血管紧张素Ⅱ诱导肾小管上皮细胞Toll样受体4和炎症因子表达

Angiotensin Ⅱ induces the expression of Toll-like receptor 4 and inflammatory factors in renal tubular epithelia cells

目的探讨Toll样受体4(Toll-like receptor4,TLR4)在血管紧张素Ⅱ(AngⅡ)诱导的大鼠肾小管上皮细胞(NRK-52E)所模拟的高血压肾损害微环境中的作用机制。方法 NRK-52E细胞按AngⅡ浓度梯度(10^(-6)~10^(-10)mol/L)培养24h以确定最佳干预浓度,按时间梯度(6~48h)分组培养选择最佳干预时间;同时建立稳定转染TLR4特异RNAi质粒的NRK-52E细胞株。免疫细胞化学及RT-PCR检测TLR4表达水平,ELISA检测上清IL-6及TNF-α水平。结果 10^(-6)~10^(-9)mol/L的AngⅡ均可诱导NRK-52E细胞中TLR4 mRNA明显上调,其中10^(-7)mol/L组作用更为显著;6h即可见TLR4 mRNA水平显著增高,高峰维持12~24h;同时IL-6、TNF-α表达亦上调。TLR4特异性RNA干扰可显著抑制NRK-52E中TLR4的mRNA表达,逆转AngⅡ对IL-6、TNF-α表达的上调作用。结论 AngⅡ可诱导NRK-52E细胞中TLR4及IL-6、TNF-α的表达,阻断TLR4可抑制相关炎症因子的表达,提示TLR4可能通过诱导微炎症反应而参与高血压肾损害。

Objective To study the working mechanism of Toll-like Receptor 4 （ TLR4） in renal tubular epithelia cell （ NRK- 52E） models of the hypertensive renal injury microenvironment induced by angiotensin II （Angll）. Methods NRK-52E cells were cul-tured in Angll of different concentrations （10^-6 - 10^-10mol/L） for 24h to determine the optimal interfering concentration, and then in Angll at the optimal concentration for 6h - 48h to determine the optimal cultivating time. TLR4-specific RNAi plasmids were transfect-ed into NRK-52E cells and the stable cell strains were selected. The expressions of TLR4 mRNA and protein were detected by reverse transcription-polymerase chain reaction （RT-PCR）, imraunocytochemistry and western blot, and the supernatant levels of IL-6 and TNF- a were tested by enzyme linked immunosorbent-assay （ELISA）. Results Angll （1 0 ^-6 - 1 0^-9M） obviously up-regulated TLR4 expres-sion, and this effect was most significant in the 10^-7mol/L group; a significant increase of TLR4 mRNA was detected after 6h of culti-vation, and the peak maintained for 12 - 24h. The levels of IL-6 and TNF_a also increased when stimulated by Angll. TLR4-specific RNAi obviously suppressed the expressions of TLR4 mRNA, and reversed the up-regulation of the expressions of IL-6 and TNF-a in-duced by Angll. Conclusions Angll can induce the expressions of TLR4 and its related inflammatory factors IL-6 and TNF-a in NRK- 52E cells. Blocking TLR4 can inhibit the expression of these inflammatory cytokines,which suggests that TLR4 may contribute to the hypertensive renal damage induced by Angll through the induction of micro-inflammation.