血管紧张素Ⅱ正向调节高糖环境下大鼠肾小球系膜细胞TLR4信号及炎性因子表达

Angiotensin Ⅱ up-regulates the release of inflammation factors via Toll-like receptor 4 signal in rat mesangial cells under high glucose

目的观察血管紧张素Ⅱ（AnglI）对高糖环境下大鼠肾小球系膜细胞Toll样受体4（TLR4）信号通路与炎性因子表达的影响，论证AngⅡ对高糖环境下肾小球系膜细胞天然免疫炎性反应的正向调节作用。方法细胞同步化后分组：正常对照（5．6mmol／L葡萄糖）组、高糖（25mmol／L葡萄糖）组、AnglI（10-7mmol／L）组、高糖（25mmol／L葡萄糖）＋AnglI（10-7mmol／L）组和AngⅡ（10-7mmol／L）＋血管紧张素1型受体（ATlR）阻断剂厄贝沙坦（10-5mmol／L）组和AngII（10-7mmol／L）＋厄贝沙坦（10-5mmol／L）＋TLR4阻断剂（10mg／L）组。分别于不同时间收集细胞，12h时采用实时定量PCR检测TLR4及其转接子髓分化因子88（MyD88）的表达，24h时免疫荧光检测TLR4蛋白表达，24h时Western印迹法检测TLR4、MyD88及核因子KB（NF—KB）蛋白的表达，同时收集24h时细胞上清液用ELISA法检测白细胞介素6（IL-6）、单核细胞趋化蛋白1（MCP-1）的水平。结果与正常对照组相比，高糖和AngⅡ组作用12h时TLR4、MyD88mRNA表达显著上调（P〈0．01），24h时TLR4、MyD88以及NF—KB蛋白表达显著上调（P〈0．01），同时炎性细胞因子IL-6、MCP-1水平亦显著上调（P〈0．01）；与高糖或AngII组相比，AngⅡ和高糖共同作用肾小球系膜细胞后TLR4、MyD88和NF—KB蛋白以及IL-6、MCP-1的水平进一步上调（P〈0．01）；ATlR、TLR4阻断剂可显著下调AnglI诱导的高糖环境下系膜细胞TLR4、MyD88表达及IL．6、MCP-1水平（P〈0．01）。结论高糖和高肾素导致大鼠肾小球系膜细胞炎性因子表达增加均与TLR4．MyD88信号通路的激活相关，TLR4信号通路在糖尿病系膜细胞天然免疫炎性反应机制中发挥关键效应。AngⅡ对高糖环境下大鼠。肾小球系膜细胞炎性因子释放起正向调节作用。

Objective To observe the regulation of Toll-like receptor 4 （TLR4） signal and the release of inflammation factors after angiotensin Ⅱ （Ang Ⅱ ） stimulation in rat mesangial cells under high glucose condition, revealing the innate immune- related mechanism of injury by Ang Ⅱ on mesangial cells under high glucose. Methods After synchronization, cells incubated with Ang H （10-7 mmo/L） and/or high glucose （25 mmol/L） were used as the stimulation group, cells without stimulation were as normal control （5.6 mmol/L glucose）. To determine the role of TLR4 and the adaptor myeloid differemiation factor 88 （Myl）88）, equal number of tlBZY-1 cells were added with 10 5 mmol/L irbesartan and/or TLR4 blocker （10 mg/L） for 1 h and then incubated with Ang [l （10-7 mmo/L） andh;r high glucose （25 retool/L） for 12 h or 24 h respectively. Real-time PCR was used to analyze TLR4 mRNA and Myl）88 mRNA expression after 12 h. hmnunofluoreseence was used to observe TLR4 protein expression after 24 h; Western blnlting was used tu observe TI,R4, MyD88 anti nut, lear factor kB（NF-kB） protein; EIJSA was used 1o deleet the eoncentralion of MCP-1, IL-6 in cell supernatant respectively. Results Compared with normal control group. TLR4 mRNA and MyD88 mRNA were highly cxpressed in high glucose or Ang It -indueed HBZY- I cells （P 〈 0.01）, TLR4, MyD88 and NF-KB protein as well as MCP-1, 11,-6 were also up-regulated significantly （P 〈 0.01）. Compared with high glucose or Ang Ⅱ group, MyD88 and NF- KB protein as well as MCP- 1, IL- 6 were further up-regulated markedly in Ang Ⅱ and high glueose costimulated group （P 〈 0.01）. In HBZY-1 cells lhal were preint.ul）aied with irbesartan and /or TLR4 blocker, TI,R4 and MyD88 protein expression were obviously inhibited, IL-6 and MCP-1 pruductiun were also decreased remarkably compared with high glucose and/or Ang Ⅱ group （P 〈 0.01）. Conclusions High glucose and Ang Ⅱ stimulate the release of prointlamnmtory factors in rat glomerular mesangial cells via TLR4-MyD88 pathway. This process is inhibited by irbesartan or TLR4 blocker via modulation of the signal. Ang Ⅱ has the posilive-regulation potential on the release of inflanmmtion factors via TLR4 signal in rat mesangial cells under high glucose conditiun.