沉默NLRP3炎性小体表达降低香烟烟雾提取物导致的人肺动脉内皮细胞凋亡

目的探讨NLPR3炎性小体(含NLR家族pyrin结构域蛋白3)对香烟烟雾提取物(CSE)诱导人肺动脉内皮细胞(HPAECs)凋亡的影响。方法用NLRP3 siRNA和NC siRNA(阴性对照)分别转染HPAECs后(分别为siNLRP3组、siNC组),再以10%CSE刺激12 h,应用annexin V/PI流式法检测这两组细胞在CSE刺激前后的凋亡水平。再将HPAECs分为对照(control)组、CSE组(加入10%CSE液)、NAC(乙酰半胱氨酸,N-acetylcysteine)+CSE组(以10 mmol/L NAC预处理1h后再以10%CSE培养液处理)及NAC组(以10 mmol/L NAC单独处理),均培养12 h后以DCFH-DA光探针标记法观察细胞内活性氧(reactive oxygen species,ROS),Western blot法测定NLRP3、caspase-1蛋白表达。结果siNC组在CSE刺激后,HPAECs存活率下降,凋亡率较CSE刺激前明显增加(P<0.05),SiNLRP3组在CSE刺激后,细胞凋亡率较siNC组同等CSE刺激后有所减少(P<0.05)。分组处理HPAECs后,CSE组细胞胞内ROS水平较对照组升高(P<0.05),NAC+CSE组细胞胞内ROS水平较CSE组有所下降(P<0.05)。CSE组NLRP3、caspase-1蛋白表达较对照组显著升高(P<0.05),而NAC+CSE组细胞NLRP3、caspase-1表达较CSE组下降(P<0.05)。结论CSE能诱导HPAECs发生凋亡及胞内ROS增加,伴有NLRP3炎性小体表达增加。NLRP3蛋白表达沉默能抑制CSE对HPAEC的促凋亡作用。

Silencing NLRP3 inflammasome suppresses apoptosis of humanpulmonary artery endothelial cells induced by cigarette smoke extract

Objective To investigate the mechanism of cigarette smoke extract(CSE)inducing apoptosis of pulmonary artery endothelial cells(HPAECs),especially focus on the role of NLRP3(NLR family pyrin domain protein 3)inflammasome.Methods After transfection with NLRP3 siRNA and NC siRNA,HPAECs from experimentalgroup and control group were stimulated by 10%CSE for 12 h,annexin V/PI flow method was used to detect the apoptosis of the two groups of cells before and after CSE stimulation.Cultured HPAECs were further divided into four groups:control group,CSE group(10%CSE solution was added),NAC+CSE group(10 mmol/L NAC pretreatment for 1 h and then 10%CSE culture solution)and NAC group(10 mmol/L NAC treatment for 1 h).After 12 h culture,intracellular reactive oxygen species(ROS)were detected by DCFH-DA fluorescent probe.The expression of NLRP3 and caspase-1 was determined by Western blot.Results After CSE stimulation,the survival rate of HPAECs in siNC group decreased,and the apoptosis was significantly increased as compared with that before CSE stimulation(P<0.05).After CSE stimulation,the apoptosis rate of siNLRP3 group decreased compared with that after the same CSE stimulation in siNC group(P<0.05).The intracellular ROS level in the CSE group was significantly increased as compared with that in the control group,which in the NAC+CSE group was decreased compared with that in the CSE group(P<0.05).In the CSE group,NLRP3 and caspase-1 were significantly increased compared with the control group,while the expression of NLRP3 and caspase-1 in NAC+CSE group was lower than that in CSE group(P<0.05).Conclusions CSE could induce apoptosis of HPAECs accompanied by increased NLRP3 inflammatory corpuscle expression.Silencing of NLRP3 protein expression can inhibit the apoptotic effect of CSE on HPAEC.