TLR4/PI3K调控内质网应激和自噬介导足细胞损伤的分子机制

Molecular mechanism of TLR4/PI3K regulating endoplasmic reticulum stress and autophagy mediated podocyte injury

目的探讨Toll样受体4(TLR4)/磷脂酰肌醇3-激酶(PI3K)通过调控内质网应激和自噬介导足细胞损伤的分子机制。方法通过转化生长因子β1(TGF-β1)刺激小鼠肾足细胞系建立足细胞损伤模型,分别用TLR4-siRNA和3-甲基腺嘌呤(3-MA)处理该模型,蛋白免疫印迹(Western blot)法检测TLR4、髓样分化因子88(MyD88)、葡萄糖调控蛋白(GRP78)、C/EBP同源蛋白(CHOP)、PI3K,以及自噬蛋白beclin-1和微管相关蛋白1轻链3B(LC3B)的水平。结果TGF-β1诱导的小鼠肾足细胞损伤后TLR4、MyD88、CHOP、PI3K和GRP78的蛋白表达在6、12、24、36、48 h时间段明显升高,在刺激24 h后达到峰值,分别为0.57±0.07、0.65±0.06、0.50±0.04、2.01±0.18及2.09±0.17;与TGF-β1联合siRNA处理组比较,TGF-β1联合siRNA-TLR4处理组小鼠肾足细胞TLR4、MyD88、CHOP、LC3-Ⅱ、beclin-1、PI3K和GRP78蛋白水平明显降低或减弱,其水平分别为0.35±0.03、0.42±0.04、0.31±0.03、0.22±0.01、0.29±0.02、1.29±0.10及1.44±0.13;与TGF-β1组比较,TGF-β1联合应用PI3K特异性抑制剂(2.5 mmol/L 3-MA)组小鼠肾足细胞后结果显示PI3K的活化水平、自噬蛋白(LC3-Ⅱ和beclin-1)及内质网应激蛋白(CHOP和GRP78)显著减弱,其水平分别为0.24±0.02、0.33±0.01、0.42±0.02、1.25±0.11及1.21±0.12,而TLR4和MyD88的表达水平无显著性变化。结论TLR4通过PI3K信号通路调控TGF-β1诱导小鼠肾足细胞损伤的内质网应激和自噬反应。

Objective To explore the molecular mechanism of Toll-like receptor 4(TLR4)and phosphatidylinositol 3-kinase(PI3K)for mediating podocyte injury by regulating endoplasmic reticulum stress(ERS)and autophagy.Methods The podocytes injury model was established by TGF-β1 stimulating mouse kidney podocytes.The model was treated by TLR4-siRNA and 3-MA.Then the levels of TLR4,MyD88,C/EBP homologous protein(CHOP),PI3K,autophage protein beclin-1 and LC3B were detected by using the Western blot.Results The protein expression levels of TLR4,MyD88,CHOP,PI3K and GRP78 were increased at 6,12,24,36,48 h after mouse kidney podocytes injury induced by TGF-β1,reaching the peak values at 24 h after stimulation,which were 0.57±0.07,0.65±0.06,0.50±0.04,2.01±0.18 and 2.09±0.17 respectively;compared with the TGF-β1 combined with small interfering RNA treatment group,the results after stimulating treating mouse kidney podocytes by in TGF-β1 combined with siRNA-TLR4 pre-treatingtreatment group TGF-β1 showed that the expression levels of TLR4,MyD88,CHOP,LC3-Ⅱ,beclin-1,PI3K and GRP78 protein were significantly decreased or weakened,which were 0.35±0.03,0.42±0.04,0.31±0.03,0.22±0.01,0.29±0.02,1.29±0.10 and 1.44±0.13 respectively;compared with the TGF-β1 group,the results after tstimulating reating mouse kidney podocytes by TGF-β1 combined with P13K specific inhibitor 2.5 mL 3-MA pre-treating TGF-β1 showed that the P13K activation level,LC3-Ⅱ,beclin-1,CHOP and GRP78 were significantly weakened,which were 0.24±0.02,0.33±0.01,0.42±0.02,1.25±0.11 and 1.21±0.12 respectively,however,the expression levels of TLR4 and MyD88 had no significant change.Conclusion TLR4 modulates TGF-β1 for inducing ERS and autophagy of mouse kidney podocytes injury via PI3K signaling pathway.