目的 探讨莫诺苷对顺铂诱导肝毒性的保护作用及其分子机制。方法 C57BL/6J小鼠ip顺铂诱导建立肝毒性模型，给予莫诺苷干预4周后，测定血清肝功能指标；苏木素-伊红（hematoxylin-eosin，HE）染色观察肝组织病理变化；普鲁士蓝染色检测肝组织内铁累积水平；ELISA法测定血清中炎症因子水平及肝脏丙二醛（malondialdehyde，MDA）和相关抗氧化酶活性；Western blotting和免疫组化法测定肝脏中核因子E2相关因子2（nuclear factor erythroid 2-related factor 2、Nrf2）和血红素加氧酶-1（heme oxygenase 1，HO-1）以及谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）等铁死亡相关蛋白表达。体外采用顺铂诱导建立AML12细胞损伤模型，给予莫诺苷、铁死亡抑制剂ferrostatin-1（Fer-1）和铁死亡诱导剂erastin诱导细胞，考察莫诺苷对AML12细胞铁死亡的影响。观察细胞形态，并用DCFH-DA荧光探针检测细胞内活性氧（reactive oxygen species，ROS）水平；采用JC-1染色检测细胞线粒体膜电位变化；采用吖啶橙/溴化乙啶（acridine orange/ethidium bromide，AO/EB）染色检测细胞死亡情况；采用免疫荧光法检测细胞GPX4和Nrf2蛋白的表达情况。结果 莫诺苷能够显著改善顺铂诱导的小鼠肝功能障碍，降低血清中丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）活性及总胆红素（total bilirubin，TBIL）水平（P＜0.05、0.01、0.001），降低肝脏中白细胞介素-1β（interleukin-1β，IL-1β）、IL-6、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平（P＜0.05、0.01、0.001），提高谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）等抗氧化酶的活性并降低MDA水平（P＜0.05、0.01、0.001）。莫诺苷显著改善了顺铂诱导的肝毒性模型小鼠的肝索排列紊乱和肝细胞质空泡化，恢复部分细胞发生的坏死，并减少炎症细胞的大量浸润。在分子机制水平，莫诺苷通过调控GPX4蛋白表达抑制铁死亡（P＜0.01），上调Nrf2和HO-1的表达减轻氧化应激损伤（P＜0.05、0.01、0.001），从而抑制顺铂诱导的肝损伤。体外实验结果与体内实验结果一致，莫诺苷显著缓解了顺铂诱导的AML12细胞氧化应激和铁死亡。结论 莫诺苷通过调控Nrf2/HO-1轴和上调GPX4蛋白表达来抑制肝脏发生氧化应激及铁死亡，从而减轻顺铂诱导的肝毒性。

Objective To investigate the protective effect and underlying molecular mechanisms of morroniside on cisplatin-induced hepatotoxicity. Methods The liver toxicity model of C57BL/6J mice was established by intraperitoneal injection of cisplatin. After four weeks of morroniside intervention treatment, the serum liver function indicators were measured. Hematoxylin-eosin (HE) stainin was used to observe liver histopathological changes; Iron accumulation in liver tissue was assessed by Prussian blue staining; Levels of inflammatory cytokine in serum were detected by ELISA; Malondialdehyde (MDA) content and activities of antioxidant enzymes in liver tissues were also determined. Western blotting and immunohistochemistry were used to detect the expressions of ferroptosis-related proteins including nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and glutathione peroxidase 4 (GPX4) in liver tissues. The AML12 cell injury model was established in vitro by cisplatin induction. Morroniside, ferroptosis inhibitor ferrostatin-1 (Fer-1), and ferroptosis inducer erastin were administered to induce cells, and the effect of morroniside on ferroptosis in AML12 cells was investigated. Cell morphology was observed; Intracellular reactive oxygen species (ROS) level was detected using DCFH-DA fluorescent probe; The changes of mitochondrial membrane potential were assessed using JC-1 staining; Cell death was evaluated using acridine orange/ethidium bromide (AO/EB) staining; Immunofluorescence staining was used to detect GPX4 and Nrf2 expressions. Results Morroniside significantly improved cisplatin-induced liver dysfunction in mice by reducing levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) in serum (P < 0.05, 0.01, 0.001), lowering levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in liver tissue (P < 0.05, 0.01, 0.001), enhancing the activities of antioxidant enzymes such as glutathione peroxidase (GSH-Px), and decreasing MDA level (P < 0.05, 0.01, 0.001). Morroniside also significantly improved the disordered arrangement of hepatic cords and vacuolation of hepatic cytoplasm in mice with cisplatin-induced hepatotoxicity, restored necrosis in some cells, and reduced the massive infiltration of inflammatory cells. At the molecular level, morroniside inhibited ferroptosis by regulating GPX4 expression (P < 0.01), and attenuated oxidative stress by up-regulating Nrf2 and HO-1 expressions (P < 0.05, 0.01, 0.001), thereby inhibiting cisplatin-induced liver injury. The results of in vitro experiments were consistent with those of in vivo experiments, morroniside significantly relieved cisplatin-induced oxidative stress and ferroptosis in AML12 cells. Conclusion Morroniside alleviates cisplatin-induced hepatotoxicity by suppressing oxidative stress and ferroptosis through the regulation of the Nrf2/HO-1 axis and up-regulation of GPX4 expression.

R285.5

浙江省中医药科技计划项目(2024ZF183);浙江省教育厅科研项目(Y202354033)