心肌缺血再灌注损伤小鼠心肌组织中长链非编码RNA Neat1的表达及其对细胞焦亡的作用机制  

Expression of long non-coding RNA Neat1 in myocardial tissue of mice with myocardial ischemia-reperfusion injury and its mechanism on pyroptosis

作　　者：柴鑫 梁政伟 张俊诗 丁菁 张倩 吕莎 邓亚竹 张荣瑞 陆德琴 CHAI Xin;LIANG Zhengwei;ZHANG Junshi;DING Jing;ZHANG Qian;LYU Sha;DENG Yazhu;ZHANG Rongrui;LU Deqin(Department of Radiology,the affiliated hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research on Common Chronic Diseases,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Pathophysiology,Guizhou Medical University,School of Basic Medical Sciences,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学附属医院放射科,贵州贵阳550004 [2]贵州省常见慢性疾病发病机制及药物防治研究重点实验室,贵州贵阳550025 [3]贵州医科大学基础医学院病理生理学教研室,贵州贵阳550025

出　　处：《贵州医科大学学报》2024年第12期1749-1759,共11页Journal of Guizhou Medical University

基　　金：贵州省科技厅基础研究计划项目(黔科合基础-ZK[2024]一般249);贵州省教育厅普通高等学校青年科技人才成长项目(黔[2021]144)。

摘　　要：目的探讨心肌缺血再灌注损伤(MIRI)对小鼠心肌组织中长链非编码RNA Neat1(Lnc Neat1)的表达及细胞焦亡的影响。方法30只C57BL/6小鼠随机均分为假手术(sham)组[左冠状动脉前降支(LAD)下穿线但不结扎]、缺血/再灌注(I/R)组(结扎LAD),抽取2组小鼠下腔静脉血,麻醉处死后取心脏,采用罗氏全自动生化分析仪检测2组小鼠血清乳酸脱氢酶(LDH)及肌酸激酶同工酶(CK-MB)的活性,采用氯化三苯基四氮唑(TTC)及苏木精-伊红(HE)染色观察2组小鼠心肌组织的心肌梗死面积和组织学特征变化;利用HL-1小鼠心肌细胞构建体外MIRI模型,将HL-1细胞随机分为缺氧4 h组(H4)组和其对照(Control 4 h组)、缺氧4 h复氧1 h(H4R1)组和其对照(Control 5 h)组、缺氧4 h复氧2 h(H4R2)组和其对照(Control 6 h)组、缺氧4 h复氧4 h(H4R4)组和其对照(Control 8 h)组,采用细胞计数试剂盒-8(CCK-8)法检测细胞活力,乳酸脱氢酶(LDH)试剂盒检测细胞培养上清液中LDH漏出率,采用碘化丙啶(PI)染色观察PI染色阳性率;采用慢病毒感染构建敲低/过表达Lnc Neat1基因,将HL-1细胞随机分为Control组、缺氧4 h复氧4 h组(H4R4)组、敲低Lnc Neat1空载对照组(sh-Lnc Neat1 NC)组、sh-Lnc Neat1 NC+H4R4组、sh-Lnc Neat1组、sh-Lnc Neat1+H4R4组、过表达Lnc Neat1空载对照组(OE-Lnc Neat1 NC)组、OE-Lnc Neat1 NC+H4R4组、OE-Lnc Neat1组及OE-Lnc Neat1+H4R4组,采用实时荧光定量PCR(qRT-PCR)法检测Lnc Neat1表达及核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、与凋亡相关斑点样蛋白(ASC)信使RNA(mRNA)表达,采用Western blot法检测NLRP3蛋白、ASC蛋白寡聚化及半胱氨酸天冬氨酸蛋白酶-1前体(pro-caspase-1)、cleaved-caspase-1、焦孔素家族蛋白D(GSDMD)、cleaved N-terminal GSDMD、白细胞介素-1β(IL-1β)及IL-18蛋白的表达。结果与sham组相比,MIRI小鼠血清中LDH、CK-MB活性增加(P<0.05),TTC染色法结果显示I/R组小鼠心肌梗死面积约33%(P<0.05),HE染色法结果显Objective To explore the expression of long non-coding RNA Neat1(Lnc RNA Neat1)in myocardial tissue of mice with myocardial ischemia-reperfusion injury(MIRI)and its mechanism on pyroptosis.Methods Thirty C57BL/6 mice were randomly divided into sham group[left anterior descending(LAD)coronary artery undergoing threaded without ligation]and ischemia/reperfusion(I/R)group(LAD ligation).Blood was withdrawn from mouse inferior vena cava in 2 groups.The hearts were collected after anesthesia.Roche automatic biochemical analyzer was used to examine the activities of mouse serum lactate dehydrogenase(LDH)and creatine kinase MB isoenzyme(CK-MB)in 2 groups.Triphenyltetrazolium chloride(TTC)and hematoxylin-eosin(HE)staining were used to detect mouse myocardial infarct size and histological features in 2 groups.MIRI model was constructed using HL-1 cardiomyocytes in vitro.HL-1 cardiomyocytes were randomly divided into 4 h hypoxia(H4 group)and its control(Control 4 h),4 h hypoxia and reoxygenation 1 h(H4R1group)and its control(Control 5 h),4 h hypoxia and reoxygenation 2 h(H4R2 group)and its control(Control 6 h group),4 h hypoxia and reoxygenation 4 h(H4R4)group and its control(Control 8 h group).Cell viability was detected by cell counting kit 8(CCK-8).LDH leakage rate was detected by LDH kit.Propyl iodide(PI)staining was used to observe the positive rate of PI staining.Lentiviral infection was used to construct cells with knockdown or overexpression of Lnc neat1.HL-1 cells were randomly divided into the following groups:control,4 h hypoxia and reoxygenation 4 h(H4R4),knockdown control(sh-Lnc Neat1 NC),sh-Lnc Neat1 NC+H4R4,sh-Lnc Neat1,sh-Lnc Neat1+H4R4,negative control for overexpression OE-Lnc Neat1 NC),OE-Lnc Neat1 NC+H4R4,OE-Lnc Neat1 and OE-Lnc Neat1+H4R4.qRT-PCR was applied to detect the expression of Lnc Neat1,the messenger RNA(mRNA)expressions of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)and apoptosis-associated speck-like protein containing a CARD(ASC).Western blo

关 键 词：心肌缺血 再灌注损伤 细胞焦亡 长链非编码RNA Neat 1 NLRP3炎症小体 HL-1小鼠心肌细胞 缺氧/复氧 

分 类 号：R363.21[医药卫生—病理学]