刘道权,宋 丹,鄢 华,苏 晞.α7nAChR通过调节miR-124对心肌梗死大鼠TNF-α转化酶、炎症反应及免疫功能的影响[J].转化医学杂志,2021,10(5):291-297
α7nAChR通过调节miR-124对心肌梗死大鼠TNF-α转化酶、炎症反应及免疫功能的影响
Effect of α7nAChR on inflammation and immune function in rats with myocardial infarction by regulating miR-124 / TNF-α converting enzyme
  
DOI:
中文关键词:  急性心肌梗死  大鼠  人α7烟碱型乙酰胆碱受体  微小RNA-124  免疫  炎症
英文关键词:Acute myocardial infarction  Rat  Human α7 nicotinic acetylcholine receptor  MicroRNA-124  Immunity  Inflammation
基金项目:
作者单位
刘道权 武汉亚洲心脏病医院 
宋 丹 武汉亚洲心脏病医院 
鄢 华 武汉亚洲心脏病医院 
苏 晞 武汉亚洲心脏病医院 
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中文摘要:
      目的 研究α7nAChR激活对急性心肌梗死大鼠免疫炎症功能的影响并探讨机制。方法 使用成年SD大鼠构建AMI模型设立为AMI组,设立假手术(sham)组为对照;AMI大鼠注射5、10、20 mg/kg α7nAChR激动剂GTS-21,并在20 mg/kg GTS-21基础上联合注射miR-124抑制剂(Inhibitor+GTS-21-20),每组10只大鼠。统计检测心肌组织梗死面积,试剂盒法检测肌酸激酶(creatine kinase, CK)、CK的MB同工酶(creatine kinase-isozyme MB, CK-MB)、乳酸脱氢酶(lactic dehydrogenase, LDH)、心肌肌钙蛋白(cardiac troponin, cTnT)水平;酶联免疫吸附试验(Enzyme Linked Immunosorbent Assay, ELISA)法检测血清肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、白介素-1β(interleukin-1β, IL)-1β、IL-6、核因子-κB(NF-κB)的水平。流式细胞术检测血液CD3+、CD4+和CD8+细胞亚群比例;实时荧光定量PCR(RTqPCR)检测心肌组织中miR-124的水平;荧光素酶报告基因检验miR-124和TNF-α转化酶(TACE)的相互作用。使用蛋白免疫印迹法检测心肌组织中TACE的蛋白表达。结果 与sham组比较,AMI组大鼠的梗死面积、CK、CK-MB、LDH、cTnT的水平均明显增加(P<0.05),而且NF-κB、TNF-α、IL-1β、IL-6分泌水平和CD3+细胞比例明显上调(P<0.05),CD4+/CD8+细胞比率下调(P<0.05)。与AMI组比较,AMI联合注射GTS-21后,miR-124的水平增加(P<0.05),大鼠的梗死面积增加,CK、CK-MB、LDH、cTnT的水平均增加(P<0.05);而且NF-κB、TNF-α、IL-1β、IL-6的水平下调,以及CD3+细胞比例减少(P<0.05),但是CD4+/CD8+细胞比率上调(P<0.05)。另外,TACE在AMI中表达上调,而GTS-21明显抑制TACE的表达,并促进miR-124的表达,还确定TACE是miR-124的靶基因。miR-124的抑制剂inhibitor明显减弱GTS-21对大鼠炎症和免疫功能的改善作用(P<0.05)。结论 激活α7nAChR通过上调miR-124从而抑制TACE并改善AMI大鼠炎症和免疫功能。
英文摘要:
      Objective To study the effect and mechanism of α7nAChR activation on immune and inflammatory function in rats with acute myocardial infarction. Methods 60 adult SD rats were used in this study in 6 groups with 10 in each group. 50 were used to construct the AMI model, and 10 were used as the sham group(control). 40 AMI rats were injected with 5, 10, and 20 mg/kg α7nAChR agonist GTS-21, miR-124 inhibitor co-injected with 20 mg/kg GTS-21(Inhibitor+GTS-21-20 group) with 10 in each group. Rats were statistically tested for infarct size of myocardial tissue, and kit method was used to detect creatine kinase (CK), creatine kinase MB isoenzyme (CK-MB), lactate dehydrogenase (LDH), and cardiac troponin (cTnT) levels. The ELISA method was used to detect the levels of serum tumor necrosis factor-α (TNF-α), interleukin (IL) -1β, IL-6, and nuclear factor-κB (NF-κB). Flow cytometry was used to detect the proportion of blood CD3+, CD4+ and CD8+cell subsets. Real-time fluorescence quantitative PCR (RTqPCR) was used to detect the level of miR-124 in myocardial tissue. Luciferase reporter gene was used to detect the interaction of miR-124 and TNF-α converting enzyme (TACE). Western blotting was used to detect TACE protein expression in myocardial tissue. Results In comparison with sham group, the infarct size, CK, CK-MB, LDH, cTnT of the AMI group were significantly increased (P<0.05); and the secretion levels of NF-κB, TNF-α, IL-1β, IL-6 and the proportion of CD3+ cells significantly increased (P<0.05). CD4+/ CD8+ cell ratio was down-regulated (P<0.05). Compared with AMI group, the level of miR-124 increased in AMI+GTS-21 groups (P<0.05). the infarct size, CK, CK-MB, LDH, cTnT were increased significantly (P<0.05). NF-κB, TNF-α, IL-1β, IL-6 secretion levels and the proportion of CD3+ cells were significantly down-regulated (P<0.05). The ratio of CD4+/CD8+ cells was up-regulated (P<0.05). In addition, the expression of TACE was up-regulated in AMI, while GTS-21 significantly inhibited the expression of TACE and promoted the expression of miR-124. TACE was identified as the target gene of miR-124 (P<0.05). Moreover, the miR-124 inhibitor significantly reduced the improvement of inflammation and immune function of GTS-21 in rats (P<0.05) . Conclusion Activation of α7nAChR inhibits TACE and improves inflammation and immune function in AMI rats by upregulating miR-124.
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