摘要
目的探讨雷帕霉素(rapamycin,Rap)对大鼠肾脏缺血再灌注损伤的保护作用及机制。方法将24只SD大鼠分为假手术组(Sham)、缺血再灌注损伤模型组(I/R)、低剂量Rap组、高剂量Rap组,术前3d开始灌胃给药,Sham组及I/R组分别给予生理盐水0.5ml/d,低剂量Rap组及高剂量Rap组每天分别给予0.5ml的1mg/kg和2mg/kg浓度的Rap,维持至术后7d,并在术后第7天给药后2h后收集肾脏组织及血液标本。血生化检测尿素氮及血肌酐;HE染色检测组织病理损伤程度;TUNEL染色检测组织细胞凋亡并计数;Western印迹及实时定量聚合酶链反应检测LC3B、P62、Beclin1、mTOR等因子mRNA水平及蛋白表达;ELISA检测谷胱甘肽(glutathione,GSH)、超氧化物歧化酶(superoxidedismutase,SOD)、丙二醛(malondialdehyde,MDA)、活性氧(reactiveoxygenspecies,ROS)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumornecrosis factor-alpha,TNF-α)表达水平。结果I/R组血清肌酐、尿素氮水平较Sham组明显上升;高剂量Rap组血清肌酐、尿素氮水平较I/R组明显下降;HE染色结果显示,Sham组大鼠肾组织结构正常,I/R组大鼠肾小管结构排列紊乱,可见上皮细胞扁平化、坏死和蛋白管型,肾小管Paller评分明显升高;对比I/R组,Rap两组损伤明显减轻;TUNEL染色结果显示,Rap两组凋亡细胞均较I/R组显著减少。I/R与Sham组相比,LC3B和Beclin1基因表达降低,P62和mTOR基因呈高表达;与I/R组比较,高剂量Rap组LC3B和Beclin1基因呈高表达,P62和mTOR基因呈低表达,同时高剂量Rap组GSH、SOD水平显著升高,MDA、ROS显著降低,且IL-6、TNF-α表达明显减少。结论Rap通过抑制细胞氧化应激及炎症反应减轻大鼠肾脏缺血再灌注损伤,其作用机制可能是Rap促进了细胞自噬水平及抑制细胞凋亡。
Objective To explore the protective effect and mechanism of rapamycin(Rap)on renal ischemiareperfusion injury in rats.Methods A total of 24 SD rats were divided into the Sham group(sham),the ischemia-reperfusion injury model group(I/R),the Rap low-dose pretreatment group(Rap-L),and the Rap high-dose pretreatment group(Rap-H).These rats were operated the intragastric administration 3 days before the surgery.Each rat in Sham group and the I/R group was given O.5 ml of normal saline daily,while each rat in Rap-L group and the Rap-H group was administered O.5 ml/(1 mg·kg)and 2 mg/kg concentrations of Rap every day.These operations was maintained and collect kidney tissues and blood samples until 7 days after the surgery.Blood biochemical tests include urea nitrogen and serum creatinine;HE staining was used to assess the degree of tissue pathological damage.TUNEL staining was employed to detect tissue cell apoptosis and count the cells.Western blotting and real-time quantitative polymerase chain reaction were used to detect the mRNA levels and protein expressions of factors such as LC3B,P62,Beclin1,and mTOR.ELISA was used to measure the expression levels of glutathione(GSH),superoxide dismutase(SOD),malondialdehyde(MDA),reactive oxygen species(ROS),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α).Results Compared with the Sham group,the levels of serum creatinine and urea nitrogen in the IRI group significantly increased.Meanwhile,compared with the IRI group,the levels of serum creatinine and urea nitrogen in the Rap-H group obviously decreased.The HE staining results showed that the renal tissue structure of rats in the Sham group was normal,while the renal tubule structure of rats in the I/R group was disordered which could be seen that epithelial cells were flat,necrosis and with protein tubule type,and the Paller score of renal tubules was significantly increased.Compared with the IRI group,tissue damage in both Rap groups was significantly alleviated.The TUNEL staining results showed that,compared with the IRI group,the number of apoptotic cells in the two Rap groups was significantly reduced.Compared with the Sham group,the expression of LC3B and Beclin1 genes was decreased in the I/R group,while the P62 and mTOR genes were highly expressed.Compared with Sham group,the I/R group showed decreased expression of LC3B and Beclin1,and increased expression of P62 and mTOR.Compared with I/R group,LC3B and Beclin1 genes were highly expressed in Rap-H group,while P62 and mTOR genes were low expressed.Meanwhile,GSH and SOD levels were significantly increased in Rap-H group,while MDA and ROS were significantly decreased,and the expressions of IL-6 and TNF-αwere significantly decreased.Conclusion Rapamycin alleviates renal IRI in rats by inhibiting the oxidative stress and the inflammatory response.The mechanism may involve the promotion of autophagy and suppression of apoptosis.
作者
张敏
范振磊
李益飞
贾云童
刘涛
Zhang Min;Fan Zhenlei;Li Yifei;Jia Yuntong;Liu Tao(Clinical Pharmacy,First Affiliated Hospital of Kuuming Medical University,Kunming,Yuaman 650032,China;Organ Transplantation Center,First Affiliated Hospital of Kaaming Medical University,Kunming,Yuuman 650032,China)
出处
《泌尿外科杂志(电子版)》
2025年第4期1-6,共6页
Journal of Urology for Clinicians(Electronic Version)
基金
云南省教育厅科学研究基金项目(2024J0194)
云南省科技厅昆明医科大学基础研究计划项目(202101AY070001-113)。
关键词
肾脏
缺血再灌注损伤
RAP
自噬
凋亡
Kidney
Ischemia-reperfusion injury
Rapamycin
Autophagy
Apoptosis
