NLRP3-CAMKⅡ-IRE-1α通路激活诱导氧化应激增强对糖尿病大鼠心室重构的影响

Effect of NLRP3-CAMKⅡ-IRE-1αpathway induced oxidative stress on ventricular remodeling in diabetic rats

目的探讨核苷酸结合寡聚结构域样受体蛋白3(NLRP3)炎性小体通过钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)激活肌醇需求激酶-1α(IRE-1α)促进氧化应激增强在糖尿病大鼠心室重构中的作用及机制。方法36只健康雄性Sprague-Dawley大鼠按随机数字表法分为对照组(CTL组)、糖尿病组(DM组)和糖尿病+格列苯脲组(GLB组),每组12只。大鼠通过单次腹腔注射55 mg/kg链脲佐菌素制备糖尿病模型,GLB组于造模成功后给予NLRP3抑制剂格列苯脲(1.25 mg/kg),连续灌胃8周。CTL组不进行任何干预。8周后记录各组血糖、血压、体质量,计算心室体质量比,测量血流动力学指标;心脏超声检查评估肺动脉血流加速时间、平均肺动脉压、收缩期与舒张期室间隔、心室前壁和心室后壁厚度;心外膜激活映射标测进行心外膜传导速度、绝对不均匀性和不均匀指数测定;采用蛋白免疫印迹检测NLRP3、胱天蛋白酶(caspase)-1、CaMKⅡ、IRE-1α、还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NOX)2、NOX4的蛋白水平;荧光染色法测定活性氧(ROS)含量;HE及Masson染色观察心室组织细胞形态和纤维化。结果与CTL组相比,DM组的血糖升高,心室体质量比增大,收缩期和舒张期室间隔厚度、左心室前壁厚度增加,体质量降低,左、右心室心外膜传导速度减慢,右心室不均匀指数增加,NLRP3、caspase-1、CaMKⅡ、IRE-1α、NOX2和NOX4蛋白表达水平升高,心室肌ROS产生增多,CVF升高(P<0.05),心室肌细胞排列紊乱,纤维化更明显;与DM组比较,GLB组收缩期和舒张期室间隔厚度、左心室前壁厚度降低,左、右心室心外膜传导速度增快,左心室绝对不均匀性和不均匀指数降低,NLRP3、caspase-1、CaMKⅡ、IRE-1α、NOX2和NOX4蛋白表达水平下降,ROS产生减少,CVF降低(P<0.05),心室肌纤维化程度减轻。结论糖尿病大鼠心室肌细胞NLRP3-CAMKⅡ-IRE-1α通路激活,促进氧化应激增强,参与心室重构,GLB可改善此变化。

Objective To explore the role and mechanism of nucleotide-binding domain-like receptor protein 3(NLRP3)inflammasome in promoting oxidative stress enhancement through triggering calmodulin-dependent protein kinaseⅡ(CaMKⅡ)to activate inositol demand kinase-1α(IRE-1α)in ventricular remodeling of diabetic rats.Methods Thirty-six healthy male Sprague-Dawley rats were randomly divided into the control group(CTL group),the diabetic group(DM group)and the diabetic+glibenclamide group(GLB group),with 12 rats in each group.Rats were injected 55 mg/kg streptozotocin intraperitoneally to prepare diabetic model.The GLB group was given 1.25 mg/kg GLB,an NLRP3 inhibitor,by gavage for 8 weeks since the successful modeling day.The CTL group received no intervention.After 8 weeks,blood glucose,blood pressure,body mass and ventricular body mass ratio of rats were recorded,and hemodynamic indexes were also measured.Pulmonary artery blood flow acceleration time,mean pulmonary artery pressure,systolic and diastolic ventricular septum,anterior and posterior ventricular wall thickness were evaluated by echocardiography.Epicardial activation mapping was used to measure epicardial conduction velocity,absolute heterogeneity and heterogeneity index.Western blot assay was used to detect NLRP3,caspase-1,CaMKⅡ,IRE-1α,niacinamide adenine dinucleotide phosphate(NOX)2 and NOX4 protein levels.The content of reactive oxygen species(ROS)was determined by fluorescence staining.The morphology and fibrosis of ventricular tissue were observed by HE and Masson staining.Results Compared with the CTL group,blood glucose,ventricular body mass ratio,systolic and diastolic septal thickness,left ventricular anterior wall thickness increased,body mass decreased,left and right ventricular epicardial conduction velocity slowed down,and right ventricular inhomogeneity index increased in the DM group.NLRP3,caspase-1,CaMKⅡ,IRE-1α,NOX2 and NOX4 protein expression levels were increased,and ROS production in ventricular muscle and CVF were increased(P<0.05).Myocardial cell arrangement was disordered,and fibrosis was more obvious in the DM group.Compared with the DM group,the thickness of ventricular septum and anterior wall of left ventricle in systolic and diastolic periods were reduced in the GLB group.Compared with the DM group,epicardial conduction velocity of left and right ventricles was increased,and absolute inhomogeneity and inhomogeneity index of left ventricle were decreased in the GLB group.The protein expression levels of NLRP3,caspase-1,CaMKⅡ,IRE-1α,NOX2 and NOX4 were lower than those of the DM group.GLB reduced the production of ROS and CVF,and the ventricular myocardial fibrosis was ameliorated(P<0.05).Conclusion The NLRP3-CAMKⅡ-IRE-1αpathway is activated in diabetic rat ventricular myocytes to promote oxidative stress and participates in ventricular remodeling,and GLB can improve this change.