血管紧张素Ⅱ和高糖环境对大鼠肾小球系膜细胞Toll样受体4信号通路及炎性因子表达的影响

Effects of AngiotensinⅡ(AngⅡ)and High Glucose on Toll-like Receptor 4(TLR4) Signaling Pathway and Inflammatory Factors in Rat Glomerular Mesangial Cells

目的探讨高糖条件下肾小球系膜细胞Toll样受体4(TLR4)及其信号通路中下游因子MyD88、核因子-κB(NF-κB)以及炎性因子的表达变化,论证高糖环境下血管紧张素Ⅱ(AngⅡ)与TLR4的对话关系,以期阐明TLR4信号通路在糖尿病肾病(DN)发病机制中的作用。方法设计3对针对大鼠TLR4基因的特异性siRNA片段以及带有绿色荧光的阴性对照,筛选出转染效率最高的siRNA,用于后续实验。第1部分实验在高糖的基础上分别加入AngⅡ、TLR4siRNA、血管紧张素Ⅱ受体阻断剂(ARB)、阴性对照siRNA,探讨高糖对各组细胞TLR4、MyD88及NF-κB表达的影响;第2部分实验在AngⅡ的基础上分别加入高糖、TLR4siRNA、ARB、阴性对照siRNA,探讨AngⅡ对各组细胞TLR4,MyD88及NF-κB表达的影响。采用荧光定量PCR检测各组TLR4、MyD88 mRNA的表达,Western blot检测TLR4、MyD88及NF-κB蛋白表达水平的变化,ELISA法检测细胞上清液中白细胞介素-6(IL-6)及单核细胞趋化因子-1(MCP-1)的表达情况。结果高糖及AngⅡ都可以上调TLR4、MyD88、NF-κB蛋白及TLR4、MyD88mRNA的表达水平(均P<0.05),也可上调IL-6及MCP-1的表达(均P<0.01)且二者协同刺激情况下上调更明显。siRNA干扰及ARB可下调TLR4、MyD88、NF-κB蛋白及TLR4、MyD88 mRNA的表达,下调IL-6及MCP-1的表达,与高糖及AngⅡ组相比差异有统计学意义(均P<0.05)。结论高糖环境和AngⅡ刺激系膜细胞TLR4/MyD88信号通路激活,特异性TLR4siRNA基因可以部分阻断由高糖及AngⅡ诱导的TLR4信号的激活,厄贝沙坦也可部分阻断TLR4信号通路,高糖和AngⅡ共刺激增强由TLR4介导的天然免疫炎症效应,TLR4/MyD88信号通路在此效应中扮演重要角色;TLR4siRNA基因沉默技术为寻找DN的治疗方法提供了新思路,也为临床上使用ARB延缓DN进程提供了新的理论依据。

Objective To observe the changes of Toll-like receptor 4（TLR4）/myeloid differentiation88（MyD88）signaling pathway as well as inflammatory factors in rat mesangial cells under angiotensinⅡ（AngⅡ）and high glucose conditions,and by applying the small RNA interference or AngⅡreceptor blocker to interfere the rat mesangial cells,to further explore the novel mechanism of AngⅡ＇s non-hemodynamic effects under high sugar environment and to clarify the pivital role of TLR4/MyD88 pathway in the pathogenesis of diabetic nephropathy（DN）.Methods Three rat TLR4-siRNA sequences were designed.The most effective siRNA was screened for following experiments.The cells in the experiment of the first part were divided into six groups[low-glucose,high-glucose,high-glucose＋AngⅡ,high-glucose＋angiotensin Ⅱ receptor blocker（ARB）,high-glucose＋TLR4siRNA,high-glucose＋ScsiRNA group],to study effect of high glucose on expression of TLR4,MyD88 and NF-κB.The cells in the experiment of the second part were also divided into six groups（low-glucose,AngⅡ,high-glucose＋AngⅡ,AngⅡ＋ARB,AngⅡ＋TLR4siRNA,AngⅡ＋ScsiRNA group）,to investigate the effect of AngⅡ on the expression of TLR4,MyD88 and NF-κB.TLR4 and MyD88 mRNA expression was detected by realtime fluorescence quantitative PCR.TLR4,MyD88 and NF-κB protein expression was detected by Western blotting.ELISA was used to detect expression of IL-6and MCP-1in supernatant.Results Compared with low-glucose groups,AngⅡ and high glucose significantly upregulated the expression of TLR4/Myd88 mRNA and protein,and NF-κB protein（P〈0.05）respectively.The expression levels of IL-6and MCP-1in cell supernatant were also increased significantly in AngⅡ group and high-glucose group（P〈0.01）,and the synergistic effect of AngⅡandhigh glucose was more significant.The RNA interference and ARB could effectively down-regulate the expression of TLR4/Myd88 mRNA and protein,NF-κB protein,and IL-6and MCP-1,as compared with high-glucose group and AngⅡ group（P〈0.05）.Conclusion TLR4/Myd88 signaling pathway can be activated by AngⅡ or high-glucose in rat mesangial cells.These effects can be partly blocked by specific TLR4 siRNA gene and AngⅡ receptor blocker.Irbesartan can partly block TLR4 signaling pathway.High glucose and AngⅡ enhance TLR4-mediated innate immune inflammation,in which TLR4/MyD88 signaling pathway plays a pivital role.TLR4 siRNA gene silencing technology provides a new strategy for the treatment of DN.