α7 nAChR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 ofα7 nAChR on inflammation and immune function in rats with myocardial infarction by regulating miR-124/TNF-αconverting enzyme

目的研究α7 nAChR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/kgα7 nAChR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(En-zyme 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CD 3^(+)、CD 4^(+)和CD 8^(+)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CD 3^(+)细胞比例明显上调(P<0.05),CD 4^(+)/CD 8^(+)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CD 3^(+)细胞比例减少(P<0.05),但是CD 4^(+)/CD 8^(+)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α7 nAChR通过上调miR-124从而抑制TACE并改善AMI大鼠炎症和免疫功能。

Objective To study the effect and mechanism ofα7 nAChR 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/kgα7 nAChR 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 tis‐sue,and kit method was used to detect creatine kinase(CK),creatine kinase MB isoenzyme(CK-MB),lactate dehydro‐genase(LDH),and cardiac troponin(cTnT)levels.The ELISA method was used to detect the levels of serum tumor ne‐crosis factorα(TNF-α),interleukin(IL)-1β,IL-6,and nuclear factor-κB(NF-κB).Flow cytometry was used to detect the proportion of blood CD 3^(+),CD 4^(+)and CD 8^(+)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-αconverting enzyme(TACE).Western blotting was used to detect TACE protein expression in myo‐cardial 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 NFκB,TNFα,IL-1β,IL-6 and the proportion of CD 3^(+)cells significantly increased(P<0.05).CD 4^(+)/CD 8^(+)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).NFκB,TNFα,IL-1β,IL-6 secretion levels and the proportion of CD 3^(+)cells were significantly down-regulated(P<0.05).The ratio of CD 4^(+)/CD 8^(+)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 promot‐ed the expression of miR 124.TACE was identified as the target gene of miR-124(P<0.05).Moreover,the miR-124 in‐hibitor significantly reduced the improvement of inflammation and immune function of GTS 21 in rats(P<0.05).Con‐clusion Activation ofα7 nAChR inhibits TACE and improves inflammation and immune function in AMI rats by upregu‐lating miR 124.