中药新药与临床药理2025，Vol.36Issue(6)：859-868,10.DOI:10.19378/j.issn.1003-9783.2025.06.003
毛蕊花糖苷通过Nrf2/HO-1通路改善小鼠肾脏缺血再灌注损伤的作用机制
Mechanism of Acteoside in Ameliorating Renal Ischemia-Reperfusion Injury in Mice via the Nrf2/HO-1 Pathway
摘要
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).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.关键词
毛蕊花糖苷/急性肾损伤/肾脏缺血再灌注损伤/Nrf2/HO-1 通路/铁死亡/肾小管上皮细胞/小鼠Key words
acteoside/acute kidney injury/renal ischemia-reperfusion injury/Nrf2/HO-1 pathway/ferroptosis/renal tubular epithelial cells/mice分类
医药卫生引用本文复制引用
冼嘉月,王思怡,蒙诗语,周玖瑶,周园..毛蕊花糖苷通过Nrf2/HO-1通路改善小鼠肾脏缺血再灌注损伤的作用机制[J].中药新药与临床药理,2025,36(6):859-868,10.基金项目
国家自然科学基金项目(82374130,82174061). （82374130,82174061）
