参芪利心汤对心力衰竭大鼠心肌细胞的保护作用及机制研究

Protective effects and mechanism of Shenqi Lixin Decoction on cardiomyocytes of rats with heart failure

目的研究参芪利心汤对阿霉素诱导心力衰竭大鼠心肌细胞的保护作用及机制。方法雄性Wistar大鼠随机分为对照组、模型组、依那普利(2.1 mg/kg)组以及参芪利心汤低、中、高剂量(9.975、19.950、39.900 g/kg)组,除对照组外,其余大鼠ip阿霉素诱导心力衰竭模型;各给药组ig相应药物,对照组和模型组ig等体积蒸馏水,1次/d,连续4周。给药结束后,采用心脏彩色多普勒超声仪测定各组大鼠左室射血分数(left ventricular ejection fraction,LVEF)、左室短轴缩短率(left ventricular fractional shortening,LVFS)以及每搏心输出量(stroke volume,SV);采用苏木素-伊红(HE)染色法观察各组大鼠心肌组织病理变化;采用透射电镜观察各组大鼠心肌细胞超微结构;采用ELISA法检测各组大鼠血浆N端B型利钠肽(N-terminal B-type natriuretic peptide,NT-pro BNP)及心肌组织三磷酸腺苷(adenosine triphosphate,ATP)含量;采用流式细胞仪检测各组大鼠心肌细胞凋亡情况;采用qRT-PCR法检测各组大鼠心肌组织B淋巴细胞瘤2(B-cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2associated X protein,Bax)、半胱氨酸蛋白酶-3(Caspase-3)和过氧化物酶体增殖物激活受体γ共激活因子-1α(peroxisome proliferator activated receptorγcoactivator-1α,PGC-1α)m RNA表达情况;采用Western blotting法检测各组大鼠心肌组织Bcl-2、Bax、Caspase-3和p53蛋白表达情况。结果与对照组比较,模型组大鼠LVEF、LVFS和SV显著降低(P<0.01),血浆NT-pro BNP含量显著升高(P<0.01),心肌组织ATP含量显著降低(P<0.01),心肌细胞凋亡率显著升高(P<0.01);心肌组织Bcl-2和PGC-1αmRNA表达水平显著降低(P<0.01),Bax和Caspase-3 mRNA表达水平显著升高(P<0.01);心肌组织Bcl-2蛋白表达显著降低(P<0.01),Bax、Caspase-3和p53蛋白表达水平显著升高(P<0.01)。与模型组比较,参芪利心汤中、高剂量组和依那普利组大鼠LVEF、LVFS和SV均显著升高(P<0.01);血浆NT-pro BNP含量显著降低(P<0.01),心肌组织ATP含量显著升高(P<0.01),心肌细胞凋亡率显著降低(P<0.01);心肌组织Bcl-2和PGC-1αmRNA表达水平均显著升高(P<0.01),Bax和Caspase-3 mRNA表达水平均显著降低(P<0.01);心肌组织Bcl-2蛋白表达水平显著升高(P<0.01),Bax、Caspase-3和p53蛋白表达水平均显著降低(P<0.05、0.01)。结论参芪利心汤能够改善心力衰竭大鼠的心肌结构、能量代谢及心功能,并减少心肌细胞凋亡,其作用机制可能与调控PGC-1α和线粒体凋亡通路相关蛋白表达有关。

Objective To study the protective effect and mechanism of Shenqi Lixin Decoction(参芪利心汤) on cardiomyocytes of rats with heart failure induced by adriamycin. Methods Male Wistar rats were randomly divided into control group, model group, enalapril(2.1 mg/kg) group, low-, medium-and high-dose Shenqi Lixin Decoction(9.975, 19.950, 39.900 g/kg) groups. In addition to control group, the rest of rats were ip adriamycin to induce heart failure model, rats in each administration group were ig corresponding drugs, rats in control group and model group were ig equal volumes of distilled water, once a day for four consecutive weeks. After administration, cardiac color doppler ultrasound was used to measure left ventricular ejection fraction(LVEF), left ventricular fractional shortening(LVFS) and stroke volume(SV) in rats of each group. Hematoxylin-eosin(HE) staining method was used to observe the pathological changes of myocardial tissue in rats;Transmission electron microscopy was used to observe the ultrastructural changes of myocardial cells in rats;N-terminal B-type natriuretic peptide(NT-proBNP) level in plasma and adenosine triphosphate(ATP) content in myocardial tissue were detected by ELISA;Flow cytometry was used to detect myocardial cell apoptosis in rats of each group;qRT-PCR method was used to detect B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), Caspase-3 and peroxisome proliferator activated receptor γ coactivator-1α(PGC-1α) m RNA expressions in myocardial tissue;Western blotting was used to detect Bcl-2, Bax, Caspase-3 and p53 protein expressions in myocardial tissue of rats in each group. Results Compared with the control group, LVEF, LVFS and SV of rats in model group were significantly reduced(P < 0.01), NT-proBNP level in plasma was increased(P < 0.01), and ATP content in myocardial tissue was reduced(P < 0.01);Cardiomyocyte apoptosis rate was increased(P < 0.01), Bcl-2 and PGC-1α mRNA levels in myocardial tissue were reduced(P < 0.01), Bax and Caspase-3 mRNA levels were increased(P < 0.01);Bcl-2 protein expression in myocardial tissue was reduced(P < 0.01), Bax, Caspase-3 and p53 protein expressions were increased(P < 0.01). Compared with model group, LVEF, LVFS and SV of rats in medium-, high-dose Shenqi Lixin Decoction group and enalapril groups were increased(P < 0.01);NT-proBNP level in plasma was decreased(P < 0.01), ATP content in myocardial tissue was increased(P < 0.01);Cardiomyocyte apoptosis rate was reduced(P < 0.01);Bcl-2 and PGC-1α m RNA levels in myocardial tissue were increased(P < 0.01), Bax and Caspase-3 m RNA levels were reduced(P < 0.01);Bcl-2 protein expression in myocardial tissue was increased(P < 0.01), Bax, Caspase-3 and p53 protein expressions were reduced(P < 0.05, 0.01). Conclusion Shenqi Lixin Decoction can improve myocardial structure, energy metabolism and cardiac function in rats with heart failure, and reduce myocardial cell apoptosis. Its mechanism may be related to the regulation expression of PGC-1α and mitochondrial apoptosis pathway-related proteins.