目的 探讨毛蕊花糖苷对缺血再灌注损伤（IRI）诱导急性肾损伤小鼠的保护作用及机制。方法 （1）体内 实验：将 56 只 C57BL/6J 雄性小鼠随机分为假手术组、假手术对照组（120 mg·kg-1毛蕊花糖苷）、模型组、铁死 亡抑制剂组（5 mg·kg-1 Fer-1）及毛蕊花糖苷低、中、高剂量组（30、60、120 mg·kg-1 ），每组 8 只。其中，假手 术对照组及毛蕊花糖苷低、中、高剂量组在肾脏 IRI 手术前 3 d 以及手术前 1 h 分别腹腔注射对应剂量的毛蕊 花糖苷；铁死亡抑制剂组仅在肾脏 IRI 手术前 1 h 腹腔注射 5 mg·kg-1 Fer-1 溶液；假手术组、模型组腹腔注射 等体积生理盐水。采用双侧肾蒂夹闭法诱导缺血 45 min 后再灌注 24 h，复制肾脏 IRI 小鼠模型。检测小鼠血 清肌酐（SCr）、尿素氮（BUN）及肾脏组织丙二醛（MDA）、谷胱甘肽（GSH）、超氧化物歧化酶（SOD）及铁离子水 平；采用 HE 染色法检测小鼠肾脏组织病理变化；RT-qPCR 法检测肾脏组织肾损伤分子 1（KIM-1）、中性粒细 胞明胶酶相关脂质运载蛋白（NGAL）、谷胱甘肽过氧化酶 4（GPX4）、铁转蛋白（CD71）mRNA 表达水平； Western Blot 法检测肾脏组织中 GPX4、xCT 蛋白表达水平。（2）体外实验：以不同浓度 Erastin（0.5、1.25、2.5、 5、10、20 µmol·L-1 ）诱导小鼠肾小管上皮细胞（mRTECs）后，采用 CCK-8 法检测细胞活性，采用 Western Blot 法检测细胞铁死亡相关蛋白 GPX4、胱氨酸/谷氨酸逆向转运蛋白（xCT）表达水平，筛选出 Erastin 诱导细胞铁死 亡的最佳浓度。采用 CCK-8 法检测不同浓度毛蕊花糖苷（0、2.5、5、10、20、40、80 µmol·L-1 ）对 mRTECs 细 胞的毒性，筛选出毛蕊花糖苷最佳干预浓度。采用 2.5 µmol·L-1 Erastin 诱导 mRTECs，并以 2.5、5、10 µmol·L-1 毛蕊花糖苷以及 5 µmol·L-1 铁死亡抑制剂 Fer-1 干预，共孵育 24 h 后采用 CCK-8 法检测细胞活力，采用 Western Blot 法检测细胞中核 Nrf2、HO-1 蛋白表达水平。结果 （1）体内实验：与假手术组比较，模型组小鼠 的肾脏指数与 SCr、BUN 水平，以及肾脏组织 KIM-1、NGAL mRNA 表达水平均显著升高（P＜0.01）；肾小管 刷状缘大部分脱落，肾小管扩张严重，可见蛋白管型和坏死组织，伴有炎性细胞浸润；肾脏组织 MDA 水平显 著升高（P＜0.01），GSH、SOD 水平均显著降低（P＜0.05，P＜0.01），铁离子水平及 CD71 mRNA 表达水平均显 著升高（P＜0.05，P＜0.01），GPX4 mRNA 及 GPX4、xCT 蛋白表达水平均显著降低（P＜0.05，P＜0.01）。与模 型组比较，各给药组小鼠的 SCr 水平明显降低（P＜0.05），毛蕊花糖苷中、高剂量组的肾脏指数显著降低（P＜ 0.01），低、中剂量组的 BUN 水平及肾脏组织 NGAL mRNA 表达水平均显著降低（P＜0.05，P＜0.01），低、高 剂量组的肾脏组织 KIM-1 mRNA 表达水平显著降低（P＜0.05，P＜0.01）；各给药组小鼠肾小管上皮细胞排列 整齐，坏死组织、蛋白管型和炎性细胞浸润减少，肾脏组织 MDA 水平显著降低（P＜0.05，P＜0.01），GSH、 SOD 水平均显著升高（P＜0.01）；毛蕊花糖苷高剂量组小鼠的肾脏组织铁离子水平显著降低（P＜0.01），GPX4 蛋白表达水平明显升高（P＜0.05）；毛蕊花糖苷各剂量组小鼠的肾脏组织 GPX4 mRNA 及 xCT 蛋白表达水平均 显著升高（P＜0.01），CD71 mRNA 表达水平显著降低（P＜0.01）。（2）体外实验：以 2.5 µmol·L-1 Erastin 作为最 佳诱导浓度，以 2.5、5、10 µmol·L-1 毛蕊花糖苷作为最佳干预浓度。与空白对照组比较，Erastin 模型组 mRTECs 的细胞活力及核 Nrf2、HO-1、GPX4 蛋白表达水平均显著下降（P＜0.01）。与 Erastin 模型组比较，各 给药组 mRTECs 的细胞活力及核 Nrf2、GPX4 蛋白表达水平均显著升高（P＜0.01），5、10 µmol·L-1毛蕊花糖苷 组 mRTECs 的 HO-1 蛋白表达水平显著升高（P＜0.01）。结论 毛蕊花糖苷可以有效改善 IRI 诱导的急性肾损伤小鼠的肾脏组织病理损伤，其机制可能与激活 Nrf2/HO-1 通路抑制铁死亡相关。

Objective To investigate the protective effect and mechanism of acteoside on acute kidney injury （AKI） induced by ischemia-reperfusion injury （IRI） in mice. Methods （1） In vivo experiments：56 male C57BL/6J mice were randomly divided into sham-operated group， sham-operated control group （120 mg·kg-1 acteoside）， model group，ferroptosis inhibitor group （5 mg·kg-1 Fer-1），and low-，medium-，and high-dose acteoside groups （30， 60， 120 mg·kg-1 ）， with 8 mice per group. The sham-operated control group and acteoside groups received intraperitoneal injections of acteoside 3 days before and 1 hour before IRI surgery. The ferroptosis inhibitor group received 5 mg·kg-1 Fer-1 solution one hour before surgery， while the sham-operated group and the model group received an equal volume of saline. Renal IRI mouse model was replicated by bilateral renal pedicle clamping for 45 minutes followed by 24 hours reperfusion. Serum creatinine （SCr）， blood urea nitrogen （BUN）， renal tissue malondialdehyde （MDA），glutathione （GSH），superoxide dismutase （SOD），and iron levels were measured. Renal histopathology was assessed by HE staining. RT-qPCR was used to detect mRNA expression of kidney injury molecule-1 （KIM-1），neutrophil gelatinase-associated lipocalin （NGAL），glutathione peroxidase 4 （GPX4），and transferrin receptor （CD71）. Western Blot was performed to analyze protein expression of GPX4 and xCT. （2） In vitro experiments： Mouse renal tubular epithelial cells （mRTECs） were treated with varying concentrations of Erastin （0.5， 1.25， 2.5， 5， 10， 20 µmol·L-1 ）. Cell viability was assessed using the CCK-8 assay，while Western Blot was performed to detect expression levels of ferroptosis-related proteins （GPX4 and the cystine/glutamate antiporter xCT） to determine the optimal Erastin concentration for inducing ferroptosis. Subsequently， the cytotoxicity of acteoside at different concentrations （0，2.5，5，10，20，40，80 µmol·L-1 ） on mRTECs was evaluated using the CCK-8 assay to identify the optimal intervention concentration. mRTECs were then treated with 2.5 µmol·L-1 Erastin and co-incubated with acteoside （2.5，5，10 µmol·L-1 ） or the ferroptosis inhibitor Fer-1 （5 µmol·L-1 ） for 24 hours. Following incubation， cell viability was measured using the CCK-8 assay， and nuclear protein expression levels of Nrf2 and HO-1 were determined by Western Blot. Results （1） In vivo experiments：Compared with the sham-operated group， the model group showed significantly increased kidney index， SCr and BUN levels， and mRNA expression levels of renal tissue KIM-1 and NGAL （P＜0.01）. The renal tubular brush border was mostly denuded with severe tubular dilation，visible protein casts and necrotic tissue，accompanied by inflammatory cell infiltration. Renal tissue MDA levels were significantly increased （P＜0.01），while GSH and SOD levels were significantly decreased （P＜ 0.05 and P＜0.01） . Iron levels and mRNA expression of CD71 were both significantly elevated （P＜0.05 and P＜ 0.01）， whereas mRNA expression of GPX4 and protein expression of GPX4 and xCT were all significantly reduced （P＜0.05 and P＜0.01） . Compared with the model group，all treatment groups showed significantly decreased SCr levels （P＜0.05）. The medium- and high-dose acteoside groups exhibited significantly reduced kidney index （P＜ 0.01）. The low- and medium-dose groups showed significantly lower BUN levels and mRNA expression levels of renal tissue NGAL （P＜0.05 and P＜0.01）. The low- and high-dose groups demonstrated significantly reduced mRNA expression level of renal tissue KIM-1 （P＜0.05 and P＜0.01） . In all treatment groups，renal tubular epithelial cells were arranged orderly with reduced necrotic tissue，protein casts and inflammatory cell infiltration. Renal tissue MDA levels were significantly decreased （P＜0.05 and P＜0.01），while GSH and SOD levels were significantly increased （P＜0.01）. The high-dose acteoside group showed significantly decreased renal tissue iron levels （P＜0.01） and significantly increased GPX4 protein expression （P＜0.05）. All acteoside dose groups exhibited significantly elevated mRNA and xCT protein expression levels of renal tissue GPX4 （both P＜0.01） and significantly reduced CD71 mRNA expression levels （P＜0.01）. （2） In vitro experiments：2.5 µmol·L-1 Erastin was selected as the optimal induction concentration， and 2.5， 5， and 10 µmol·L-1 acteoside were determined as the optimal intervention concentrations. Compared with the blank control group，the mRTECs cell viability and protein expression levels of nuclear Nrf2，HO-1， and GPX4 in the Erastin model group were significantly decreased （P＜0.01）. Compared with the Erastin model group， mRTECs cell viability and protein expression levels of nuclear Nrf2 and GPX4 in all treatment groups were significantly increased （P＜0.01）. The protein expression level of HO-1 in mRTECs in the 5 and 10 µmol·L-1 acteoside groups were significantly elevated （P＜0.01）. Conclusion Acteoside can effectively ameliorate renal histopathological damage in mice with AKI induced by IRI，and its mechanism may be related to the activation of the Nrf2/HO-1 pathway to inhibit ferroptosis.

R285.5

国家自然科学基金项目（82374130，82174061）