【作者】 高宗桂；

【导师】 李国彰；

【作者基本信息】 北京中医药大学 ， 生理学， 2014， 博士

【摘要】 中风是严重危害人类健康和生命安全的常见难治性疾病。祖国医学将其列为“风、痨、臌、膈”四大疑难病之首,存在着明显三高(发病率高、致残率高、死亡率高)现象。因而,提高中风的治疗与预防水平、降低中风的发病率,致残率和死亡率是当务之急。缺血性中风占所有中风的85%,因此研究缺血性中风的发病原因、机制及其防治对策,既有极其重要的社会现实意义,又具有较高的理论价值和临床实际意义。论文工作包括综述和实验研究两个部分。一、综述缺血性中风及其中药治疗相关研究的进展(一)系统概述了缺血性中风发病中能量代谢障碍、梗塞周围去极化、氧化应激损伤、炎症反应和细胞凋亡的发生、发展机制。(二)重点介绍了缺血性中风发病过程中由炎症反应和氧化应激损伤介导的信号转导通路研究进展,(三)重点介绍了川芎嗪在缺血性中风临床和基础研究方面的进展。明确这些研究领域的进展,将为本研究奠定坚实的理论基础,并为实验研究设计提供前提条件。二、实验研究(一)研究方法采用病理组织学方法、免疫组织化学方法、现代分子生物学方法。(二)技术手段采用流式细胞仪分析、定量RT-PCR基因检测、免疫印迹、免疫荧光双标法和化学比色法等技术。(三)检测指标1.脑损伤指标：脑梗塞面积、血脑屏障通透性、脑皮质含水量2.氧化应激相关因子：MDA、NO、CAT、GSH-Px。3.免疫指标(1)免疫细胞：中性粒细胞和T淋巴细胞数量、巨噬细胞/小胶质细胞的静息与活化的比值。(2)炎症介质：MPO、TNF-α和IL-1β。,4.信号转导分子：即早基因c-fos和c-jun、p-c-jun、Fos蛋白、Jun蛋白、JNK、AP-1、HO-1、Nrf2。(四)结果与结论1实验一川芎嗪对缺血性中风大鼠神经组织损伤及其功能的影响(1)实验制备的缺血性中风大鼠模型可靠：在实验大鼠脑组织发生了明确的脑组织损伤(脑梗塞、血脑屏障破坏和脑水肿),并伴发神经行为学改变；(2)川芎嗪对缺血性中风大鼠发生了明确的药理学干预作用：表现为减小脑梗塞的面积,减轻脑水肿和血脑屏障损伤程度,改善大鼠神经行为异常。提示：川芎嗪具有一定的神经保护作用。2实验二川芎嗪对缺血性中风大鼠氧化应激反应的影响(1)缺血性中风大鼠永久性脑缺血受损皮质组织发生了过氧化反应：MDA含量增加,中性粒细胞NO含量升高,CAT、GSH-Px活性有增高趋势；(2)川芎嗪可降低缺血性中风大鼠脑缺血受损皮质组织过氧化程度：川芎嗪可下调MDA含量和中性粒细胞NO含量；川芎嗪可上调抗氧化物质CAT、GSH-Px活性提示：川芎嗪干预缺血性中风机制中,抑制氧化应激损伤可能是其机制之一。3实验三川芎嗪对缺血性中风炎症反应的影响的实验研究(1)缺血性脑中风大鼠产生了明显的炎症反应：伤害侧皮质组织内CD45阳性的免疫细胞数量增多,巨噬细胞/小胶质细胞被激活数量增多；伤害侧皮质组织内MPO活性及CD68表面抗原蛋白表达量增多；(2)川芎嗪可干预缺血性中风大鼠的炎症反应：下调免疫细胞数量和活化的巨噬细胞/胶细胞数量,下调脑缺血造成受损皮质组织MPO活性及CD68表面抗原蛋白表达量。提示：川芎嗪干预缺血性中风机制中,抑制炎症反应可能是其重要机制之一4实验四川芎嗪对缺血性中风大鼠缺血诱导激活的信号转导通路的影响的实验研究(1)证实缺血性中风大鼠缺血脑组织启动的炎症反应信号转导通路之一是JNK/AP-1信号转导通路；川芎嗪干预缺血性中风大鼠过程中的抗炎作用机制：抑制AP-1DNA结合活性,限制或减弱转录蛋白c-Fos蛋白质c-Jun蛋白质和JNK蛋白磷酸化。提示：川芎嗪阻断或削弱JNK/AP-1炎症反应信号转导通路,从而实现抗炎和保护脑组织作用。(2)证实缺血性中风大鼠缺血脑组织启动氧化应激信号转导通路之一是Nrf2/ARE通路。川芎嗪干预缺血性中风大鼠的抗氧化应激的作用机制：进一步上调HO-1和Nrf2免疫荧光细胞数量、上调Nrf2和HO-1蛋白表达量。提示：川芎嗪可激活Nrf2/ARE通路,促使Ⅱ相酶HO-1释放,发挥清除自由基抗氧化作用,以减少脑组织的损伤。三、本课题研究的创新点(一)通过“川芎嗪干预缺血性中风作用的信号转导通路机制研究”,证实川芎嗪干预缺血性中风作用过程中,炎症反应和氧化应激损伤所介导的信号转导通路包括JNK/AP-1通路和Nrf2/ARE通路。(二)以缺血性中风氧化应激损伤为目标,重点观察了川芎嗪对缺血性中风大鼠受损皮质组织MDA、NO、Catalase和GPx作用的影响。证实川芎嗪可降低永久性脑缺血所引发的过氧化程度。(三)以缺血性中风炎症反应为目标,重点研究了川芎嗪对缺血性中风大鼠永久性缺血受损皮质组织免疫细胞(中性粒细胞、T淋巴细胞、巨噬细胞／小胶质细胞等)和炎症因子(TNF-α、IL-1β、MPO等)的影响。证实川芎嗪通过下调免疫细胞和炎症因子的方式来发挥抗炎作用。更多还原

【Abstract】 Stroke is a common refractory disease and a serious hazard to human health and safety. It is even considered as one of the four incurable diseases including "stroke, consumption, bloating, dysphagia" in Chinese medicine. Due to its characteristics of high incidence, high morbidity and high mortality, it has become a priority for this disease for reducing the incidence of stroke morbidity/mortality, and improving prevention and treatment of stroke. Because85percent of all stroke cases are due to ischemic event, it is becoming more important to explore pathogenesis, mechanisms and counter measures of ischemic stroke in theoretical value and clinical practice.The work of this paper includes two parts, review and experimental research.1. Ischemic stroke and the research progress in the treatment of ischemic stroke with Chinese medicineThis study systemically reviewed the development of the pathogenesis of ischemic stroke, the development of the signal transduction pathways involved in ischemic cerebral stroke from ischemic injury and the research progress in the treatment of ischemic stroke with Chinese medicine. It systemically summarized the obstacle of energy metabolism during ischemic stroke, peri-infarct depolarization, oxidative stress, inflammation and mechanism of apoptosis. It focused on the research progress of the inflammation and oxidative stress-mediated signaling transduction pathways in the pathogenesis of ischemic stroke, the clinical and basic research of tetramethylpyrazine (TMP) progress in ischemic stroke. Clarifying all of these progresses in the research was to lay a solid theoretical foundation for this study and provided preconditions for the experimental design.2Experimental research2.1Research methodsExperimental study using histopathological method, immunohistochemical method, modern molecular biological techniques.2.2Technical manipulationsFlow cytometry, Real-time RT-PCR for gene detection, quantitative immunoblotting, immunofluorescence method and chemical colorimetry technique.2.3Detection index2.3.1Brain injury index:the area of cerebral infarction, blood-brain barrier permeability, cerebral water content. 2.3.2Oxidative stress related factors:MDA, NO, CAT, GSH-Px.2.3.3Immune index2.3.3.1Immune cells, neutrophils and T lymphocytes, macrophage/microglial resting and activated ratio.2.3.3.2Inflammatory mediators:MPO, TNF-α and IL-1β.2.3.4Signal transduction molecule:the immediate early genes c-fos and c-jun, p-c-jun, Fos protein, Jun protein, JNK, AP-1, HO-1, Nrf2.2.4The research results2.4.1Experiment1Effect of Tetramethylpyrazine on experimental neuron-tissue injury and function of ischemic stroke in rats2.4.1.1A reliable Ischemicstroke rat model from experimental preparation:Clear and definite brain tissue injury in experimental rats (cerebral infarction, blood-brain barrier damage and brain edema), and concomitant neurological behavioral changes;2.4.1.2Tetramethylpyrazine has clear and definite pharmacological intervention on ischemic stroke rats:To express the reduction of cerebral infarction area, reduce brain edema and blood-brain barrier damage, improve the abnormal neurological behavior of rat. Suggestion:Tetramethylpyrazine has a neuroprotective effect.2.4.2Experiment2Effect of Tetramethylpyrazine on oxidative stress reaction of rats with ischemic stroke2.4.2.1The peroxidation of permanent cerebral ischemia of ischemic stroke in the rat with damaged cortical tissueThe increase of MDA content, increased content of NO in neutrophils, CAT, GSH-Px activity had increased trend;2.4.2.2Tetramethylpyrazine can reduce peroxidation of damaged cortical tissue of rat with cerebral ischemic stroke:Tetramethylpyrazine may reduce the content of MDA and content of NO in neutrophils.Tetramethylpyrazine may increase the activity of antioxidant CAT, GSH-Px.Suggestion:intervention mechanism of Tetramethylpyrazine on ischemic stroke, inhibiting oxidative stress injury may be one of its mechanisms.2.4.3Experimental3Study of on inflammatory reaction of ischemic stroke2.4.3.1Ischemic stroke of rats produced obvious inflammation:2.4.3.1.1On injury cortex tissue, the number of CD45positive immune cells increased and the number of macrophages/microglia activated increased;2.4.3.1.2On injury cortex tissue, the expression increased of MPO activity and CD68surface antigen protein; 2.4.3.2Tetramethylpyrazine interventing in inflammation of ischemic stroke in rats:To down-regulation of immune cells number and activated macrophages/microglia number.To down-regulation of MPO activity and CD68surface antigen protein expression of impaired cortical tissue caused by cerebral ischemia.Suggestion:intervention mechanism of Tetramethylpyrazine in ischemic stroke, inhibit inflammatory reaction may be one of the important mechanisms.2.4.4Experimental4Experimental study of Tetramethylpyrazine on signal transduction pathway induced and activated by ischemia in rats with ischemic stroke.2.4.4.1Confirmed one of the inflammatory signal transduction pathway initi ated by ischemic brain tissue in rats with ischemic stroke is JNK/AP-1pathway.The anti-inflammatory mechanism of Tetramethylpyrazine intervention of ischemic stroke in rats:the inhibition of AP-1DNA binding activity, restricted or decreased transcription protein c-Fos protein, c-Jun protein and JNK protein phosphorylation.Suggestion:Tetramethylpyrazine block or weaken the JNK/AP-1inflamm ation signal transduction pathway, so as to achieve the effect of anti-inflammati on and protect the brain tissue.2.4.4.2Confirmed one of the oxidative stress signal transduction pathway initiated by ischemic brain tissue in rats with ischemic stroke is Nrf2/ARE pathway.Effect and mechanism of antioxidant stress of Tetramethylpyrazine invent ischemia rats with ischemic stroke:further up regulation of immunofluor escence cells number of HO-1and Nrf2, up-regulating expression of Nrf2and HO-1protein.Suggestion:Tetramethylpyrazine may activate Nrf2/ARE pathway, the phase Ⅱ enzymes of HO-1release, antioxidation and clear the free radical, in order to reduce the injury of brain tissue.3. Innovation of the research3.1It is the first time to study the mechanisms of signal transduction pathway of TMP intervening in ischemic stroke.It confirmed that the pathways of TMP intervening in ischemic stroke are JNK/AP-1and Nrf2/ARE pathway in inflammation and oxidative stress-mediated signal transduction.3.2This study observed the effects of TMP focus on MDA, NO, Catalase and GPx role of damaged cortical tissue of ischemic stroke in rats with injury of oxidative stress as a target. It determined that TMP can reduce peroxidation caused by permanent brain ischemia.3.3This study focused on the effects of TMP on damaged cortical tissue immune cells of permanent ischemic stroke in rat (neutrophils, T lymphocytes, macrophages/microglia) and inflammatory cytokines (TNF-α, IL-1β, MPO, etc.) with inflammatory response of ischemic stroke as a target.It confirmed that TMP anti-inflammatory effect is by down-regulating immune cells and inflammatory cytokines way.更多还原