替格瑞洛通过akt/AMPK/eNOS信号通路调节AMI后小鼠血管新生及其机制研究

Tegretol regulation for post-AMI neovascularisation in mice through the akt/AMPK/eNOS signalling pathway and its mechanism

目的 探究替格瑞洛通过akt/AMPK/eNOS通路调节腺苷浓度,进而促进急性心肌梗死(AMI)后血管新生的作用,并探讨其潜在机制。方法 采用冠脉结扎法建立AMI的小鼠模型,使用替格瑞洛灌胃处理建立实验组,通过akt抑制剂Perifosine、AMPK抑制剂Dorsomorphin和腺苷受体抑制剂MRS1523建立对应蛋白抑制的模型,采用HE染色、Masson染色评估心肌损害情况,采用免疫组化、rt-PCR、Western blot检测VEGF蛋白水平和mRNA水平表达情况,采用酶标法检测AMI小鼠体内腺苷表达情况,随后采用Western blot检测akt/AMPK/eNOS通路蛋白及其磷酸化形式的表达情况。结果 替格瑞洛可明显上调p-akt/akt和p-eNOS/eNOS比值,下降p-AMPK/AMPK比值,并增加AMI后小鼠体内腺苷水平,进而促进心肌梗死后局部心肌细胞内VEGF蛋白水平和mRNA水平表达。结论 替格瑞洛可通过活化akt/AMPK/eNOS系统来调节AMI后小鼠体内腺苷浓度,进而促进局部心肌细胞内VEGF蛋白水平和mRNA水平表达。

Objective To investigate the role of tegretol in regulating adenosine concentration through the akt/AMPK/eNOS pathway,thereby promoting post-MI angiogenesis,and explore the underlying mechanisms.Methods A mouse model of AMI was established by coronary ligation.The experimental group was established by Tegretol gavage treatment,and the model corresponding to protein inhibition was established by akt inhibitor perifosine,AMPK inhibitor dorsomorphin and adenosine receptor inhibitor MRS1523.HE staining and Masson’s staining were used to assess myocardial damage.Immunohistochemistry,rt-PCR and Western blot were used to detect the expression of VEGF protein and mRNA levels.Adenosine expression in AMI mice was detected by enzyme labelling.Western blot was then used to detect the expression of akt/AMPK/eNOS pathway proteins and their phosphorylated forms.Results Tegretol could significantly upregulate p-akt/akt and p-eNOS/eNOS ratio,decrease p-AMPK/AMPK ratio and increase adenosine level in vivo in post-AMI mice,which in turn promoted the expression of VEGF protein level and mRNA level in local cardiomyocytes after myocardial infarction.Conclusion Tegretol can regulate adenosine concentration in vivo in post-MI mice through activation of the akt/AMPK/eNOS system,thereby promoting the expression of VEGF protein level and mRNA level in local intracardiac myocytes.