NLRP3炎症小体激活介导的巨噬细胞极化在糖尿病小鼠缺血性脑卒中后心肌损伤中的作用

Role of NLRP3 inflammasome activation-mediated macrophage polarization in myocardial injury after ischemic stroke in diabetic mice

目的评价NOD样受体蛋白3(NLRP3)炎症小体激活介导的巨噬细胞极化在糖尿病小鼠缺血性脑卒中后心肌损伤中的作用。方法4~6周龄雄性野生型C57BL/6J小鼠和NLRP3^(-/-)小鼠,通过高糖高脂饮食联合链脲佐菌素注射的方法构建糖尿病模型。取糖尿病野生型C57BL/6J小鼠24只和NLRP3-/-小鼠23只,采用随机数字表分为野生型假手术组(WT_(D)-SHAM组,n=9)、野生型缺血性脑卒中组(WT_(D)-MCAO组,n=15)、NLRP3^(-/-)假手术组(NLRP3^(-/-)_(D)-SHAM组,n=9)和NLRP3^(-/-)缺血性脑卒中组(NLRP3^(-/-)_(D)-MCAO组,n=14)。采用大脑中动脉阻塞法建立缺血性脑卒中模型。于建模后3、7、14和28 d时行超声心动图和心电图检查。于建模后28 d时,深麻醉下处死小鼠,取心肌组织,采用实时荧光定量PCR法检测巨噬细胞标志物F4/80和M2型巨噬细胞标志物CD206的mRNA表达。结果与WT_(D)-SHAM组相比,WT_(D)-MCAO组建模后28 d时心排血量、左心室质量和左心室校正质量均下降,建模后14和28 d时QT间期和QTc间期均延长(P<0.05)。与NLRP3^(-/-)_(D)-SHAM组相比,NLRP3^(-/-)_(D)-MCAO组建模后3 d时心排血量、左心室质量和左心室校正质量均下降,QT间期和QTc间期均延长(P<0.05)。WT_(D)-SHAM组与WT_(D)-MCAO组、NLRP3^(-/-)_(D)-SHAM组与NLRP3^(-/-)_(D)-MCAO组,心肌组织CD206和F4/80的mRNA表达比较差异无统计学意义(P>0.05)。与WT_(D)-MCAO组相比,NLRP3^(-/-)_(D)-MCAO组建模后14和28 d时QT间期和QTc间期均缩短,建模后28 d时心肌组织F4/80 mRNA表达下调,CD206 mRNA表达上调(P<0.05)。结论NLRP3炎症小体激活介导的巨噬细胞向M2型极化,参与了糖尿病小鼠缺血性脑卒中后心肌损伤的过程。

Objective To evaluate the role of NOD-like receptor 3(NLRP3)inflammasome activation-mediated macrophage polarization in myocardial injury after ischemic stroke in diabetic mice.Methods Wild-type C57BL/6J mice and NLRP3^(-/-)mice,aged 4-6 weeks,were fed a high fat diet combined with streptozotocin administration to develop the diabetic model.Twenty-four diabetic wild type C57BL/6J mice and 23 diabetic NLRP^(3-/-)mice were divided into wild type sham operation group(WT_(D)-SHAM group,n=9),wild type ischemic stroke group(WT_(D)-MCAO group,n=15),NLRP3^(-/-)sham operation group(NLRP3^(-/-)_(D)-SHAM group,n=9)and NLRP3^(-/-)ischemic stroke group(NLRP3^(-/-)_(D)-MCAO group,n=14).The ischemic stroke model was developed by middle cerebral artery occlusion in the animals anesthetized with isoflurane.Echocardiography and electrocardiography were carried out at 3,7,14 and 28 days after developing the model.Mice were sacrificed under deep anesthesia,and myocardial tissues were taken at 28 days after surgery for determination of the expression of macrophage marker F4/80 and M2 type macrophage marker CD206 mRNA(by real-time fluorescence quantitative polymerase chain reaction).Results Compared with WT_(D)-SHAM group,the cardiac output,mass of left ventricle and corrected mass of left ventricle were significantly decreased at 28 days after surgery,and QT interval and QTc interval were prolonged at 14 and 28 days after developing the model in WT_(D)-MCAO group(P<0.05).Compared with NLRP3^(-/-)_(D)-SHAM group,the cardiac output,mass of left ventricle and corrected mass of left ventricle were significantly decreased,and QT interval and QTc interval were prolonged at 3 days after surgery in NLRP3^(-/-)_(D)-MCAO group(P<0.05).There was no significant difference in CD206 and F4/80 mRNA expression between WT_(D)-SHAM group and WT_(D)-MCAO group and between NLRP3^(-/-)_(D)-SHAM group and NLRP3^(-/-)_(D)-MCAO group(P>0.05).Compared with WT_(D)-MCAO group,the QT interval and QTC interval were significantly shortened at 14 and 28 days after developing the model,and the expression of F4/80 mRNA was down-regulated and the expression of CD206 mRNA was up-regulated at 28 days after developing the model in NLRP3^(-/-)_(D)-MCAO group(P<0.05).Conclusions NLRP3 inflammasome activation-mediated polarization of macrophages to M2 phenotype is involved in myocardial injury after ischemic stroke in diabetic mice.