基于内质网应激的灸预处理对大鼠脑缺血/再灌注损伤的保护作用研究

Ischemis cerebrovascular disease is the frequently-occurring and common diseases with high incidence,high morbidity,high mortality. Studies have shown that cerebral ischemia / reperfusion injury is a common pathological process of acute cerebral infarction after recanalization . In recent years, found that endoplasmic reticulum stress involved in cerebral ischemia / reperfusion injury and become a research hotspot .studies have proven that acupuncture on cerebral ischemia / reperfusion injury in rats , This experiment observed in early to moxibustion preventive treatment can effectively inhibit the basis of cerebral ischemia-reperfusion injury,model for cerebral ischemia/reperfusion injury in rats,by means of moxibustion preventive treatment.Use the perspective of endoplasmic reticulum stress applied within immunohistochemistry and quantitative real-time PCR technology dynamic observation of endoplasmic reticulum stress -inducible protein GADDl53, marker protein GRP78, Caspase -12 protein expression inherent and to explore whether the moxibustion preconditioning has protective effect of the brain concerned with weakened ERS confirmed as a benign stress . So that the body produces appropriate ERS, which play a protective role of endogenous brain Further analysis of differences in gene expression related to time and space-time，hoping to initially determin the protection of moxibustion on time windown. Initially identified moxibustion Provide reliable experimental and theoretical basis for clinical moxibustion prevention of cerebral ischemic cerebrovascular disease . initially identified moxibustion Provide reliable experimental and theoretical basis for clinical moxibustion prevention of cerebral ischemic cerebrovascular disease and build up a basis for acupuncture career advancement in the future.

缺血性脑血管病是具有高发病率、高致残率、高死亡率的常见病，研究表明，脑缺血/再灌注损伤是急性脑梗死再通复苏后常见的病理过程。近年发现内质网应激参与脑缺血/再灌注损伤，已成为研究的热点，研究证实，针灸对脑缺血/再灌注损伤具有保护作用，但针灸影响内质网应激对脑缺血/再灌注损伤的研究尚未见报道，本实验在我们前期工作的基础上，以脑缺血/再灌注大鼠为模型，以灸预处理为干预手段，从内质网应激的角度，应用免疫组化和实时定量PCR技术动态观察内质网应激诱导蛋白GADDl53、标志蛋白GRP78、固有蛋白Caspase一12的表达,探讨灸预处理脑保护作用是否与减弱ERS有关，证实灸预处理作为一种良性应激刺激,使机体产生适宜的ERS，而发挥内源性脑保护作用。进一步分析相关基因的的差异表达、时空性等， 确定艾灸的保护时间窗，为临床灸疗防治脑缺血性脑血管病提供可靠的实验依据和理论基础。发展中医针灸事业。

主要研究内容：.以脑缺血/再灌注大鼠为模型，以灸预处理为干预手段，测试动物的神经行为学评分，计算脑梗死体积、脑含水量。从内质网应激的角度，对实验样本的病理学、免疫组化、 蛋白免疫印迹（Western blot）、PCR等相关指标进行检测，并对数据进行统计分析。动态观察内质网应激诱导蛋白GADDl53、标志蛋白GRP78、固有蛋白Caspase一12的表达,并探索灸预处理与内质网应激启动效应、凋亡效应密切相关的PERK、IREI通路之间联系。.重要结果：.1.灸预处理能明显改善I/R大鼠神经功能缺损体征和大鼠梗死灶体积。.2.灸预处理能减轻脑组织病理学损伤。.3.灸预处理可通过上调GRP78蛋白及其mRNA表达，提高ER的蛋白处理能力，下调神经元CHOP和Caspase-12蛋白表达，抑制神经元凋亡，维持内质网的稳定性，起到神经元保护作用。本实验显示灸预处理组GRP78蛋白低于同一时间点的模型组，差异具有统计学意义(P< 0.05 )，提示预先的灸预处理增加了大鼠对损伤的应激能力，在脑缺血后产生的未折叠蛋白少于模型组。.4.灸预处理靶向性阻断UPR通路eIF2α信号通路，提高PERK通路的活跃程度，上调PERK蛋白表达有关，维持和恢复细胞自噬及细胞内稳态，使细胞内蛋白质合成减少，缓解了ERS，.5.灸预处理对脑缺血/再灌注损伤的保护作用具有时效性，在脑缺血/再灌注24h和72h检测的指标差异性较明显，这部分数据分析结果暂未全部完成，故对于保护时间窗的结论，将在后续讨论。.科学意义：. 内质网应激（ERS）对决定应激细胞的结局、适应、损伤或凋亡有重要作用。本项目以内质网（ER）为靶点研究灸预处理对脑缺血/再灌注损伤的保护作用，将为针灸防治脑血管疾病研究提供新思路。为阐释针灸的作用特点及防治相关重大疾病可能机制提供参考。具有重要的科学和现实意义。