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    基于PI3K/Akt信号通路探讨伊伐布雷定对冠心病大鼠血小板活性的影响

    Investigate the Effect of Ivabradine on Platelet Activity in Rats with Coronary Heart Disease Based on PI3K/Akt Signaling Pathway

      摘要
    • 摘要:
      目的  基于PI3K/Akt信号通路探讨伊伐布雷定对冠心病大鼠血小板活性的影响。
      方法 雄性Wistar大鼠经不同处理后分为对照组、模型组、伊伐布雷定组(10 mg·kg−1)和阿司匹林组(10 mg·kg−1)，每组6只，每天灌胃1次，持续4周。采用超声心动图技术评估左心室射血分数(left ventricular ejection fraction，LVEF)、左心室短轴缩短率(left ventricular fraction shortening，LVFS)；检测血浆血小板聚集率；采用流式细胞术检测血小板表面蛋白CD62p、PAC-1的表达量；采用酶联免疫吸附试验检测血浆β血小板球蛋白(plasma beta platelet globulin，β-TG)、血小板因子4(platelet factor 4，PF4)、白细胞介素6(interleukin 6，IL-6)，白细胞介素1β(interleukin 1β，IL-1β)、肿瘤坏死因子α(tumor necrosis factor α，TNF-α)水平；采用生化测试盒检测大鼠血清中总胆固醇(total cholesterol，TC)、甘油三酯(triglyceride，TG)、低密度脂蛋白胆固醇(low density lipoprotein cholesterin，LDL-C)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol，HDL-C)水平，采用比色法测定血清肌酸激酶(creatine kinase，CK)、肌酸激酶MB同工酶(creatine kinase MB isoenzyme，CK-MB)、乳酸脱氢酶(lactic dehydrogenase，LDH)、心肌肌钙蛋白T(cardiac troponin，cTnT)水平；采用Western blotting检测血小板中PI3K/Akt通路蛋白的变化；采用苏木素-伊红染色和Masson染色观察各组心肌组织病理学变化和纤维化。
      结果 治疗4周后，与模型组大鼠相比，伊伐布雷定组大鼠的LVEF、LVFS显著增加，血小板聚集率降低；血小板表面蛋白CD62p、PAC-1的表达降低，血清β-TG、PF、IL-6、IL-1β、TNF-α水平降低；血清TC、TG、LDL-C水平降低，HDL-C水平升高；血清CK、CK-MB、LDH、cTnT水平降低；且伊伐布雷定激活血小板细胞中PI3K/Akt通路蛋白的磷酸化；减轻心肌组织纤维化，改善心肌组织病理学变化。
      结论 伊伐布雷定可通过抑制冠心病大鼠血浆中血小板聚集，抑制血小板表面蛋白CD62p、PAC-1的表达和β-TG、PF4分泌，降低血浆中炎性因子水平和血脂水平，改善心肌组织病理学变化，改善心功能，其机制可能与激活血小板细胞中PI3K/Akt通路蛋白的磷酸化相关。

       

      Abstract:
      OBJECTIVE  To investigate the effect of ivabradine on platelet activity in rats with coronary heart disease based on PI3K/Akt signaling pathway.
      METHODS  After different treatments, male Wistar rats were divided into control group, model group, ivabradine hydrochlorid group(10 mg·kg−1) and aspirin group(10 mg·kg−1), 6 rats in each group were gavaged once a day for 4 weeks. Echocardiography techniques were used to evaluate the left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS). The plasma platelet aggregation rate was measured, and the expression levels of platelet surface proteins CD62p and PAC-1 were detected by flow cytometry; plasma levels of plasma beta platelet globulin(β-TG), platelet factor 4(PF 4), interleukin 6(IL-6), interleukin 1β(IL-1β), tumor necrosis factor α(TNF-α) were measured by enzyme-linked immunosorbent assay; using a biochemical test kit to detect the levels of total cholesterol(TC), triglyceride(TG), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C) in rat serum. The levels of serum creatine kinase(CK), creatine kinase MB isoenzyme(CK-MB), lactate dehydrogenase(LDH), and cardiac troponin T(cTnT) were measured using colorimetric method; Western blotting was used to detect changes in PI3K/Akt pathway proteins in platelets; Using hematoxylin eosin staining and Masson staining to observe the pathological changes and fibrosis of myocardial tissue in each group.
      RESULTS  After 4 weeks of treatment, compared with the rats in the model group, LVEF and LVFS were increased, platelet aggregation rate was decreased, the expression of the platelet surface proteins CD62p, PAC-1 were decreased, serum levels of β-TG, PF, IL-6, IL-1β and TNF-α were decreased; serum levels of TC, TG, LDL-C were decreased, and serum levels of HDL-C was increased; serum levels of CK, CK-MB, LDH, and cTnT were decreased in the ivarabradine group. And ivabradine activated the phosphorylation of PI3K/Akt pathway proteins in platelet cells; it reduced myocardial tissue fibrosis and improved myocardial histopathological changes.
      CONCLUSION  Ivarbredin can inhibit platelet aggregation in the plasma of rats with coronary heart disease, suppress the expression of platelet surface proteins CD62p and PAC-1, as well as the secretion of β-TG and PF4, and reduce the levels of inflammatory factors and blood lipid, alleviate myocardial fibrosis and improve pathological changes in myocardial tissue, improve heart function. The mechanism may be related to the phosphorylation of PI3K/Akt pathway proteins in activated platelet cells.

       

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