摘要
目的探讨哺乳动物雷帕霉素靶蛋白(mTOR)在六价铬[Cr(Ⅵ)]所致大鼠急性肾损伤(AKI)中的作用。方法42只雄性SD大鼠随机分为正常对照组(单次ip给予生理盐水)、不同剂量Cr(Ⅵ)处理组〔单次ip给予Cr(Ⅵ)5或10 mg·kg-1〕、雷帕霉素(Rap)干预组〔隔日1次(共4次)ip给予Rap 2 mg·kg-1,而后ip给予Cr(Ⅵ)5或10 mg·kg-1〕和NAC干预组〔连续3 d,每日1次ip NAC 300 mg·kg-1,而后ip给予Cr(Ⅵ)5或10 mg·kg-1〕。Cr(Ⅵ)处理48 h后,分别称取大鼠体质量和肾质量,计算肾系数;HE染色后,在显微镜下采用AutoCAD软件计算肾小管损伤面积百分比;ELISA法检测血清肌酐(Scr)和尿中性粒细胞明胶酶相关脂质运载蛋白(NGAL)水平;紫外分光光度法检测肾组织中谷胱甘肽(GSH)和丙二醛(MDA)水平;Western印迹法检测肾组织中磷酸化mTOR(p-mTOR)、磷酸化p70核糖体蛋白S6激酶(p-p70S6K)和活化的胱天蛋白酶3蛋白表达水平。结果与正常对照组相比,Cr(Ⅵ)10 mg·kg-1组大鼠肾质量和肾系数明显增加(P<0.05);Cr(VI)5和10 mg·kg-1组Scr、尿NGAL水平、肾小管损伤平均面积百分比和MDA含量明显升高(P<0.05),肾组织中p-mTOR、p-p70S6K和活化胱天蛋白酶3蛋白表达水平明显上调(P<0.05),GSH含量明显降低(P<0.05)。与同剂量Cr(Ⅵ)处理组相比,Rap或NAC干预组Scr、肾小管损伤面积百分比、肾组织p-mTOR、p-p70S6K和活化的胱天蛋白酶3表达水平均明显下调(P<0.05)。与NAC干预组比较,Rap干预组Scr、肾小管损伤平均面积百分比和活化的胱天蛋白酶3表达水平明显降低(P<0.05),而氧化应激标志物MDA水平无明显差异。结论Cr(Ⅵ)可诱导mTOR磷酸化激活导致AKI,mTOR抑制剂Rap可有效拮抗Cr(Ⅵ)引起的AKI。
OBJECTIVE To explore the role of the mammalian target of rapamycin(mTOR)protein in acute kidney injury(AKI)induced by hexavalent chromium[Cr(Ⅵ)]in rats.METHODS Forty-two male SD rats were randomly divided into normal control(single ip with saline),different-dose Cr(Ⅵ)groups〔single ip with Cr(Ⅵ)5 or 10 mg·kg-1〕,Rap intervention group〔ip with Rap 2 mg·kg-1 once every other day(totally four times)before being treated with Cr(Ⅵ)5 or 10 mg·kg-1〕,and NAC intervention group〔ip with NAC 300 mg·kg-1 for 3 d(once per day),followed by treatment with Cr(Ⅵ)5 or 10 mg·kg-1〕.After 48 h of Cr(Ⅵ)treatment,body mass and kidney mass of rats were detected to calculate the kidney coefficient.HE staining of renal tissues was performed,and the percentage of the renal tubular injury area was calculated by AutoCAD software under the microscope.The levels of serum creatinine(Scr)and urinary neutrophil gelatinase-associated lipocalin(NGAL)were detected by ELISA,while the levels of glutathione(GSH)and malondialdehyde(MDA)in kidney tissues were measured by spectrophotometric colorimetry assay.The protein levels of phosphorylated mTOR(p-mTOR),phosphorylated p70 ribosomal protein S6 kinase(p-p70 S6 K)and cleaved-caspase 3 in renal tissues were analyzed by Western blotting.RESULTS After treatment with Cr(Ⅵ)10 mg·kg-1 for 48 h,rats showed not only an elevation of kidney mass and kidney coefficient,but also higher levels of Scr and urinary NGAL,as well as higher percentage of the renal tubular injury area and MDA compared with normal control(P<0.05).Also,Cr(Ⅵ)induced significant upregulation of p-mTOR,p-p70 S6 K or cleaved-caspase 3,but a significant decrease in GSH content in kidney tissues compared with that of normal control(P<0.05).Further more,compared with single Cr(Ⅵ)treatment of 5 or 10 mg·kg-1,the corresponding intervention of Rap or NAC decreased the levels of Scr and the percentage of the renal tubular injury area,and significantly down-regulated the expressions of p-mTOR,p-p70 S6 K or cleaved-caspase 3 protein in kidney tissues(P<0.05).Rap intervention reduced the levels of Scr,the percentage of the renal tubular injury area and cleaved-caspase 3 protein levels more significantly than NAC intervention(P<0.05).CONCLUSION Cr(Ⅵ)induces the activation of p-m TOR protein,and m TOR inhibitor Rap can antagonize effectively renal tubular damage elicited by Cr(Ⅵ)in rats.
作者
张英进
宋梅芳
易宗娓
张在其
申向东
费嫦
杨渊
ZHANG Ying-jin;SONG Mei-fang;YI Zong-wei;ZHANG Zai-qi;SHEN Xiang-dong;FEI Chang;YANG Yuan(Department of Integrated Traditional&Western Medicine,the Second Xiangya Hospital,Central South University,Changsha 410011,China;Dong Medicine Key Laboratory of Hunan Province,Department of Laboratory Medicine,Hunan University of Medicine,Huaihua 418000,China;Department of Nephrology,Changzhi Peace Hospital,Changzhi Medical College,Changzhi 046000,China;Department of Pathology,Changzhi Peace Hospital,Changzhi Medical College,Changzhi 046000,China)
出处
《中国药理学与毒理学杂志》
CAS
北大核心
2019年第8期601-607,共7页
Chinese Journal of Pharmacology and Toxicology
基金
侗医药研究湖南省重点实验室平台建设项目（2017CT5025）~~
关键词
六价铬
急性肾损伤
哺乳动物雷帕霉素靶蛋白
hexavalent chromium
acute kidney injury
mammalian target of rapamycin
作者简介
张英进,博士,主治医师,主要从事中西医肾病学研究;通讯作者:杨渊,E-mail:yang1977yuan@126.com。
