基于4HNE氧化损伤途径探讨丹参总酚酸心肌内源性保护机制

Myocardial ischemia-reperfusion injury is mainly caused by oxidative stress. Reactive oxygen species(ROS) is the activator of oxidative stress and 4-hydroxynonenal (4HNE) is one of peroxide products of oxidative stress and 4HNE is far more toxic and stabile.Both of them interacted with each other in the course of myocardial ischemia-reperfusion injury resulting in myocardial dysfunction, cell apoptosis and signal transduction blocking. The metabolic pathway of ROS has been clarified. Aldehyde dehydrogenase(ALDH-2) is one of the endogenous antioxidant enzymes and also the key enzyme to metabolize 4HNE. Increasing the activity of ALDH-2 can effectively attenuate ischemia-reperfusion injury. Our prefer study found that salvianolic acids anti-reperfusion injury effects were close related with its effect on increasing ALDH-2 activity. In order to study the molecular mechanism of anti-oxidative injury effects of salvianolic acids we establish ischemic reperfusion injury models in animal rat,isolated heart and cultured myocardial cell.And then we combined observing organ and cell structures and functions with modern molecular biology technology to thoroughly study the molecular mechanism and multi-targets of anti- oxidative injury effects of salvianolic acids. Generation of ROS was detect using 5-(6)-chloromethyl-2, 7-dichlorodihydrofluorescein diacetate. ALDH-2 and 4HNE protein adducts formation was studied by Wester blot. qRT-PCR was used to analysis genes translation, To reveal multitargets of salvianolic acids alleviating myocardial reperfusion injury, expound molecular mechanism of enhancing ALDH-2 activity and clarify effct of salvianolic acids on time sequential relationship among ROS generation,cell apoptosis and MAPKs signaling pathways activation induced by 4HNE.From both promoting 4HNE metabolism and enhancing myocardial cells resistance to 4HNE to expound endogenous protective mechanisms of total salvianolic acids to reperfusion myocardial oxidative injury.This study shed a new light to the research of traditional Chinese medicine antioxidant injury.

缺血再灌注产生的过氧化产物4-HNE是导致心肌氧化损伤的主要物质之一。ALDH2是4HNE代谢的关键酶。前期研究证明丹参总酚酸（Sals）抗再灌注损伤疗效确切，其效用与增加ALDH2活性密切相关。为了探讨Sals抗再灌注损伤与4HNE/ALDH-2相关的分子机制，本研究拟①在整体动物和离体心脏缺血再灌注模型上观察Sals心肌保护作用与ALDH-2活性大小、蛋白表达、基因转录及ALDH-2激活关键因子PKCε活化、移位、与ALDH-2相互作用的关系，阐释Sals提高ALDH-2活性、减少4HNE毒性的分子机制；② 观察Sals对激活或阻断ALDH-2通路后，4HNE诱导心肌细胞ROS产生、MAPKs活化、心肌凋亡的影响，明确Sals增强心肌细胞对4HNE损伤耐受力的作用机制。从促进4HNE代谢和增强心肌细胞对4HNE损伤耐受两条途径阐释Sals抗再灌注氧化损伤的内源性保护的分子机制。

心肌缺血再灌注引起的心肌损伤严重影响了再灌注的治疗效果，是造成再灌注病情恶化、死亡率增加的主要原因。激发和提高心肌内源性抗氧化保护能力成为再灌注损伤防治的一个研究方向。.本课题利用在体、离体、细胞缺血再灌注模型借助于心肌功能检测和现代分子生物学技术，以造成再灌注氧化损伤关键物质为核心，分析Sals对心肌缺血再灌注大鼠心肌组织尤其是线粒体多种抗氧化体系物质及氧化产物MDA、4HNE含量、ALDH-2活性影响，观察Sals对再灌注离体心脏ALDH-2 激活关键因子PKCε活化、移位、与ALDH-2相互作用的影响，观察Sals对阻断ALDH-2通路后4HNE诱导的心肌细胞线粒体增殖的影响，从整体动物、离体心脏、心肌细胞层层深入阐释Sals抗再灌注氧化损伤细胞保护的分子机制。.目前研究结果表明：⑴ 丹参总酚酸能够明显抑制大鼠离体和在体缺血再灌注心肌收缩功能的下降、心脏系数的增加，维持心肌组织形态结构和线粒体的数量和结构的完整性；丹参总酚酸能够明显降低心肌组织XO、NADPH等多种氧化酶活性，进而减少氧自由基的产生，增加心肌组织内源性抗氧化酶GSH-Px和ALDH-2的活性，清除氧自由基，减少心肌组织脂质过氧化物MDA和4-HNE的形成以及清除脂质过氧化物。（2）丹参总酚酸对大鼠离体心肌内源性保护机制可能与其通过作用于ALDH-2活化途径上的PKC-e，之后促进ALDH-2的磷酸化，进而ALDH-2磷酸化后促进毒物4-HNE的代谢有关。⑶ 丹参总酚酸促进线粒体相关功能蛋白PGC-1α、NRF1和mtTFA的mRNA和蛋白水平表达，增加mtDNA的拷贝数，促进线粒体生物合成而防御心肌线粒体功能障碍，保护心肌缺血再灌注心脏。作用机制值得进一步深入研究（具体数据参见正文）。从心肌线粒体氧化途径初步阐释了丹参总酚酸在心肌缺血再灌注防治领域应用的作用机制。