基于P62-Keap1-NRF2通路探讨铁死亡在慢性肝衰竭中的作用与机制研究

Study on the Role and Mechanism of Ferroptosis in Chronic Liver Failure Based on the P62-Keap1-NRF2 Pathway

摘要: 目的：探讨P62-Keap1-NRF2通路介导铁死亡在慢性肝衰竭中的作用，并观察铁死亡抑制剂Ferrostatin-1的干预效应。方法：体外实验采用BRL3A大鼠肝细胞，分为对照组、CCl₄模型组(2.5 μmol/L, 12 h)和Fer-1干预组(10 μmol/L)。CCK-8法筛选最佳造模条件，免疫荧光检测细胞内Fe²⁺和活性氧(ROS)，生化法检测丙二醛(MDA)，qPCR和Western blot检测P62、Keap1、NRF2的mRNA及蛋白表达。体内实验采用SD大鼠，随机分为对照组、CLF模型组(50% CCl₄橄榄油腹腔注射22周)和Fer-1干预组(造模后另使用尾静脉注射Fer-1共2周)。HE及Masson染色观察肝组织病理变化，qPCR和Western blot检测肝组织中上述指标。结果：CCK-8确定2.5 μmol/L CCl₄处理12 h为最佳造模条件。体外实验显示，与对照组相比，模型组细胞内Fe²⁺、ROS、MDA水平显著升高(均P < 0.05)，P62和NRF2的mRNA及蛋白表达降低，Keap1表达升高(均P < 0.05)；Fer-1干预可显著逆转上述变化(均P < 0.05)。体内实验显示，模型组大鼠肝组织出现典型假小叶、胶原沉积及坏死，P62和NRF2表达下降，Keap1表达升高；Fer-1干预可减轻病理损伤，恢复通路分子表达(均P < 0.05)。结论：铁死亡在CLF中发挥重要作用，P62-Keap1-NRF2通路是其关键调控轴，Fer-1可通过恢复该通路稳态逆转铁死亡效应，为CLF的靶向治疗提供了新思路。

Abstract: Objective: To investigate the role of the P62-Keap1-NRF2 pathway in mediating ferroptosis in chronic liver failure (CLF) and to observe the interventional effect of the ferroptosis inhibitor Ferrostatin-1 (Fer-1). Methods: For in vitro experiments, BRL3A rat hepatocytes were divided into control group, CCl₄ model group (2.5 μmol/L, 12 h) and Fer-1 intervention group (10 μmol/L). The optimal modeling conditions were determined by CCK-8 assay. Intracellular Fe²⁺ and reactive oxygen species (ROS) levels were detected by immunofluorescence, malondialdehyde (MDA) content was measured by biochemical assay, and the mRNA and protein expression levels of P62, Keap1, and NRF2 were detected by qPCR and Western blot, respectively. For in vivo experiments, SD rats were randomly divided into control group, CLF model group (intraperitoneal injection of 50% CCl₄ in olive oil for 22 weeks), and Fer-1 intervention group (tail vein injection of Fer-1 for an additional 2 weeks after modeling). Liver histopathological changes were observed by HE and Masson staining, and the above indicators in liver tissues were detected by qPCR and Western blot. Results CCK-8 assay determined that treatment with 2.5 μmol/L CCl₄ for 12 h was the optimal modeling condition. In vitro experiments showed that compared with the control group, the model group exhibited significantly elevated levels of intracellular Fe²⁺, ROS, and MDA (all P < 0.05), decreased mRNA and protein expression of P62 and NRF2, and increased expression of Keap1 (all P < 0.05). Fer-1 intervention significantly reversed these changes (all P < 0.05). In vivo experiments showed that the liver tissues of rats in the model group displayed typical pseudolobule formation, collagen deposition and necrosis, with decreased expression of P62 and NRF2 and increased expression of Keap1. Fer-1 intervention alleviated the pathological damage and restored the expression of pathway molecules (all P < 0.05). Conclusion: Ferroptosis plays an important role in CLF, and the P62-Keap1-NRF2 pathway is a key regulatory axis. Fer-1 can reverse the ferroptosis effect by restoring the homeostatic function of this pathway, providing a new strategy for targeted therapy of CLF.