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氢溴酸加兰他敏介导AMPKα1/Nrf2/HO-1通路对大鼠心肌缺血再灌注损伤的保护作用

The Regulation of AMPKα1/Nrf2/ HO-1 Pathway Mediated by Galantamine Hydrobromide Lycoremine in Myocardial Ischemia Reperfusion Rats

    摘要
  • 摘要:
      目的  研究氢溴酸加兰他敏（galantamine hydrobromide lycoremine，Gal）介导腺苷酸激活蛋白激酶（AMPK）α1/Nrf2/血红素加氧酶 (heme oxygenase-1, HO-1)通路对心肌缺血再灌注（I/R）大鼠内质网应激型凋亡、心肌凋亡和纤维化的影响。
      方法  构建心肌I/R损伤大鼠模型，将大鼠随机分为5组：对照组、I/R 模型组、Gal 1 mg/kg组、Gal 2 mg/kg组、Gal 4 mg/kg组进行后续实验。多普勒超声检测各组大鼠左室射血分数（LVEF）、左心室舒张末期容积（LVEDV）、左心室收缩末期容积（LVESV）、左心室壁厚度（LVWT）、左室短轴缩短率（FS）；HE染色检测心肌组织病理损伤程度；免疫组化检测Caspase-3阳性表达率；RT-PCR检测心肌Caspase-3 mRNA表达；蛋白免疫印迹检测内质网应激因子CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein，CHOP)、cleaved Caspase-12、生长抑制和DNA损伤诱导蛋白(growth arrest and DNA damageinducible protein 34,GADD34)、免疫球蛋白重链结合蛋白(immunoglobulin heavy-chain-binding protein, BiP)、α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA )、Collagen Ⅰ、AMPKα1、Nrf2、HO-1蛋白表达水平；并加入AMPK的抑制剂Compound C进行验证。
      结果  与I/R 模型组比较，Gal 各组心肌组织病理损伤程度明显改善，cleaved Caspase-3阳性表达率和Caspase-3 mRNA水平表达降低（P<0.05）；Gal 2 mg/kg、Gal 4 mg/kg组LVWT、FS、LVEF升高（P<0.05），LVEDV、LVESV降低（P<0.05），CHOP、cleaved Caspase-12、α-SMA、Collagen Ⅰ、AMPKα1、Nrf2、HO-1蛋白表达降低（P<0.05），GADD34、BiP蛋白表达升高（P<0.05）。加入AMPK抑制剂Compound C后，与I/R模型组相比较，CC 组AMPKα1、Nrf2、HO-1、BiP蛋白水平降低（P<0.05），CHOP、α-SMA、Collagen Ⅰ蛋白水平升高（P<0.05），LVESV、FS降低（P<0.05），Caspase-3 mRNA水平升高（P<0.05）。与CC组比较，CC+Gal 4mg/kg组AMPKα1、Nrf2、HO-1、BiP蛋白水平升高（P<0.05），CHOP、α-SMA、Collagen Ⅰ蛋白水平降低（P<0.05），LVESV、FS升高，Caspase-3 mRNA水平降低（P<0.05）。
      结论  氢溴酸加兰他敏介导AMPKα1/Nrf2/HO-1通路可调节心肌缺血再灌注大鼠内质网应激型凋亡、心肌凋亡和纤维化。

     

    Abstract:
      Objective  To investigate the effects of AMPKα1/Nrf2/heme oxygenase-1 (HO-1) pathway mediated by galantamine hydrobromide lycoremine (Gal) on endoplasmic reticulum stress apoptosis, myocardial apoptosis and fibrosis in rats with myocardial ischemia reperfusion (I/R).
      Methods  A myocardial ischemia reperfusion injury rat model was established, and the rats were randomly divided into 5 groups: Control group, I/R model group, Gal 1 mg/kg group, Gal 2 mg/kg group and Gal 4 mg/kg group. Left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular wall thickness (LVWT), and left ventricular short-axis shortening rate (FS) were detected by doppler ultrasound. Hematoxylin eosin staining was used to detect the pathological damage of myocardial tissue. The expression of Caspase-3 was detected by immunohistochemistry. Protein expression levels of CCAAT/enhancer-binding protein homologous protein (CHOP), cleaved Caspase-12, growth arrest and DNA damageinducible protein 34 (GADD34), immunoglobulin heavy-chain-binding protein (BiP), α-smooth muscle actin (α-SMA), Collagen Ⅰ, AMPKα1, Nrf2, and HO-1 were measured by western blot, and AMPK inhibitor Compound C was added for verification.
      Results  Compared with the I/R model group, the grade of pathological damage of myocardial tissue in each group of Gal was improved, and cleaved Caspase-3 positive expression rate and Caspase-3 mRNA level were significantly reduced (P<0.05) as well. The results showed that LVWT, FS and LVEF in Gal 2 mg/kg and Gal 4 mg/kg groups were significantly increased (P<0.05), LVEDV and LVESV were significantly reduced (P<0.05) compared with I/R model group. CHOP, cleaved Caspase-12, α-SMA, Collagen Ⅰ, AMPKα1, Nrf2, HO-1 protein levels were significantly reduced (P<0.05), and GADD34 and BiP protein levels were significantly increased (P<0.05) in Gal 2 mg/kg and Gal 4 mg/kg groups.
      Conclusion  The regulation of AMPKα1/Nrf2/HO-1 pathway mediated by Gal on endoplasmic reticulum stress apoptosis, myocardial apoptosis and fibrosis in myocardial ischemia reperfusion rats.

     

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