摘要目的 观察血管紧张素Ⅱ(AngⅡ)刺激肾小管上皮细胞(NRK-52E)后肿瘤坏死因子α(TNF-α)和热休克蛋白47(HSP47)的表达,分析Toll样受体4(TLR4)信号通路的变化与上述因子的关联,探讨AngⅡ促进NRK-52E细胞炎性反应、纤维化的天然免疫机制.方法 细胞同步化后,将其分为4组:对照组、AngⅡ(10-7 mmol/L)组、坎地沙坦(10-5 mmol/L)+AngⅡ组、TLR4阻断剂(20 mg/L)+AngⅡ组,培养6 h后RT-PCR法检测TLR4及转接信号髓分化因子88(MyD88)mRNA表达水平;12 h后免疫荧光法检测细胞表面TLR4蛋白表达;24 h后以ELISA法检测细胞上清液TNF-α及HSP47的浓度.结果 与对照组相比,AngⅡ显著上调NRK-52E细胞TLR4、MyD88 mRNA和TLR4蛋白表达(均P＜0.01),并诱导细胞TNF-α和HSP47的释放(均P＜0.01).与AngⅡ组相比,TLR4阻断剂和坎地沙坦干预均显著抑制AngⅡ对细胞TLR4、MyD88的刺激效应(均P＜0.01);坎地沙坦抑制AngⅡ诱导的细胞TNF-α、HSP47的释放(均P＜0.01),TLR4阻断剂对细胞TNF-α、HSP47的下调呈剂量依赖性.结论 AngⅡ对NRK-52E细胞天然免疫信号TLR4、MyD88具有激活效应,该信号激活可能是AngⅡ促进肾小管细胞炎性相关因子释放的重要机制之一.坎地沙坦抑制肾小管细胞炎性因子的体外效应也与其调节该信号通路有关.

abstractsObjective To observe the release of inflammation-related factors after angiotensin Ⅱ (Ang Ⅱ ) stimulation in rat tubular epithelial cells (NRK-52E), to analyze whether these effects were mediated by TLR4-MyD88 pathway, and to reveal the novel mechanism of injury by Ang Ⅱ on NRK-52E cells. Methods After synchronization, cells incubated with AngⅡ (10-7 mmol/L) were used as the stimulation group, cells without stimulation were as normal control. To determine the role of TLR4 and the adaptor MyD88, equal number of NRK-52E cells was added with 10-5 mmol/L candesartan or 20 mg/L TLR4 blocking peptide for 1 h and then incubated with Ang Ⅱ (10-7 mmol/L) respectively. RT-PCR was used to analyze TLR4 mRNA and MyD88 mRNA expression. Immunofluorescence and confocal microscopy were used to observe TLR4 protein expression. ELISA was used to detect the concentration of tumor necrosis factor-alpha (TNF-α) and heat shock protein 47(HSP47) in cell supernatant respectively. Results TLR4 and MyD88 were highly expressed in Ang Ⅱ-induced NRK-52E cells (P＜0.01), and the TNF-α and HSP47 levels were also increased markedly compared with control group (P＜0.01). In NRK-52E cells that were pre-incubated with candesartan, TLR4 and MyD88 expression were obviously inhibited,subsequently, HSP47 and TNF-α production decreased remarkably compared with Ang Ⅱ group (P＜0.01). TLR4 blocking peptide had the similar effect in a dose-dependent manner, in which its effect was dependent on inhibiting TLR4-MyD88 expression. Conclusion The mechanism of Ang Ⅱ -induced injury effect on NRK-52E cells is related to the increase of TLR4-MyD88 activity,which is followed by the enhance of TNF-α and HSP47 expression. This process is inhibited by candesartan via modulation of innate immune pathway.