芪参益气滴丸对急性心梗大鼠α7nAChR抗炎通路的影响

Effect of Qishen Yiqi dripping pills on α7nAChR anti-inflammatory pathway in rats with AMI

目的探讨芪参益气滴丸对急性心肌梗死(AMI)大鼠α7烟碱型乙酰胆碱能受体(α7nAChR)抗炎通路的影响,分析其作用机制。方法选用45只成年雄性(Sprague-Dawley,SD)大鼠,随机分为假手术组,模型组及治疗组,每组15只。采用结扎左冠状动脉前降支建立AMI动物模型,假手术组仅手术不结扎血管。术后第2 d,治疗组灌芪参益气滴丸40 mg/(kg.d),假手术组及模型组给予等量生理盐水,连用28 d,并记录各组大鼠的生存情况。在第28 d,超声心动图评估大鼠心功能,苏木精-伊红(HE)及三色(Masson)染色分析心肌组织形态学变化、原位末端标记法(TUNEL)分析心肌细胞凋亡变化,酶联免疫吸附试验法分析血清中白介素-6(IL-6)及肿瘤坏死因子-α(TNF-α)炎性因子的变化,蛋白印迹法及聚合酶链式反应分析心肌组织中IL-6、TNF-α、α7nAChR、NF-κB p50及NF-κB p65蛋白及mRNA的表达水平的变化。结果模型组与治疗两组大鼠存活率无明显差异(P> 0.05);超声心动图检测显示,治疗组大鼠的心功能明显恢复,左室射血分数(LVEF)升高(P <0.05),左室舒张末期内径(LVDd)、左室收缩末期内径(LVDs)降低(P <0.05);HE及Masson染色分析显示,心肌组织恢复好,细胞核肥大减轻较为清晰,有少量炎性细胞浸润及成纤维细胞增生,胶原纤维增生不明显;TUNEL分析心肌细胞凋亡表明,细胞凋亡个数明显降低(P <0.05);酶联免疫吸附试验法分析表明,血清中IL-6、TNF-α炎性因子明显降低(P <0.05);蛋白印迹法分析表明,心肌组织中α7nAChR蛋白明显提高(P <0.05),IL-6,TNF-α、NF-κB p50及NF-κB p65蛋白明显降低(P <0.05);聚合酶链式反应分析表明,心肌组织中IL-6,TNF-α、NF-κB p50及NF-κB p65mRNA水平明显降低(P <0.05)。结论芪参益气滴丸可以改善AMI后心功能、减轻AMI损伤引发的炎症反应、细胞凋亡及心室重构,其机制可能与α7nAChR抗炎通路有关。

Objective To explore the effect of Qishen Yiqi dripping pills on α7 nicotinic acetylcholine receptor(α7 n AChR) anti-inflammatory pathway in rats with acute myocardial infarction(AMI) and analyze its mechanism. Methods A total of 45 adult male(Sprague-Dawley, SD) rats were selected and randomized into the sham operation group, model group and treatment group, 15 rats for each group. An AMI animal model was established by ligating the left anterior descending coronary artery(LADCA). In the sham operation group, operation was performed and the artery was not ligated. On Day 2 after operation, the treatment group took Qishen Yiqi dripping pills(40 mg/kg.d), and the sham operation group and the model group were given the same amount of normal saline, for 28 consecutive days. The survival of rats in each group was recorded. On Day28, the heart function of rats was evaluated by echocardiography(UCG);the morphological change of myocardia was analyzed by hematoxylin-eosin(HE) staining and Masson’s trichrome staining, and the change of myocardial apoptosis was analyzed by TUNEL;ELISA was used to analyze changes of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in serum;Western blotting and polymerase chain reaction(PCR) were used to analyze changes in the expression level of IL-6, TNF-α, α7 nAChR,NF-κBp50 and NF-κBp65 protein, and mRNA in myocardium. Results There was no significant difference in the survival rate between the model group and the treatment group(P > 0.05). UCG showed that the treatment group had significantly recovered heart function, increased left ventricular ejection fraction(LVEF)(P < 0.05), and decreased left ventricular end diastolic diameter(LVDd) and left ventricular end systolic diameter(LVDs)(P < 0.05). HE and Masson staining analysis showed well recovered myocardium, clearly reduced cell nuclear hypertrophy, a small amount of inflammatory cell infiltration and fibroblast proliferation, and unobvious collagen fiber proliferation. TUNEL analysis on myocardial apoptosis suggested that apoptotic cells decreased markedly(P < 0.05). ELISA analysis revealed that IL-6 and TNF-α inflammatory factor in serum declined obviously(P < 0.05). Western blotting analysis suggested that α7 nAChR protein in myocardium increased significantly(P < 0.05), and the IL-6, TNF-α, NF-κBp50 and NF-κBp65 protein decreased significantly(P < 0.05). PCR analysis showed that IL-6, TNF-α,NF-κBp50 and NF-κBp65, and mRNA level in myocardium lowered significantly(P < 0.05). Conclusion Qishen Yiqi dripping pills can improve heart function after occurrence of AMI and reduce inflammatory response, apoptosis and ventricular remodeling caused by AMI injury. Its mechanism may be associated with α7 nAChR anti-inflammatory pathway.