基于线粒体相关凋亡及PKC信号转导通路探讨八味沉香散干预心肌缺血再灌注损伤作用机制研究

Ischemic heart disease is a common disease that seriously damages human health. With the wide application of interventional cardiology in its treatment, on the prevention and treatment of myocardial ischemia reperfusion injury (MIRI) drugs is significant. Tibetan BaweiChenxiang Powde has been listed in China for the treatment of ischemic heart disease clinical efficacy is very significant. Basic research shows that it has good anti myocardial ischemia effect, and it can reduce the damage of mitochondria and inhibit the apoptosis of myocardial cells in MIRI. Based on the previous research, our research group put forward the hypothesis : it can inhibit the apoptosis of mitochondrial related three factors (ROS, intracellular calcium overload and mitochondrial damage) to protect mitochondrial function. It can interfere with cardiomyocyte apoptosis in MIRI. The mechanism is to activate the PKC pathway related mitochondrial to initiate drug preconditioning and regulate the downstream apoptotic factors. According to this hypothesis, the study will establish MIRI model with human cardiac myocytes. Furthermore, ransmission electron microscopy, flow cytometry, laser confocal microscopy, Western blot, real-time quantitative RT-PCR were utilized to clarify the specific mechanism of Baweichenxiang Powder’s intervention on caridianl cell apopsis by conserving mitochondrial function from aspects of its influence on apoptosis factor through mitochondria-related apoptotic factors and PKC signal pathways. It provides experimental basis for the treatment of heart disease under the guidance of Tibetan medicine theory.

缺血性心脏病是严重危害健康的常见病，随着介入性心脏病学在其治疗中广泛应用，防治心肌缺血再灌注损伤（MIRI）的研究意义重大。我国已上市的藏药八味沉香散治疗缺血性心脏病临床疗效显著，前期基础研究表明其具有较好的抗心肌缺血作用，可减轻MIRI线粒体损伤，抑制心肌细胞凋亡。本课题组基于前期研究提出假说：八味沉香散能通过抑制线粒体相关细胞凋亡三因素（活性氧生成、细胞内钙超载、线粒体损伤）保护线粒体功能，抗MIRI心肌细胞凋亡，机制是通过激活线粒体相关的PKC通路启动药物预适应，调控下游凋亡因子实现的。本研究拟针对此假说，以人心肌细胞建立MIRI模型，采用透射电镜、流式细胞术、激光共聚焦、蛋白免疫印迹、荧光实时定量PCR等方法，从线粒体相关凋亡因素、PKC通路相关凋亡因子等方面研究，探讨八味沉香散在线粒体相关细胞凋亡及信号转导通路方面干预MIRI的作用机制。通过本研究为藏医药治疗心痛病提供科学依据。

缺血性心脏病是严重危害健康的常见病，随着介入性心脏病学在其治疗中广泛应用，防治心肌缺血再灌注损伤的研究意义重大。我国已上市的藏药八味沉香散治疗缺血性心脏病临床疗效显著，前期基础研究表明其具有较好的抗心肌缺血作用。本研究首先进行八味沉香散水提、30%、60%、95%醇提工艺制备。以3因素3水平正交试验法分别得到9个水提物和9个醇提物，成功建立细胞缺血再灌注损伤（I/R损伤）模型，用LDH检测法、CCK8法评估不同提取物对H9C2 I/R损伤的细胞毒性和细胞活率，以正交设计因素水平与细胞活率、细胞毒性、出膏率计算综合评分，得到最佳提取工艺：水提物为8倍水量提取3次每次1h；醇提物为8倍量60%的乙醇提取3次，每次2h。然后成功培养传代人心肌细胞HCM，流式细胞法检测其水提、醇提物均能不同程度抑制HCM I/R损伤细胞凋亡，对早期凋亡抑制作用最好。通过流式细胞法、荧光酶标法、透射电镜法检测八味沉香散对线粒体相关细胞凋亡三因素（活性氧生成、细胞内钙超载、线粒体损伤）的作用，结果显示八味沉香水提、醇提物均可抑制HCM的I/R损伤中活性氧生成、胞浆和线粒体内钙离子浓度、MPTP的开放和膜电位降低，电镜检测可使肿胀和空泡化的线粒体数量减少，并抑制凋亡小体形成。最后以WB和qPCR检测发现I/R损伤时，八味沉香散水提、醇提物可促进PKC通路的PKCε因子基因转录和蛋白合成，上调其下游抑制凋亡的Bcl-2蛋白表达和基因转录水平，抑制下游凋亡相关因子bax、caspase-3蛋白表达和基因转录水平。本研究阐明了心肌细胞在缺血再灌注损伤刺激下，八味沉香散可以通过抑制线粒体相关细胞凋亡三因素，保护线粒体的结构功能，并揭示其抗心肌细胞凋亡的机制是通过激活PKC通路启动药物预适应，调控线粒体相关凋亡因子的基因转录和蛋白表达实现的。基于线粒体功能和藏医理论中“隆”的特点高度契合，以及八味沉香散治疗“隆血失调”心痛病中“隆”的关键作用，本研究为此理论提供了可靠的基础实验依据，为其现代化发展和推广产生较为重要的影响。