Nrf2/HO-1信号通路在人脐带间充质干细胞来源的外泌体减轻小鼠肾缺血再灌注损伤中的作用

Role of Nrf2/HO-1 signaling pathway in human umbilical cord mesenchymal stem cells-derived exosomes-induced reduction of renal ischemia-reperfusion injury in mice

目的评价核因子E2相关因子2(Nrf2)/血红素氧化酶-1(HO-1)在人脐带间充质干细胞来源的外泌体(hucMSCs-exo)减轻小鼠肾缺血再灌注损伤中的作用。方法培养人脐带间充质干细胞,提取外泌体,通过透射电子显微镜、纳米颗粒跟踪分析和Western blot法进行鉴定。雄性SPF级C57BL/6小鼠36只,体质量20~25 g,取30只小鼠,采用随机数字表法分为5组(n=6):假手术组(Sham组)、假手术+Nrf2抑制剂ML385组(Sham+ML385组)、肾缺血再灌注组(I/R组)、肾缺血再灌注+外泌体组(I/R+EXO组)、肾缺血再灌注+外泌体+Nrf2抑制剂ML385组(I/R+EXO+ML385组)。采用夹闭两侧肾蒂45 min后再灌注的方法制备肾缺血再灌注损伤模型。Sham+ML385组和I/R+EXO+ML385组于造模前45 min腹腔注射ML38530 mg/kg,I/R+EXO组和I/R+EXO+ML385组于再灌注前15 min尾静脉注射hucMSCs-exo 100μg。再灌注24 h时检测血清BUN和Cr浓度;取肾组织,观察病理学变化,检测IL-6、TNF-α和MDA含量、SOD活性和ROS水平,分别采用Western blot法和RT-qPCR法检测Nrf2、HO-1及其mRNA的表达。取6只小鼠采用随机数字表法分为2组(n=3):假手术组(Sham-IM组)和肾缺血再灌注组(I/R-IM组),进行VISQUE活体成像观察。结果透射电镜下观察外泌体具有典型的杯状形态,纳米颗粒跟踪分析外泌体平均直径96.7 nm,Western blot法检测其表面标志CD9、CD63、TSG101表达阳性。与Sham-IM组比较,I/R-IM组小鼠肾脏荧光强度值升高(P<0.05)。与Sham组比较,I/R组血清BUN和Cr浓度升高,肾组织IL-6、TNF-α、MDA含量和ROS水平升高,SOD活性降低,Nrf2、HO-1及其mRNA表达下调(P<0.05),肾组织病理损伤加重;Sham+ML385组血清BUN和Cr浓度比较差异无统计学意义(P>0.05)。与I/R组比较,I/R+EXO组血清BUN和Cr浓度降低,肾组织IL-6、TNF-α、MDA含量和ROS水平降低,SOD活性升高,Nrf2、HO-1及其mRNA表达上调(P<0.05),肾组织病理损伤减轻;与I/R+EXO组比较,I/R+EXO+ML385组血清BUN和Cr浓度升高,肾组织IL-6、TNF-α、MDA含量和ROS水平升高,SOD活性降低,Nrf2、HO-1及其mRNA表达下调(P<0.05),肾组织病理损伤加重。结论hucMSCs-exo减轻小鼠肾缺血再灌注损伤的机制可能与激活Nrf2/HO-1信号通路有关。

Objective To evaluate the role of nuclear factor E2-related factor 2(Nrf2)/heme oxidase-1(HO-1)in reduction of renal ischemia-reperfusion(I/R)injury by the human umbilical cord mesenchymal stem cells(hucMSCs)-derived exosomes(hucMSCs-exo)in mice.Methods The hucMSCs were cultured,and exosomes were extracted and identified by transmission electron microscopy,nanoparticle tracking analysis and Western blot.Thirty-six male SPF-grade C57BL/6 mice,weighing 20-25 g,were used.Thirty mice were selected and divided into 5 groups(n=6 each)by a random number table method:sham operation group(Sham group),sham operation+Nrf2 inhibitor ML385 group(Sham+ML385 group),renal I/R group(I/R group),renal I/R+exosome group(I/R+EXO group),and renal I/R+exosome+Nrf2 inhibitor ML385 group(I/R+EXO+ML385 group).A model of renal I/R injury was prepared by clamping the bilateral renal pedicles for 45 min followed by perfusion in anesthetized animals.ML38530 mg/kg was intraperitoneally injected at 45 min before preparing the model in Sham+ML385 group and I/R+EXO+ML385 group,and hucMSCs-exo 100μg was injected via the tail vein at 15 min before reperfusion in I/R+EXO group and I/R+EXO+ML385 group.Serum blood urea nitrogen(BUN)and creatinine(Cr)concentrations were detected at 24 h of reperfusion.The renal tissues were obtained for examination of the pathological changes and for determination of contents of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α)and malondialdehyde(MDA),superoxide dismutase(SOD)activity and reactive oxygen species(ROS)levels and expression of Nrf2 and HO-1 protein and mRNA(by Western blot and quantitative real-time polymerase chain reaction).The left 6 mice were allocated to sham operation group(Sham-IM group,n=3)and renal I/R group(I/R-IM group,n=3)by a random number table method for VISQUE in living imaging observation.Results The exosomes showed a typical cup-shaped morphology with a transmission electron microscope,the nanoparticles tracked and analyzed the average diameter of the exosome,with an average diameter of 96.7 nm,and the positive expression of surface markers CD9,CD63 and TSG101 was detected using Western blot.The renal fluorescence intensity value was significantly increased in I/R-IM group as compared with Sham-IM group(P<0.05).Compared with Sham group,the serum BUN and Cr concentrations were significantly increased,the contents of IL-6,TNF-αand MDA and ROS levels were increased,the activity of SOD was decreased,the expression of Nrf2 and HO-1 protein and mRNA was down-regulated(P<0.05),and the pathological changes of renal tissues were aggravated in I/R group,and no significant change was found in serum BUN and Cr concentrations in Sham+ML385 group(P>0.05).Compared with I/R group,the serum BUN and Cr concentrations were significantly decreased,the contents of IL-6,TNF-αand MDA and ROS levels were decreased,the activity of SOD was increased,the expression of Nrf2 and HO-1 protein and mRNA was up-regulated(P<0.05),and the pathological changes of renal tissues were significantly attenuated in I/R+EXO group.Compared with I/R+EXO group,the serum BUN and Cr concentrations were significantly increased,the contents of IL-6,TNF-αand MDA and ROS levels were increased,the activity of SOD was decreased,the expression of Nrf2 and HO-1 protein and mRNA was down-regulated(P<0.05),and the pathological changes of renal tissues were aggravated in I/R+EXO+ML385 group.Conclusions The mechanism by which hucMSCs-exo reduces renal I/R injury may be related to activation of the Nrf2/HO-1 signaling pathway in mice.