【摘要】 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、20μmol·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、80μmol·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2.5μmol·L^-1 Erastin诱导mRTECs，并以2.5、5、10μmol·L^-1毛蕊花糖苷以及5μmol·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2.5μmol·L^-1 Erastin作为最佳诱导浓度，以2.5、5、10μmol·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、10μmol·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。更多还原

【Abstract】 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）. ^Conclu^sion 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.更多还原