刺五加注射液对急性心肌梗死后心力衰竭大鼠心肌保护作用的研究

Protective effect of Ciwujia Injection on heart failure after acute myocardial infarction in rats

关键词:  急性心肌梗死  心力衰竭  刺五加注射液  心功能  Nrf2/HO-1信号通路  氧化应激

Key words:  acute myocardial infarction  heart failure  Ciwujia Injection  cardiac function  Nrf2/HO-1 signaling pathway  oxidative stress

摘要:

[目的]探讨刺五加注射液(CWJI)对急性心肌梗死后心力衰竭(HF-AMI)大鼠心肌组织的保护作用及可能机制。[方法]通过结扎左冠状动脉前降支复制HF-AMI大鼠模型,设模型(Model)组、卡托普利(CPT,10 mg/kg)组和CWJI低(CWJI-L,15 mg/kg)、中(CWJI-M,30 mg/kg)、高剂量(CWJI-H,60 mg/kg)组,另设假手术(Sham)组,每组16只。各组分别1次/日连续给药治疗4周后,通过多普勒超声和生理信号采集系统检测大鼠心功能[左室射血分数(LVEF)、左室短轴缩短率(LVFS)、左室内压最大上升速率和下降速率(+dp/dtmax、-dp/dtmax)],酶联免疫吸附法(ELISA)检测血清心肌肌钙蛋白(cTnI)、血管紧张素Ⅱ(AngⅡ)、醛固酮(ALD)水平,计算左室心肌肥厚指数(LVHI),通过苏木精-伊红(HE)、TUNEL、Masson染色观察心肌组织病理学改变、细胞凋亡和组织纤维化状况,透射电子显微镜观察心肌细胞线粒体超微结构变化,分光光度法检测心肌组织总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)、活性氧(ROS)含量,RT-PCR法检测核因子E2相关因子2(Nrf2)、血红素加氧酶1(HO-1)mRNA表达,Western Blot法检测Nrf2、HO-1蛋白表达。[结果]与Model组比较,CPT组和CWJI-M、CWJI-H组大鼠LVEF、LVFS、+dp/dtmax、-dp/dtmax、血清cTnI、AngⅡ、ALD水平、LVHI显著降低(P＜0.05);心肌组织病理学改变、细胞凋亡和组织纤维化状况均明显改善,细胞凋亡指数(AI)和胶原容积分数(CVF)显著降低(P＜0.05);心肌细胞线粒体超微结构变化明显改善;心肌组织T-SOD、GSH-Px活性显著升高,MDA、ROS含量显著降低(P＜0.05);Nrf2、HO-1 mRNA和蛋白表达量显著升高(P＜0.05)。CWJI上述效应呈现一定剂量依赖性,且CWJI-H组对除LVFS外其他指标的作用优于CPT组(P＜0.05)。[结论] CWJI可减轻HF-AMI大鼠心肌组织病变、细胞凋亡、组织纤维化和线粒体超微结构病变,改善心功能,其机制可能与激活Nrf2/HO-1信号通路,抑制氧化应激有关。

Abstract:

[Objective] To investigate the protective effect and possible mechanism of Ciwujia Injection(CWJI) on myocardial tissue of rats with heart failure after acute myocardial infarction(HF-AMI). [Methods] The rat model with HF-AMI was established by ligation of the anterior descending branch of the left coronary artery,and model group,Captopril(CPT,10 mg/kg) group,CWJI low-dose(CWJI-L,15 mg/kg),medium-dose(CWJI-M,30 mg/kg),high-dose(CWJI-H,60 mg/kg) group,and the Sham group were set up,with 16 rats in each group. After 4 weeks of continuous administration once a day in each group,the cardiac function [left ventricular ejection fraction(LVEF),left ventricular short axis shortening rate(LVFS),maximum rate of rise and fall of left indoor pressure(+dp/dtmax,-dp/dtmax)]was detected by Doppler ultrasound and physiological signal acquisition system. The level of cardiac troponin(cTnI),angiotensin Ⅱ(AngⅡ) and aldosterone(ALD) in serum were determined by enzyme-linked immunosorbent assay(ELISA). The left ventricular myocardial hypertrophy index(LVHI) was calculated. The myocardial histopathological changes,apoptosis and tissue fibrosis were observed by HE,TUNEL or Masson staining,the mitochondrial ultrastructure changes was observed by transmission electron microscopy. The activity of total superoxide dismutase(T-SOD),glutathione peroxidase(GSH-Px) and the content of malondialdehyde(MDA),reactive oxygen species(ROS) were detected by spectrophotometry. The mRNA expression of nuclear factor E2-related factor 2(Nrf2) and heme oxygenase 1(HO-1) were detected by RT-PCR. The protein expression of Nrf2 and HO-1 were detected by Western Blot. [Results] Compared with the model group,the LVEF,LVFS,+dp/dtmax,-dp/dtmax,the level of cTnI,Ang Ⅱ,ALD in serum and LVHI in CPT group,CWJI-M,CWJI-H groups were significantly decreased(P＜0.05). The myocardial histopathological changes,apoptosis and tissue fibrosis were significantly improved,and the apoptosis index(AI) and collagen volume fraction(CVF) were significantly decreased(P＜0.05). The ultrastructural changes of myocardial cell mitochondria were significantly improved. The activity of T-SOD,GSH-Px in myocardial tissue were significantly increased,while the content of MDA,ROS were significantly decreased(P＜0.05). The mRNA and protein expression of Nrf2,HO-1 were significantly increased(P＜0.05). The above effects of CWJI were dose-dependent,and the effect of CWJI-H group on other indexes except LVFS was better than those of CPT group(P＜0.05). [Conclusion] CWJI can alleviate myocardial tissue lesion,cell apoptosis,tissue fibrosis and mitochondrial ultrastructure lesion in rats with HF-AMI,and improve cardiac function,which may be related to activation of Nrf2/HO-1 signaling pathway and inhibition of oxidative stress.