目的 探讨金丝桃苷对脓毒症相关急性肾损伤（sepsis-associated acute kidney injury，SAKI）小鼠的干预作用及相关机制。方法 采用盲肠结扎穿孔术（cecal ligation and puncture，CLP）建立SAKI小鼠模型，设置假手术组、模型组、地塞米松（1 mg/kg）组及金丝桃苷高、低剂量（50、25 mg/kg）组和金丝桃苷（50 mg/kg）＋核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）抑制剂ML385（30 mg/kg）组。给予药物干预7 d后，绘制小鼠生存曲线，检测肾功能、炎症因子和氧化应激水平；采用苏木素-伊红（hematoxylin-eosin，HE）染色观察肾组织病理变化；采用TUNEL染色检测肾组织细胞凋亡情况；免疫组化检测肾组织Nrf2的表达；Western blotting检测肾组织Nrf2、PTEN诱导激酶1（PTEN induced kinase 1，Pink1）、Parkin蛋白表达；免疫荧光分析肾组织微管相关蛋白1轻链3B（microtubule-associated protein 1 light chain 3B，LC3B）和线粒体外膜转运酶20同源物（translocase of the outer mitochondrial membrane 20，TOMM20）的共定位情况。体外采用脂多糖（lipopolysaccharide，LPS）诱导人肾小管上皮细胞（human kidney-2，HK-2）损伤，给予金丝桃苷干预后，检测细胞活力、凋亡和活性氧水平；透射电镜观察线粒体超微结构；流式细胞仪检测线粒体膜电位（mitochondrial membrane potential，MMP）和三磷酸腺苷（adenosine triphosphate，ATP）水平以评估线粒体功能；Western blotting检测Nrf2、血红素加氧酶-1（heme oxygenase-1，HO-1）以及线粒体自噬蛋白Pink1、Parkin、p62的表达。结果 与模型组比较，金丝桃苷可显著提高SAKI小鼠存活率（P＜0.05），减轻体质量下降和肾脏病理损伤（P＜0.05、0.01），改善肾功能和氧化应激反应（P＜0.05、0.01），抑制炎症因子水平（P＜0.05、0.01），上调肾组织Nrf2、Pink1、Parkin的表达（P＜0.01），增加肾组织LC3B与TOMM20的共定位。体外实验结果显示，金丝桃苷（40 μmol/L）可抑制LPS诱导的HK-2细胞凋亡和氧化应激（P＜0.01），提高MMP及ATP水平（P＜0.01），上调Nrf2、HO-1、Pink1、Parkin蛋白表达（P＜0.01），下调p62的表达（P＜0.01），促进线粒体自噬，缓解线粒体损伤。此外，Nrf2抑制剂ML385显著逆转金丝桃苷对CLP诱导的SAKI小鼠的保护作用（P＜0.05、0.01），同时减弱了金丝桃苷诱导的HK-2细胞线粒体自噬。结论 金丝桃苷可能通过激活Nrf2/HO-1信号通路调控Pink1/Parkin介导的线粒体自噬，缓解线粒体功能障碍，减轻SAKI小鼠的氧化应激与炎症反应，从而发挥肾脏保护作用。

ObjectiveTo investigate the intervention effect and related mechanism of hyperoside on sepsis-associated acute kidney injury (SAKI) in mice. Methods A mouse model of SAKI was established by cecal ligation and puncture (CLP). Sham group, model group, dexamethasone (1 mg/kg) group, hyperoside high-, low-dose (50, 25 mg/kg) groups and hyperoside (50 mg/kg) + nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor ML385 (30 mg/kg) group were set up. After 7 d of drug intervention, the survival curve of mice was plotted. The levels of renal function, inflammatory factors and oxidative stress were detected. The pathological changes of renal tissue were observed by hematoxylin-eosin (HE) staining. TUNEL staining was used to detect apoptosis in renal tissue cells. Immunohistochemical was used to detect Nrf2 expression in renal tissue. Western blotting was used to detect the expressions of Nrf2, PTEN induced kinase 1 (Pink1) and Parkin proteins in renal tissue. Immunofluorescence was used to detect the co-localization of microtubule associated protein 1 light chain 3B (LC3B) and translocase of the outer mitochondrial membrane 20 (TOMM20) in renal tissue. Lipopolysaccharide (LPS) was used in vitro to induce injury in human tubular epithelial cells (HK-2). After intervention with hyperoside, cell viability, apoptosis and reactive oxygen species levels were detected. Mitochondrial ultrastructure was observed by transmission electron microscopy. Flow cytometry was used to detect mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels to evaluate mitochondrial function. Western blotting was used to detect the expressions of Nrf2, heme oxygenase-1 (HO-1) and mitochondrial autophagy proteins Pink1, Parkin, p62. Results Compared with model group, hyperoside could significantly improve the survival rate of SAKI mice (P < 0.05), alleviate weight loss and renal pathological damage (P < 0.05, 0.01), improve renal function and oxidative stress response (P < 0.05, 0.01), inhibit inflammatory factor levels (P < 0.05, 0.01), up-regulate the expressions of Nrf2, Pink1, Parkin in renal tissue (P < 0.01), and increase the co-ocalization of LC3B and TOMM20 in renal tissue. In vitro experimental results showed that hyperoside (40 μmol/L) could inhibit LPS-induced apoptosis and oxidative stress in HK-2 cells (P < 0.01), increase MMP and ATP levels (P < 0.01), up-regulate Nrf2, HO-1, Pink1, Parkin protein expressions (P < 0.01), down-regulate p62 expression (P < 0.01), promote mitochondrial autophagy, and alleviate mitochondrial damage. In addition, Nrf2 inhibitor ML385 significantly reversed the protective effect of hyperoside on CLP-induced SAKI mice (P < 0.05, 0.01), while weakening hyperoside induced mitochondrial autophagy in HK-2 cells.Conclusion Hypericin may regulate Pink1/Parkin mediated mitochondrial autophagy by activating Nrf2/HO-1 signaling pathway, alleviate mitochondrial dysfunction, alleviate oxidative stress and inflammatory response in SAKI mice, and thus exert renal protective effects.

R285.5

国家自然科学基金面上项目（81873055）;南京中医药大学自然科学基金项目（XZR2023058）;南京市卫生科技发展专项（YKK20125）;江苏省中医药科技发展计划重点项目（ZD202415）