Tongxinluo Improves Apolipoprotein E-Deficient Mouse Heart Function

: 通心络改善载脂蛋白E基因敲除小鼠心功能摘要背景：既往众多研究表明，作为一种复方中药，通心络（TXL）可以减少心肌缺血-再灌注损伤，保护毛细血管内皮功能，抑制动物模型心室重构。本研究目的在于阐明通心络是否可以通过内皮功能保护或增加心肌微血管数量，从而改善高胆固醇血症诱发的心功能障碍，并对其潜在的分子机制做深入探讨。 方法：本实验研究共计8周。实验开始后，通心络组小鼠（n=8）给予0.5%羧甲基纤维素钠（CMC-Na)注悬的通心络超微粉（1.52g 生药/ kg/d）口服灌胃(0.1 ml/10g 体重)，每日1次；8只特征匹配的野生型C57BL/6J小鼠作为对照组。模型组（n=8）与对照组小鼠饲以同等剂量的CMC-Na口服灌胃，每日1次。实验结束后测定各组小鼠血清总胆固醇、高密度脂蛋白胆固醇、极低密度脂蛋白胆固醇、甘油三酯和血糖水平；通过超声心动图检查，采集各组小鼠心率（HR）、左室舒张末期内径（LVDED）、左室收缩末期内径(LVSED)等参数，计算左室射血分数（LVEF）、每搏输出量（SV）、心输出量（CO）及左室短轴缩短率（LVFS）等指标以评价心功能、同时测量小鼠胸主动脉收缩期峰值流速（PSV）、舒张末血流速度(EDV)与平均血流速度(MFV)，计算搏动指数（PI）与阻力指数（RI）以评价血管弹性与顺应性；采用体外乙酰胆碱诱导主动脉环舒张反应方法评价各组小鼠内皮依赖性血管舒张反应；体外血管新生实验在一周内完成；开展小鼠心肌CD34免疫组织化学检测；分别采用蛋白印迹分析与RT-PCR方法测定各组小鼠心肌组织中血管内皮生长因子（VEGF）蛋白与基因表达表达水平。本研究的数据采用单因素方差分析(ANOVA)，P < 0.05为有统计学意义。 结果：与模型组比较，通心络组小鼠血清胆固醇水平无显著改变 (F = 2.300,P = 0.240)，但通心络可以使模型小鼠的甘油三酯和极低密度脂蛋白胆固醇水平降低(F = 9.209,P =0.024;F = 9.786,P = 0.020)；通心络可以改善模型小鼠的心脏功能，增加LVEF、SV、CO和LVFS(P < 0.05)；通心络可增强模型小鼠主动脉PSV和MFV(F = 10.774,P =0.024; F = 11.354,P = 0.020),减少模型组小鼠主动脉的PI(1.41±0.17 vs 1.60±0.17;F = 14.270,P = 0.037)。本研究中，乙酰胆碱可剂量依赖性(0.1-10 μmol/L)诱导正常组小鼠主动脉环的舒张反应（51% ± 9%），模型组小鼠内皮依赖性血管舒张则明显受损(23% ± 2%)；与模型组比较，通心络组小鼠血管舒张率明显改善（44% ± 3%，F=9.280, P=0.040)；实验证实通心络可以抑制模型小鼠主动脉血管生成(F = 21.223,P = 0.010)；与对照组比较，模型组小鼠心肌组织微血管密度（MVD）显著减少(54.0 ± 3.0/mm2 vs 75.0 ± 2.0/mm2; F=16.054, P=0.010)；与模型组比较，通心络小鼠心肌MVD则明显增加(65.0 ± 5.0/mm2 vs 54.0 ± 3.0/mm2; F=11.929, P=0.020)。通心络明显增加模型组小鼠VEGF蛋白表达(F = 20.247,P = 0.004)，使VEGF mRNA表达增加4.59倍。 结论: 通心络可多方面改善ApoE-/-模型小鼠心功能，除降低血脂以延缓动脉粥样硬化进程外，对血管尤其是微血管的保护在其作用机制中十分关键，这包括提高主动脉血供能力及弹性、改善内皮依赖性血管舒张功能、抑制含斑块动脉组织血管新生以及增加心肌组织微血管密度，后者深层分子机制可能与其增强VEGF基因与蛋白表达有关。. Methods: After intragastric administration of TXL (0.1 ml/10 g body weight) to C57BL/6J wild-type mice (n = 8) and ApoE-/- mice (n = 8), total cholesterol, high-density lipoprotein-cholesterol, very-low-density lipoprotein (VLDL)-cholesterol, triglyceride, and blood glucose levels in serum were measured. The parameters of heart rate (HR), left ventricular diastolic end diameter, and left ventricular systolic end diameter were harvested by ultrasonic cardiogram. The left ventricular ejection fraction, stroke volume, cardiac output, and left ventricular fractional shortening were calculated. Meanwhile, aorta peak systolic flow velocity (PSV), end diastolic flow velocity, and mean flow velocity (MFV) were measured. The pulsatility index (PI) and resistant index were calculated in order to evaluate the vascular elasticity and resistance. The endothelium-dependent vasodilatation was evaluated by relaxation of aortic rings in response to acetylcholine. Western blotting and real-time quantitative reverse transcription polymerase chain reaction were performed for protein and gene analyses of vascular endothelial growth factor (VEGF). Immunohistochemical detection was performed for myocardial CD34 expression. Data in this study were compared by one-way analysis of variance between groups. A value of P < 0.05 was considered statistically significant. Results: Although there was no significant decrease of cholesterol level (F = 2.300, P = 0.240), TXL inhibited the level of triglyceride and VLDL (F = 9.209, P = 0.024 and F = 9.786, P = 0.020, respectively) in ApoE-/- mice. TXL improved heart function of ApoE-/- mice owing to the elevations of LVEF, SV, CO, and LVFS (all P < 0.05). TXL enhanced aortic PSV and MFV (F = 10.774, P = 0.024 and F = 11.354, P = 0.020, respectively) and reduced PI of ApoE-/- mice (1.41 ± 0.17 vs. 1.60 ± 0.17; P = 0.037). After incubation with 10 μmol/L acetylcholine, the ApoE-/- mice treated with TXL aortic segment relaxed by 44% ± 3%, significantly higher than control group mice (F = 9.280, P = 0.040). TXL also restrain the angiogenesis of ApoE-/- mice aorta (F = 21.223, P = 0.010). Compared with C57BL/6J mice, the MVD was decreased in heart tissue of untreated ApoE-/- mice (54.0 ± 3.0/mm2 vs. 75.0 ± 2.0/mm2; F = 16.054, P = 0.010). However, TXL could significantly enhance MVD (65.0 ± 5.0/mm2 vs. 54.0 ± 3.0/mm2; F = 11.929, P = 0.020) in treated ApoE-/- mice. In addition, TXL obviously increased the expression of VEGF protein determined by Western blot (F = 20.247, P = 0.004). Conclusions: TXL obviously improves the ApoE-/- mouse heart function from different pathways, including reduces blood fat to lessen atherosclerosis; enhances aortic impulsivity, blood supply capacity, and vessel elasticity; improves endothelium-dependent vasodilatation; restraines angiogenesis of aorta-contained plaque; and enhances MVD of heart. The molecular mechanism of MVD enhancement maybe relate with increased VEGF expression.