活血化瘀中药山楂叶活性成分抗程序性坏死作用及分子机制研究

The finding of necroptosis in 2005 makes the modulation on necrosis feasible,and brings a revolutionary impact on the medicine research and clinical therapy.However,until now,the effective inhibitory approaches to necroptosis are still in shortage.According to the theory of Chinese Medicine,the essence of necroptosis is "the organ destruction resulted from blood clotting in the vessel",which should be treated with the approach of activating blood and dissolving stasis.This study is planned to observe the effect of procyanidins which is the main active component of hawthorn leaf, a Chinese Medicine herb having the ability of activating blood and modulating qi, and further explore the mechanism of procyanidins'effect.Briefly,in the study project,at first,several calssical necroptosis models,such as L929 cell injury induced with TNFα,Jurkatt cell injury induced with fasL,and 3T3 cell injury induced with TNFα and Z-VAD,etc.,are to be duplicated; then the effects of procyanidins and its main active components on these necroptosis models will be observed,in order to value the inhibitory potential of procyanidins on necroptosis; afterwards, the direct effects of procyanidins and its main active components on each step of the necroptosis pathway including the combination of TNFα and TNFα receptor 1,the combination of TNFα receptor 1 and RIP1,the formation of RIP1-RIP3 complex,the burst of free radicals,and the membrane injury induced by free radicals,are to be observed,aiming to identify the directly intenvented part by procyanidins and clarify the molecular mechanism of the inhibitory effect of procyanidins on necroptosis;and at last, the effects of procyanidins on necroptosis in myocardium ischemia and cerebral ischemia which have been found most relevant to necroptosis are to be valued, for the purpose of validating the therapeutic potential of procyanidins on clinically necroptosis-related diseases. Totally,this study may be significant for the research of necroptosis,and the mechanism research of the therapy of Chinese Medicine herbs on necroptosis, as well as enriching the Chinese Medicine theory of activating blood and dissolving stasis, improving the therapy of the major disedases such as myocardium ischemia and cerebral ischemia,etc.

2005年美国科学家发现程序性坏死，使调控坏死成为可能，对医学研究和疾病治疗产生革命性影响。目前对于程序性坏死缺乏有效抑制手段。从中医理论来看，程序性坏死的发生与"瘀阻脉络，脏体败坏"的病机密切相关，应治以活血化瘀。本课题选用活血理气中药山楂叶的活性成分原花青素，观察它对程序性坏死的治疗作用，并深入探讨作用机制。拟复制多种经典程序性坏死模型，包括TNFα诱导L929细胞损伤、fasL诱导Jurkat细胞损伤等，观察山楂叶原花青素及其主要成分的作用；再观察药物对程序性坏死通路各环节的直接影响，包括对RIP1-RIP3复合物形成、氧自由基爆发性生成与损伤等过程的影响，明确药物作用环节；最后，选用急性心脑缺血模型，对山楂叶原花青素治疗临床疾病中细胞程序性坏死的作用进行验证。这一课题对于程序性坏死研究，探讨中药治疗程序性坏死机制，以及丰富中医活血化瘀理论，改进心脑缺血等重大疾病的治疗具有重要意义。

大量细胞爆发性坏死是大多数重大难治性疾病发病、致死的关键。程序性坏死的发现，使坏死调控成为可能。当前药物调控程序性坏死的研究刚刚起步，在中药领域更是一片空白。本研究选择重要的活血化瘀中药山楂叶，观察其主要成分原花青素的抗程序性坏死作用，探讨分子机制，并研究山楂叶原花青素的抗心肌缺血作用，以及程序性坏死在心肌缺血发病中的作用。本研究复制4种经典程序性坏死模型，观察山楂叶原花青素及其6种主要成分，和另外十余种活血化瘀、清热解毒中药主要活性成分的抗程序性坏死作用；最终明确了山楂叶原花青素及其主要成分EGCG的抗程序性坏死作用。本研究进一步在最经典的程序性坏死模型上，探讨了山楂叶原花青素和EGCG治疗作用的分子机制；结果显示山楂叶原花青素和EGCG均可以显著抑制启动程序性坏死、在程序性坏死发生中具有核心地位的RIP通路的活化的，阻断细胞及线粒体氧自由基生成爆发，从而达到对抗程序性坏死的效果。本研究观察山楂叶原花青素及其6种主要成分抗心肌细胞缺血的作用，明确山楂叶原花青素及其4种成分单体的功效，发现一种较强的抗心肌缺血单体-儿茶素。本研究还探讨了程序性坏死与心肌缺血损伤的关系，实验结果不支持心肌细胞程序性坏死在缺血损伤发病中占主要地位，此外，研究还发现程序性坏死通路成分PGAM5可能在心肌缺血中起保护作用。总之，本研究在中药领域首次明确了山楂叶原花青素抗程序性坏死的作用，对于阐明山楂叶活血化瘀作用实质，丰富、发展活血化瘀药物研究，完善活血化瘀理论具有重要意义，对于寻找抗程序性坏死新药，亦是一个重要的探索；本研究阐明了山楂叶原花青素抗程序性坏死的分子机制，对于阐明山楂叶、原花青素的细胞、分子药理机制具有重要意义；本研究探索了程序性坏死与心肌细胞缺血损伤的关系，这对于明确心肌缺血性损伤机制，研究抗心肌缺血药物具有一定的意义，也是从程序性坏死―这一最新的病理生理学进展角度对心肌缺血损伤的机制进行阐释的一次探索。