TLR4/NLRP3炎症复合体介导对比剂诱导的肾小管上皮细胞炎症和损伤

Involvement of TLR4/NLRP3 inflammasome in contrast medium-induced inflammation and injury in renal tubular epithelial cells

目的:探讨Toll样受体4(TLR4)/Nod样受体蛋白3(NLRP3)炎症复合体是否介导了对比剂(CM)引起的肾小管上皮细胞炎症和损伤。方法:本研究运用碘普罗胺作用于大鼠肾小管上皮细胞NRK-52E建立损伤模型。应用CCK-8法测定细胞存活率;Western blot测定TLR4、NLRP3、凋亡相关斑点样蛋白(ASC)、caspase-1和cleaved caspase-3的蛋白水平;ELISA法检测炎症因子白细胞介素1β(IL-1β)和IL-18的水平;Hoechst 33258核染色法检测凋亡率;JC-1染色法测定线粒体膜电位。用小干扰RNA沉默NLRP3表达。结果:CM可降低NRK-52E细胞的存活率并上调cleaved caspase-3的蛋白水平(P<0.05);此外,CM可上调细胞TLR4/NLRP3炎症复合体的表达并促进炎症因子IL-1β和IL-18的分泌(P<0.05)。沉默NLRP3可以对抗CM诱导的炎症因子分泌;TLR4抑制剂TAK-242及沉默NLRP3能减轻CM引起的细胞凋亡和线粒体功能损伤。结论:TLR4/NLRP3炎症复合体参与了CM致急性肾损伤的发病机制,并介导了CM诱导的肾小管上皮细胞损伤和炎症。

AIM ： To investigate whether Toll-like receptor 4 （ TLR4 ） and Nod-like receptor protein 3 （NLRP3） inflammasome were involved in contrast medium （CM）-induced inflammation and injury in renal tubular epithe-lial cells. METHODS ： Iopromide was used to injure NRK-52E cells in the study. The cell viability was measured by CCK-8 assay. The protein levels of TLR4, NLRP3, apoptosis-associated speckle-like protein （ ASC） , caspase-1 and cleaved caspase-3 were determined by Western blot. The releases of interleukin （IL） - lp and IL-18 were detected by ELISA. The apoptotic rate was evaluated by Hoechst staining, and mitochondrial membrane potential （MMP） was analyzed by JC-1 staining. siRNA was transfected into the NRK-52E cells to silence NLRP3 expression. RESULTS： CM decreased the viability of NRK-52E cells （ P 〈 0. 05 ） . CM also elevated the protein levels of cleaved caspase-3 , TLR4 , NLRP3 , IL- lp and IL-18 （ P 〈 0. 05 ）. Silencing NLRP3 attenuated CM-induced releases of inflammatory cytokines. Moreover, treat-ment with TLR4 inhibitor TAK-242 or knockdown of NLRP3 by siRNA transfection both attenuated cell apoptosis and loss of MMP caused by CM. CONCLUSION： TLR4/NLRP3 inflammasome takes part in the pathogenesis of CM-induced acute kidney injury, and mediates CM-induced injury and inflammation in renal tubular epithelial cells.