CAJ | 학술논문

目的观察血管紧张素Ⅱ(AngⅡ)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(5.6 mmol/L葡萄糖)组、高糖(25 mmol/L葡萄糖)组、AngⅡ(10-7mmol/L)组、高糖(25 mmol/L葡萄糖)+AngⅡ(10-7mmol/L)组和AngⅡ(10-7 mmol/L)+血管紧张素1型受体(AT1R)阻断剂厄贝沙坦(10-5mmol/L)组和AngⅡ(10-7 mmol/L)+厄贝沙坦(10-5 mmol/L)+TLR4阻断剂(10 mg/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,24 h时免疫荧光检测TLR4蛋白表达,24 h时Western印迹法检测TLR4、MyD88及核因子κB(NF-κB)蛋白的表达,同时收集24 h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、MyD88 mRNA表达显著上调(P＜0.01),24 h时TLR4、MyD88以及NF-κB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-κB蛋白以及IL-6、MCP-1的水平进一步上调(P＜ 0.01);AT1R、TLR4阻断剂可显著下调AngⅡ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.
목적 관찰혈관긴장소Ⅱ(AngⅡ)대고당배경하대서신소구계막세포Toll양수체4(TLR4)신호통로여염성인자표체적영향,론증AngⅡ대고당배경하신소구계막세포천연면역염성반응적정향조절작용.방법 세포동보화후분조:정상대조(5.6 mmol/L포도당)조、고당(25 mmol/L포도당)조、AngⅡ(10-7mmol/L)조、고당(25 mmol/L포도당)+AngⅡ(10-7mmol/L)조화AngⅡ(10-7 mmol/L)+혈관긴장소1형수체(AT1R)조단제액패사탄(10-5mmol/L)조화AngⅡ(10-7 mmol/L)+액패사탄(10-5 mmol/L)+TLR4조단제(10 mg/L)조.분별우불동시간수집세포,12h시채용실시정량PCR검측TLR4급기전접자수분화인자88(MyD88)적표체,24 h시면역형광검측TLR4단백표체,24 h시Western인적법검측TLR4、MyD88급핵인자κB(NF-κB)단백적표체,동시수집24 h시세포상청액용ELISA법검측백세포개소6(IL-6)、단핵세포추화단백1(MCP-1)적수평.결과 여정상대조조상비,고당화AngⅡ조작용12h시TLR4、MyD88 mRNA표체현저상조(P＜0.01),24 h시TLR4、MyD88이급NF-κB단백표체현저상조(P＜0.01),동시염성세포인자IL-6、MCP-1수평역현저상조(P＜0.01);여고당혹AngⅡ조상비,AngⅡ화고당공동작용신소구계막세포후TLR4、MyD88화NF-κB단백이급IL-6、MCP-1적수평진일보상조(P＜ 0.01);AT1R、TLR4조단제가현저하조AngⅡ유도적고당배경하계막세포TLR4、MyD88표체급IL-6、MCP-1수평(P＜0.01).결론 고당화고신소도치대서신소구계막세포염성인자표체증가균여TLR4-MyD88신호통로적격활상관,TLR4신호통로재당뇨병계막세포천연면역염성반응궤제중발휘관건효응.AngⅡ대고당배경하대서신소구계막세포염성인자석방기정향조절작용.
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 Ⅱ (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 differentiation factor 88 (MyD88),equal number of HBZY-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 Ⅱ (10-7 mmo/L) and/or high glucose (25 mmol/L) for 12 h or 24 h respectively.Real-time PCR was used to analyze TLR4 mRNA and MyD88 mRNA expression after 12 h.Immunofluorescence was used to observe TLR4 protein expression after 24 h; Western blotting was used to observe TLR4,MyD88 and nuclear factor κB (NF-κB) protein; ELISA was used to detect the concentration of MCP-1,IL-6 in cell supernatant respectively.Results Compared with normal control group,TLR4 mRNA and MyD88 mRNA were highly expressed in high glucose or Ang Ⅱ-induced HBZY-1 cells (P ＜ 0.01),TLR4,MyD88 and NF-κB protein as well as MCP-1,IL-6 were also up-regulated significantly (P ＜ 0.01).Compared with high glucose or Ang Ⅱ group,MyD88 and NF-κB protein as well as MCP-1,IL-6 were further up-regulated markedly in Ang Ⅱ and high glucose costimulated group (P ＜ 0.01).In HBZY-1 cells that were preincubated with irbesartan and/or TLR4 blocker,TLR4 and MyD88 protein expression were obviously inhibited,IL-6 and MCP-1 production 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 proinflammatory 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 positive-regulation potential on the release of inflammation factors via TLR4 signal in rat mesangial cells under high glucose condition.