目的 探究麝香酮改善顺铂诱导的急性肾损伤（acute kidney injury，AKI）的作用机制。方法 C57BL/6J小鼠随机分为对照组、模型组、地塞米松（2 mg/kg）组和麝香酮低、中、高剂量（24、36、48 mg/kg）组，建立顺铂诱导的AKI小鼠模型，给予药物干预后，采用苏木素-伊红（hematoxylin-eosin，HE）染色观察肾脏组织病理变化；免疫组化检测肾脏中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin，NGAL）和肾损伤分子-1（kidney injury molecule-1，Kim-1）的表达和分布；检测血尿素氮（blood urea nitrogen，BUN）和血肌酐（serum creatinine，SCr）水平；免疫荧光染色检测肾脏组织中剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved cystein-asparate protease-3，cleaved Caspase-3）的表达和分布。采用顺铂诱导建立NRK-52E细胞损伤模型，MTT法检测顺铂、麝香酮、p53抑制剂pifithrin-μ对细胞活力的影响；倒置显微镜观察细胞形态变化；Calcein AM/PI染色观察细胞死亡情况；Hoechest 33342染色观察细胞核碎裂情况；透射电子显微镜观察细胞死亡类型；免疫荧光染色检测p53的表达和分布；Western blotting检测Caspase-3、cleaved Caspase-3和p53的表达；MitoSOX染色检测线粒体活性氧（mitochondrial reactive oxygen species，mtROS）水平；免疫共沉淀检测p53泛素化修饰水平。结果 麝香酮显著减轻顺铂引起的肾脏病理损伤，恢复肾小管形态（P＜0.01），降低肾脏组织NGAL、Kim-1、cleaved Caspase-3的表达和分布（P＜0.01），降低BUN、SCr水平（P＜0.01）；麝香酮和pifithrin-μ显著抑制顺铂诱导的NRK-52E细胞凋亡及mtROS水平（P＜0.01），上调Caspase-3蛋白表达（P＜0.05、0.01），下调cleaved Caspase-3、p53蛋白表达（P＜0.05、0.01），促进p53泛素化修饰（P＜0.05）。结论 麝香酮通过促进p53泛素化修饰抑制肾小管上皮细胞凋亡，从而改善顺铂诱导的AKI。

Objective To explore the mechanism by which muscone improves cisplatin-induced acute kidney injury (AKI). Methods C57BL/6J mice were randomly divided into control group, model group, dexamethasone (2 mg/kg) group, muscone low-, medium-, and high-dose (24, 36, 48 mg/kg) groups, a cisplatin-induced AKI mouse model was established. After drug intervention, renal tissue pathological changes were observed using hematoxylin-eosin (HE) staining. Immunohistochemistry was used to detect the expressions and distribution of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (Kim-1) in kidney. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were detected. Immunofluorescence staining was used to detect the expression and distribution of cleaved cysteine aspartate protease-3 (cleaved Caspase-3) in renal tissue. An NRK-52E cell injury model induced by cisplatin was established, the effects of cisplatin, muscone and p53 inhibitor pifithrin-μ on cell viability were detected using MTT assay. The morphological change of cells was observed under an inverted microscope. Calcein AM/PI staining was used to observe cell death. Hoechest 33342 staining was used to observe the fragmentation of cell nuclei. Cell death types was observed using transmission electron microscopy. Immunofluorescence staining was used to detect the expression and distribution of p53. Western blotting was used to detect the expressions of Caspase-3, cleaved Caspase-3 and p53. MitoSOX staining was used to detect the levels of mitochondrial reactive oxygen species (mtROS). Immunoprecipitation was used to detect the level of p53 ubiquitination modification. Results Muscone significantly reduced the renal pathological damage caused by cisplatin, restored the morphology of renal tubules (P < 0.01), decreased the expressions and distribution of NGAL, Kim-1 and cleaved Caspase-3 in renal tissue (P < 0.01), reduced the levels of BUN and SCr (P < 0.01). Muscone and pifithrin-μ significantly inhibited cisplatin-induced apoptosis and mtROS level in NRK-52E cells (P < 0.01), upregulated Caspase-3 protein expression (P < 0.05, 0.01), downregulated cleaved Caspase-3 and p53 protein expressions (P < 0.05, 0.01), and promoted p53 ubiquitination modification (P < 0.05). Conclusion Muscone inhibits apoptosis of renal tubular epithelial cells by promoting p53 ubiquitination modification, thereby improving cisplatin-induced AKI.

R285.5

秦创原中医药产业创新聚集区项目（L2024-QCY-ZYYJJQ-202）；陕西省秦创原“科学家＋工程师”队伍建设项目（2023KXJ-128）