes-associated protein 1 during liver regeneration[J]. Regen Ther, 2023, 24: 256-266. doi: 10.1016/j.reth.2023.07.004

基金资助

国家自然科学基金(81800524)