优化新生脉散方通过NMDAR通路抑制心衰心肌细胞凋亡的机制研究

Cardiomyocyte apoptosis is closely related to ventricular remodeling and plays an important role in the development of heart failure (HF). The ionotropic glutamate receptor NMDAR is specifically expressed in cardiomyocytes, and the continuous openness pathway can lead to apoptosis. Previous studies suggest that Youhua Xinshengmaisan can improve ventricular remodeling, reduce cardiomyocyte apoptosis in rats with HF; improve heart function and quality of life, as well as increase exercise tolerance in patients with HF. The potential target is related to the NMDA receptor pathway. The study uses rat model of HF to observe the effect of Youhua Xinshengmaisan on cardiac function and structure. Loading drug-containing serums into cardiomyocytes of HF rats and H9C2 cardiomyocytes injured by H2O2, detecting apoptosis by TUNEL and PI & Annexin V double staining, Ca2+ concentration and ROS content by fluorescence colorimetric, expression of Caspase-12, 9, 3 active fragments, cytochrome C and Bcl-2/Bax protein by Western Blot, and mRNA by RT-PCR. The main objective for the study is to verify the key role and molecular biological mechanism of NMDAR in cardiomyocyte apoptosis in HF, and explore the possible mechanism of Youhua Xinshengmaisan to improve ventricular remodeling by inhibiting cardiomyocyte apoptosis.

心肌细胞凋亡与心室重构密切相关，在心衰的发生发展中起重要作用。离子型谷氨酸受体NMDAR在心肌细胞中特异性表达，其通路持续性开放可导致细胞凋亡。前期研究提示优化新生脉散方可改善心衰大鼠心室重构、减少心肌细胞凋亡，改善心衰患者心功能及生存质量、增加运动耐量；其潜在作用靶点与NMDA受体通路相关。本项目采用心衰大鼠模型，观察优化新生脉散方对心脏功能及结构的影响；加载含药血清于心衰大鼠心肌细胞及H2O2损伤的H9C2心肌细胞，采用TUNEL及PI及Annexin V双重染色法检测细胞凋亡，荧光比色法检测Ca2+浓度及ROS含量，Western Blot检测Caspase-12、9、3活性片段、细胞色素C及Bcl-2/Bax蛋白的表达，RT-PCR测定mRNA，验证NMDAR在心衰心肌细胞凋亡过程中的关键作用及分子生物学机制，探讨优化新生脉散方通过抑制心肌细胞凋亡而改善心室重构的可能机制。

心肌细胞凋亡是导致心衰发生发展的重要原因。心肌损伤激活NMDAR信号通路，使得Ca2+超载，继而发生氧化应激，导致细胞凋亡。优化新生脉散方在临床治疗心衰方面取得一定的疗效。我们通过三个实验验证了优化新生脉散方作用于NMDAR信号通路，实现抑制细胞凋亡，治疗心衰的作用。首先我们通过制备心衰大鼠模型，灌胃给药治疗后采用TUNEL法检测细胞凋亡率，Western Blot、qRT-PCR法检测Caspase-3、Caspase-9、Caspase-12、Cyt-c、Bax及Bcl-2等凋亡蛋白及mRNA的表达，ELISA法检测Ca2+和ROS水平，上述实验证实了优化新生脉散方及艾芬地尔（NMDAR抑制剂）在改善心脏功能及病理结构的作用，能显著降低心肌细胞凋亡率，减少凋亡蛋白的表达以及Ca2+和ROS水平。接着我们通过H2O2诱导体外H9c2心肌细胞损伤，在分别加载优化新生脉散方及NMDAR抑制剂含药血清治疗后，采用Annexin V-FITC/PI双染法检测细胞凋亡，余实验同在体实验一致，其结果均能显著降低心肌细胞凋亡率，减少凋亡蛋白的表达以及Ca2+和ROS水平，进一步证实了优化新生脉散方的有效性以及在心肌细胞损伤时存在NMDAR信号通路激活导致Ca2+超载，继而发生氧化应激，导致细胞凋亡。为了进一步验证优化新生脉散方是否通过抑制NMDAR信号通路来实现对心衰的治疗，我们使用NMDA对体外H9c2心肌细胞进行诱导损伤，同样加载优化新生脉散方及NMDAR抑制剂含药血清进行治疗，余实验检测指标同上，结果表明优化新生脉散方可以显著改善NMDA诱导的细胞凋亡。通过以上实验说明了NMDAR信号通路的过度激活，可以促使心室重构及细胞凋亡进一步发生发展为心衰，优化新生脉散方可通过抑制NMDAR信号通路的激活，通过减少Ca2+及ROS水平，抑制Caspase级联的表达，从而抑制心肌细胞凋亡、减缓心室重构。因此，优化新生脉散方通过作用于NMDAR信号通路可能是治疗心衰、改善患者临床症状的有效途径。