shRNA干扰NLRP3对晚期糖基化终末产物诱导的心肌细胞炎症反应的影响

The Effect of Interference of NLRP3 with shRNA on AGEs-induced Inflammatory Response in Myocardial Cell

目的探讨沉默NOD样受体家族pyrin域3(NOD-like receptor family,pyrin domain containing 3,NLRP3)炎性小体对晚期糖基化终末产物(advanced glycation end products,AGEs)诱导的心肌细胞损伤的调节作用及机制。方法 H9c2心肌细胞分为4组:对照组、AGEs组、AGEs+sh-Ctrl组、AGEs+sh-NLRP3组,后两组细胞首先将短发夹RNA(shRNA)对照(sh-Ctrl)和shRNA干扰NLRP3(sh-NLRP3)质粒分别转染入H9c2细胞,然后后3组细胞用100mg/L AGEs处理24h,建立H9c2损伤模型,对照组细胞用溶剂处理24h。Western blot检测NLRP3、凋亡相关微粒蛋白(apoptosis-associated speck-like protein containing CARD,ASC)、Caspase-3、Caspase-9、核转录因子κB(nuclear factorκB,NF-κB)P65和磷酸化P65(p-P65)的表达,酶联免疫吸附实验(ELISA)检测白细胞介素(interleukin,IL)-6、IL-18和IL-1β的水平,流式细胞术检测细胞凋亡,免疫荧光染色检测细胞核中NF-κB P65的水平。结果 AGEs组和AGEs+sh-Ctrl组NLRP3、ASC、Caspase-3和Caspase-9的表达均高于对照组(P<0.05)。与AGEs组相比,AGEs+sh-NLRP3组NLRP3、ASC、Caspase-3和Caspase-9的表达均下降(P<0.05)。与对照组相比,AGEs组和AGEs+sh-Ctrl组IL-6、IL-18、IL-1β水平升高,细胞凋亡率上升(P<0.05)。AGEs+sh-NLRP3组IL-6、IL-18、IL-1β水平和细胞凋亡率低于AGEs组(P<0.05)。AGEs组和AGEs+sh-Ctrl组NF-κB P65磷酸化水平及细胞核的NF-κB P65水平高于对照组(P<0.05)。与AGEs组相比,AGEs+sh-NLRP3组NF-κB P65磷酸化水平及细胞核的NF-κB P65水平降低(P<0.05)。结论沉默NLRP3可通过抑制NF-κB P65的活化减轻AGEs诱导的心肌细胞凋亡及炎症反应。

Objective This study aims to explore the effect of silence of NOD-like receptor family,pyrin domain containing 3(NLRP3)inflammasome on advanced glycation end products(AGEs)-induced myocardial injury.Methods The myocardial injury model was indued by AGEs.NLRP3 was silenced by shRNA.H9c2 cells were divided into four groups:H9c2(control group);AGEs group;AGEs+sh-Ctrl group;AGEs+sh-NLRP3 group.The latter two groups of cells will first shRNA control(sh-Ctrl)and shRNA-NLRP3(sh-NLRP3)plasmids were transfected into H9c2 cells,the last 3cells were then treated for 24 h with 100 mg/L AGEs,establishment of H9c2 damage model,control cells were treated with solvent for 24h;Apoptosis was measured by Hoechst33258 staining.The levels of interleukin(IL)-6,IL-18 and IL-1β were detected by enzyme-linked immunosorbent assay(ELISA).The protein levels of NLRP3,apoptosis-associated speck-like protein containing CARD(ASC),Caspase-3,Caspase-9,nuclear factorκB(NF-κB)P65and p-P65 were tested by Western blot.The nuclear NF-κB P65 levels were detected by immunofluorescent staining.Results The expressions of NLRP3,ASC,Caspase-3 and Caspase-9in AGEs group and AGEs+sh-Ctrl group was higher than control group(P<0.05).Compared with AGEs group,the expressions of NLRP3,ASC,Caspase-3 and Caspase-9 in AGEs+sh-NLRP3 group was decreased(P<0.05).Compared with control group,the apoptosis and the levels of IL-6,IL-18 and IL-1β in AGEs group and AGEs+sh-Ctrl group were elevated(P<0.05).The apoptosis and the levels of IL-6,IL-18 and IL-1β in AGEs+sh-NLRP3 group were lower than those of AGEs group(P<0.05).The phosphorylation of NF-κB P65 and nuclear NF-κB P65 in AGEs group and AGEs+sh-Ctrl group were higher than control group(P<0.05).Compared with AGEs group,the phosphorylation of NF-κB P65 and nuclear NF-κB P65 in AGEs+shNLRP3 group were reduced(P<0.05).Conlusion Silence of NLRP3 inflammasome alleviates AGEs-induced apoptosis and inflammatory response in myocardial cell via inhibiting NF-κB P65 activation.