伊伐布雷定通过调控PI3K/Akt转导途径对冠心病大鼠模型血小板活性的影响

The effect of ivabradine on platelet activity in a rat model of coronary heart disease by regulating the PI3K/Akt transduction pathwayGuo Hao1, Wang Yan1, Wang Jianmei 2, Liu Zhenghao2

关键词:  伊伐布雷定  PI3/AKT通路  冠心病  血小板

Key words:  ivabradine  PI3/AKT pathway  Coronary heart disease  platelet

摘要:

目的：探讨伊伐布雷定通过调控PI3K/Akt转导途径对冠心病大鼠模型血小板活性的影响。方法：雄性Wistar大鼠分别给予低脂低胆固醇饲料和高脂高胆固醇饲料，饲养8周后，给予高脂高胆固醇饲料的大鼠腹腔注射垂体后叶素，2天后，采用超声心动图技术评估大鼠冠心病模型。取6只低脂低胆固醇饮食的大鼠为对照组（Control，LFCD+ saline），选取18只冠心病大鼠，随机分为3组，每组6只，分别为模型组（Model组，HFCD+ saline），伊伐布雷定组（Ivabradine hydrochlorid，HFCD+IVH，10mg/kg）和阿司匹林组（Aspirin，HFCD+ Aspirin，10mg/kg），每天灌胃1次，持续4周。治疗结束后，采用超声心动图技术评估各组大鼠的左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）；比较大鼠血浆的血小板聚集率，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（ELISA）检测血浆β血小板球蛋白（β-TG）、血小板因子4（PF4）的含量，炎性因子白细胞介素6（IL-6），白细胞介素1β（IL-1β），和肿瘤坏死因子α（TNF-α)含量；采用生化测试盒检测大鼠血清中总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C），高密度脂蛋白胆固醇（HDL-C）的含量，采用比色法测定血清肌酸激酶（CK）、肌酸激酶MB同工酶（CK-MB）、乳酸脱氢酶（LDH）和心肌肌钙蛋白（cTnT）的含量；采用western blot检测血小板中PI3K/Akt通路蛋白的变化；采用苏木素-伊红（H&E）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：高脂饲料饲养大鼠8周腹腔注射了30U/kg的垂体后叶素2天后可诱导大鼠冠心病模型。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；显著抑制血小板聚集；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IL-6，IL-1β，和TNF-α水平；降低血脂参数TC，TG，LDL-C水平，且升高HDL-C水平；降低血清CK、CK-MB、LDH和cTnT的含量；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参数，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 a rat model of coronary heart disease by regulating the PI3K/Akt transduction pathway. Method: Male Wistar rats were fed with low-fat and low cholesterol feed and high-fat and high cholesterol feed, respectively. After 8 weeks of feeding, rats fed with high-fat and high cholesterol feed were intraperitoneally injected with posterior pituitary hormone. Two days later, the rat coronary heart disease model was evaluated using echocardiography techniques. Six rats on a low-fat and low cholesterol diet were selected as the control group (Control, LFCD+saline), and 18 rats with coronary heart disease were randomly divided into three groups, with six rats in each group: Model group (HFCD+saline), Ivabridine hydrochloride (HFCD+IVH, 10mg/kg), and Aspirin group (Aspirin, HFCD+ Aspirin，10mg/kg）， Administer orally once a day for 4 weeks. After treatment, echocardiography techniques were used to evaluate the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of each group of rats. The platelet aggregation rate in rat plasma was compared, and the expression levels of platelet surface proteins CD62p and PAC-1 were detected by flow cytometry; Enzyme linked immunosorbent assay (ELISA) was used to detect the contents of plasma β - thromboglobulin (β - TG), platelet factor 4 (PF4), inflammatory factor interleukin-6 (IL-6), interleukin-1 β (IL-1 β), and tumor necrosis factor alpha (TNF - α); Using a biochemical test kit to detect the levels of total cholesterol (TC), triglycerides (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 blot was used to detect changes in PI3K/Akt pathway proteins in platelets; Using hematoxylin eosin (H&E) staining and Masson staining to observe the pathological changes and fibrosis of myocardial tissue in each group. Result: After 8 weeks of feeding rats with high-fat diet, intraperitoneal injection of 30U/kg of posterior pituitary hormone for 2 days can induce a rat model of coronary heart disease. After 4 weeks of treatment, compared with the model group rats, the LVEF and LVFS of the ivabradine group rats were significantly increased; Inhibit platelet aggregation; Inhibit 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 IL-6, IL-1 β, and TNF - α in plasma; Reduce blood lipid parameters TC, TG, LDL-C levels, and increase HDL-C levels; Reduce the levels of serum CK, CK-MB, LDH, and cTnT; Activate the phosphorylation of PI3K/Akt pathway proteins in platelet cells; Reduce myocardial fibrosis and improve pathological changes in myocardial tissue. Conclusion: Ivar Bredin 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 parameters in the plasma, 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.