摘要
目的:研究组蛋白去乙酰化酶沉默信息调节因子1(silent information regulator 1,SIRT)对心肌缺血再灌注介导的心肌细胞焦亡的影响及相关机制。研究对象:选择95只SD雄性大鼠,体重250-300 g,其中80只采用随机数字表法分为5组:假手术组、模型组、SIRT1高表达组、SIRT1低表达组和单磷酸腺苷激活的蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)信号通路抑制组,每组16只;另15只大鼠采用随机数字表法分为3组:对照组,SIRT1高表达组和SIRT1低表达组,每组5只。干预措施:80只大鼠每组处理方法如下:(1)假手术组:仅开胸暴露心脏,不做心肌缺血再灌注处理;(2)模型组:心肌内注射空白载体慢病毒7 d后,构建心肌缺血再灌注模型;(3)SIRT1高表达组:心肌内注射SIRT1高表达慢病毒7 d后,构建心肌缺血再灌注模型;(4)SIRT1低表达组:心肌内注射SIRT1小干扰RNA(small interfering RNA,siRNA)表达慢病毒7 d后,构建心肌缺血再灌注模型;(5)AMPK信号通路抑制组:心肌内注射SIRT1高表达慢病毒7 d后,给予AMPK信号通路抑制剂compound C,构建心肌缺血再灌注模型。SD大鼠麻醉后,通过慢病毒注射技术,按每只老鼠1×10^(7)TU/30μl的量,将空白载体慢病毒、过表达SIRT1慢病毒或SIRT1 siRNA慢病毒,在左心室前壁分别取5个部位进行心肌注射。在注射慢病毒后第7天,阻断左前降支冠状动脉40 min再松开120 min,构建心肌缺血再灌注模型。另外15只大鼠用于检测SIRT1慢病毒感染效率,分为:(1)对照组:心肌注射空白载体慢病毒7 d;(2)SIRT1高表达组:心肌内注射SIRT1高表达慢病毒7 d;(3)SIRT1低表达组:心肌内注射SIRT1 siRNA表达慢病毒7 d。观测指标与测量方法:待构建模型后对大鼠行安乐死,取缺血区心肌组织,采用生化检测以评价血清心肌酶活性;采用Westernblot法检测焦亡相关蛋白核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain-like recep
Objective:To investigate the effects and mechanism of the histone deacetylase silent information regulator 1(SIRT1)on pyroptosis mediated by myocardial ischemia-reperfusion.Subjects:Ninty-five male Sprague Dawley(SD)rats,weighing 250-300 g,and eighty of them were randomly assigned to five groups:sham surgery group,model group,high SIRT1 expression(SIRT1^(+))group,low SIRT1 expression(SIRT1^(-))group,and adenosine monophosphate-activated protein kinase(AMPK)pathway inhibition group(n=16 rats per group).The other fifteen rats were randomly assigned to three groups:control group,high SIRT1 expression(SIRT1^(+))group,low SIRT1 expression(SIRT1^(-))group(n=5 rats per group).Interventions:Sham surgery group:thoracotomy was conducted to expose the heart,but myocardial ischemia-reperfusion was not performed.Model group:lentiviruses carrying an empty vector were injected intramyocardially,followed by the induction of myocardial ischemia-reperfusion 7 days later.SIRT1^(+)group:lentiviruses carrying the SIRT1 gene were injected intramyocardially,followed by the induction of myocardial ischemia-reperfusion 7 days later.SIRT1^(-)group:lentiviruses carrying small interfering RNAs(siRNAs)targeting SIRT1 were injected intramyocardially,followed by the induction of myocardial ischemia-reperfusion 7 days later.AMPK pathway inhibition group:lentiviruses carrying the SIRT1 gene were injected intramyocardially,followed by treatment with compound C,an AMPK pathway inhibitor,and the induction of myocardial ischemia-reperfusion 7 days later.Main Outcomes and Measurements:The rats were euthanized after model construction.Ischemic myocardial tissue was collected,and the activity of serum myocardial enzymes was measured via biochemical testing.Western blot analysis was employed to examine the expression levels of pyroptosis-associated proteins,including nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing a caspase activation and recruitment domain(ASC),caspase-1 P10,an
作者
钟泽
罗秀英
相鹏
季红慧
吴新东
崇爱国
胡新央
Zhong Ze;Luo Xiuying;Xiang Peng;Ji Honghui;Wu Xindong;Chong Aiguo;Hu Xinyang(Department of Cardiology,Jiande First People’s Hospital,Jiande,Zhejiang 311600,China;Department of Cardiology,the Second affiliated Hospital of Zhejiang University School of Medicine,Hangzhou 310000,China)
基金
2018浙江省医药卫生科技计划项目(2018KY661)
2017杭州市科技计划引导项目(20171226Y278)
