强心汤保护心血管内皮治疗慢性心力衰竭的作用机制

Mechanism of Qiangxin Decoction protecting cardiovascular endothelium in the treatment of chronic heart failure

目的探讨强心汤调控miR⁃125b⁃5p、抑制核因子κB(NF⁃κB)介导的炎性因子的表达以保护心血管内皮祖细胞,治疗慢性心力衰竭(CHF)的作用机制。方法42只SPF级SD大鼠按照随机数字表法分为对照组、模型组、强心汤低剂量组(7.45 g/kg)、强心汤中剂量组(14.90 g/kg)、强心汤高剂量组(29.80 g/kg)、沙库巴曲缬沙坦组(10.42 mg/kg),每组7只。除对照组外,其他各组大鼠均采用结扎冠状动脉左前降支方法建立CHF模型。造模后第2天分别给予相应药物灌胃,连续给药28 d。采用心脏彩超检测各组大鼠心功能指标[左室舒张末期内径(LVEDd)、左室收缩末期内径(LVEDs)、左室射血分数(LVEF)]。HE染色观察大鼠左心室心肌组织病理学变化。Masson染色观察各组大鼠心脏心肌纤维化情况。荧光原位杂交(FISH)检测大鼠左心室心肌组织miR⁃125b⁃5p在内皮祖细胞的表达情况。蛋白质印迹法检测大鼠左心室心肌组织脑源性神经营养因子(BDNF)、NF⁃κB p65、磷酸化NF⁃κB p65(p⁃NF⁃κB p65)蛋白表达。酶联免疫吸附测定法检测炎性因子肿瘤坏死因子⁃α(TNF⁃α)、白细胞介素(IL)⁃1β和IL⁃6的含量。结果与对照组比较,模型组大鼠LVEDd、LVEDs升高,LVEF降低;心肌组织病理损伤严重,组织结构异常改变,横纹肌模糊,心肌细胞排列紊乱且部分变性坏死;心肌组织呈蓝色的胶原纤维、黏液沉积增多;左心室心肌组织miR⁃125b⁃5p平均光密度明显升高;左心室心肌组织BDNF蛋白表达减少,p⁃NF⁃κB p65/NF⁃κB p65值增加;TNF⁃α、IL⁃1β和IL⁃6含量明显增加(P<0.05)。与模型组比较,强心汤中、高剂量组和沙库巴曲缬沙坦组大鼠LVEDd、LVEDs均降低,LVEF均升高;左心室心肌组织的病理损伤改善明显,横纹肌清晰,心肌细胞排列整齐有序,细胞坏死减少;心肌组织呈蓝色的胶原纤维、黏液沉积减少,心肌纤维化情况明显减少;左心室心肌组织miR⁃125b⁃5p平均光密度明显降低;左心室心肌组织BDNF蛋白表达增加,p⁃NF⁃κB p65/NF⁃κB p65值降低;TNF⁃α、IL⁃1β和IL⁃6含量明显降低(P<0.05)。结论强心汤能够降低miR⁃125b⁃5p表达,抑制NF⁃κB通路的激活,降低促炎因子TNF⁃α、IL⁃1β和IL⁃6的表达,减轻心肌炎性反应、病理损伤及减少心肌纤维化,最终保护心血管内皮,改善心功能,从而治疗CHF。

Objective To investigate the mechanism of Qiangxin Decoction treating chronic heart failure(CHF)by regulating miR⁃125b⁃5p and thus inhibiting the expression of nuclear factor⁃κB(NF⁃κB)⁃mediated inflammatory factors in order to protect the cardiovascular endothelial progenitor cells.Methods According to the random number table method,42 SPF⁃grade SD rats were divided into the control group,the model group,the Qiangxin Decoction low⁃dose group(7.45 g/kg),the Qiangxin Decoction medium⁃dose group(14.90 g/kg),the Qiangxin Decoction high⁃dose group(29.80 g/kg),and the sacubitril valsartan group(10.42 mg/kg),with 7 rats in each group.Except for the control group,the CHF model was established by ligating the left anterior descending branch of the coronary artery in each group.Two days after modeling,the rats were treated with the corresponding drugs by gavage for 28 days.Cardiac function indexes,including left ventricular end⁃diastolic internal diameter(LVEDd),left ventricular end⁃systolic internal diameters(LVEDs),and left ventricular ejection fraction(LVEF),were examined by cardiac ultrasound examination,and the pathological changes in the myocardial tissue of the left ventricle of the rats were observed by HE staining.Myocardial fibrosis was observed in each group by Masson staining.Fluorescence in situ hybridization(FISH)was performed to determine the expression of miR⁃125b⁃5p in endothelial progenitor cells of the left ventricular myocardial tissue.The protein expressions of brain⁃derived neurotrophic factor(BDNF),NF⁃κB p65 and p⁃NF⁃κB p65 in the left ventricular myocardial tissue were detected by Western blotting.Enzyme⁃linked immunosorbent assay was used to detect the expression of inflammatory factor tumor necrosis factor⁃α(TNF⁃α),interleukin(IL)⁃1βand IL⁃6.Results Compared with the control group,LVEDd and LVEDs were elevated and LVEF was reduced in the model group;the pathological damage of myocardial tissue was severe with abnormal changes in the tissue structure,striated muscle became fuzzy,and cardiomyocytes were disordered in their arrangement and partially denatured and necrotic;blue collagen fibers and mucus deposition in the myocardial tissues were increased;the average optical density of miR⁃125b⁃5p was increased in the left ventricular myocardial tissue;the protein expression of BDNF was decreased,and the ratio of p⁃NF⁃κB p65/NF⁃κB p65 was increased;the contents of TNF⁃α,IL⁃1β,and IL⁃6 were significantly increased(P<0.05).Compared with the model group,LVEDd and LVEDs were reduced,and LVEF was increased in the Qiangxin Decoction medium⁃,high⁃dose groups and the sacubitril valsartan group;the pathological damage of myocardial tissues in the left ventricle was improved,with clear striated muscle,neat and orderly arrangement of cardiomyocytes,and reduction of cellular necrosis;blue collagen fibers and mucus deposition were reduced,and myocardial fibrosis was significantly reduced;the average optical density of miR⁃125b⁃5p in the left ventricular myocardial tissue was significantly reduced;the protein expression of BDNF was increased,and the ratio of p⁃NF⁃κB p65/NF⁃κB p65 was reduced;the contents of TNF⁃α,IL⁃1β,and IL⁃6 were significantly reduced(P<0.05).Conclusion Qiangxin Decoction can reduce miR⁃125b⁃5p expression,inhibit the activation of NF⁃κB pathway,reduce the expression of the pro⁃inflammatory factors TNF⁃α,IL⁃1β,and IL⁃6,attenuate the myocardial inflammatory response,pathological injury and reduce myocardial fibrosis,ultimately protect the cardiovascular endothelium,and improve cardiac function to treat CHF.