高糖调节PINK1/Parkin途径刺激大鼠肾小球系膜细胞氧化应激和炎症反应的研究

High glucose stimulates oxidative stress and inflammatory response of glomerular mesangial cells in rats by regulating PINK1/Parkin pathway

目的探讨高糖刺激对大鼠肾小球系膜细胞(RMCs)的潜在影响。方法采用5.5、30.0 mmol/L葡萄糖处理RMCs,采用实时荧光定量PCR(RT-q PCR)检测PINK1和Parkin mRNA水平;采用蛋白免疫印迹法检测PINK1、Parkin、线粒体外膜转位酶20(Tomm20)和LC3-Ⅱ蛋白水平;采用免疫荧光技术检测LC3-Ⅱ与Tomm20的共定位;采用流式细胞术测定RMCs中活性氧(ROS)水平和凋亡情况;采用酶联免疫吸附试验和RT-q PCR检测炎症因子的释放情况;应用蛋白免疫印迹法检测核因子κB(NF-κB)通路的调节作用。结果经30.0 mmol/L葡萄糖处理后,在RMCs中发现了PINK1和Parkin mRNA呈低表达,PINK1和Parkin蛋白水平也降低;同时,RMCs中LC3-Ⅱ和Tomm20蛋白水平升高,而Tomm20和LC3-Ⅱ的共表达信号显著减少。经30.0 mmol/L葡萄糖处理后,RMCs中ROS水平、凋亡率和肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、转化生长因子-β(TGF-β)的浓度与mRNA水平均升高。在30.0 mmol/L葡萄糖处理的RMCs中,NF-κB通路被激活。结论高糖刺激促进ROS的产生和炎症反应,同时也抑制了RMCs中的PINK1/Parkin信号通路。高糖刺激能够通过调节PINK1/Parkin介导的线粒体自噬途径影响糖尿病肾病(DN)的进展。

Objective To explore the potential impact of high glucose stimulation on rat glomerular mesangial cells(RMCs).Methods RMCs were treated with 5.5 and 30.0 mmol/L glucose,and the levels of PINK1 and Parkin mRNA were detected by real-time fluorescence quantitative PCR(RT-q PCR).The protein levels of PINK1,Parkin,translocase of outer mitochondrial membrane 20(Tomm20)and LC3-Ⅱwere detected by Western blot.The colocalization of LC3-Ⅱand Tomm20 was detected by immunofluorescence technique.The level of reactive oxygen species(ROS)and apoptosis in RMCs were measured by flow cytometry.The release of inflammatory factors was detected by enzyme-linked immunosorbent assay and RT-q PCR.Regulating effect of nuclear factorκB(NF-κB)pathway was detected by Western blot.Results After treatment of 30.0 mmol/L glucose,the PINK1 and Parkin mRNA had lower expression in RMCs,and the levels of PINK1 and Parkin proteins were also decreased.Meanwhile,the levels of LC3-Ⅱand Tomm20 proteins in RMCs were increased,while the co-expression signals of Tomm20 and LC3-Ⅱwere decreased significantly.After treatment by 30.0 mmol/L glucose,the ROS level,apoptosis rate and the concentrations and mRNA levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and transforming growth factor-β(TGF-β)were increased in RMCs.In RMCs treated with 30.0 mmol/L glucose,NF-κB pathway was activated.Conclusion High glucose stimulation can not only promote the production of ROS and inflammatory response,but also can inhibit the PINK1/Parkin signaling pathway in RMCs.High glucose stimulation can affect the progression of diabetic nephropathy(DN)by regulating PINK1/Parkin mediated autophagy pathway.