清开灵有效组分干预缺血性中风细胞粘附机制的研究

【作者】 徐丽荣；

【导师】 唐启盛； 李澎涛；

【作者基本信息】 北京中医药大学 ， 中医内科， 2005， 博士

【摘要】 在临床脑血管病中,70%以上为缺血性脑血管病,缺血性脑卒中的发病率、死亡率和致残率都很高,已经成为我国主要病死原因之一。人们传统认为中枢神经系统是受血脑屏障保护的免疫特免器官,随着对缺血、缺氧性脑损伤机制研究的不断深入,发现脑组织也能产生细胞因子和化学因子,表达粘附分子,引起炎症反应。炎症反应在脑损伤中起着重要作用,脑缺血后继发炎症损伤,以其预后差,死亡率高,而日益受到人们的关注,已成为该领域的研究热点。而循环中的白细胞与微血管内皮细胞异常粘附是引发炎症反应的前提条件。清开灵注射液为安宫牛黄丸的改良剂型,具有清热解毒、化痰通窍的功效,应用于临床以来,初期广泛用于治疗各种高热、感染以及各类流行性疾病。上世纪八十年代初,开始应用于以中风急性期为主的脑血管疾病的治疗,取得了较好的临床疗效。清开灵注射液的主要功效与中风病“毒损脑络”的中医病机学说在一定程度上相吻合,其在缺血性脑血管病中发挥作用的机制、环节有待明确,进而可望从微观的层面发掘“毒”、“络”、“毒损脑络”病机及“解毒通络”治法的现代生物学内涵。本课题为国家重点基础研究发展规划(973) 项目“祛邪扶正方剂的研究--清开灵阻抑中风病缺血级联反应的药效物质与作用机理”(课题号G1999054404) 的一部分,基于以上理论,在前面工作的基础上,选择缺血后12h、24h两个时段,以白细胞与内皮细胞的粘附过程为研究中心,以清开灵有效组分为干预手段,以脑缺血后血管内皮细胞及神经元的损伤程度为主要药效评价因素,检测相关细胞因子、粘附因子的表达水平。具体包括以下五部分内容及相关实验:一、清开灵有效组分对引发炎症及血管内皮细胞损伤始动因子NF-κB的干预作用胆酸在缺血12h阻抑内皮细胞NF-κB表达;栀子苷在此时段显著阻抑其在神经元表面的表达,至缺血后24h该作用进一步加强,对其在内皮细胞的表达亦发挥阻抑作用;黄芩苷在缺血后不同时段均使内皮细胞NF-κB表达减弱,作用持续、稳定;合方对其在内皮细胞、神经元、胶质细胞上的表达均有阻抑作用。总之除珍珠母外各药物在缺血后不同时段阻抑NF-κB表达,进而减轻血管内皮细胞损伤及缺血后炎症反应。二、清开灵有效组分对细胞粘附不同阶段的干预作用在本部分生化实验中,脑缺血后不同时段不同有效组分发挥作用的程度虽有差异,但总体来说,除珍珠母外各有效组分均能显著降低MCAO大鼠脑组织中E-selectin水平;珍珠母、黄芩苷、合方在缺血12h可降低ICAM-1表达水平,至缺血24h栀子苷、胆酸也开始显效,免疫组化图像也显示了相似的作用趋势,各药物通过以上作用阻抑白细胞与内皮细胞的初级、牢固粘附。本部分免疫组化结果显示珍珠母、栀子苷、黄芩苷、合方对缺血24h内皮细胞表面P-selectin表达具有显著抑制作用,胆酸也有一定阻抑作用,从而抑制白细胞与内皮细胞的初级粘附;除胆酸以外其他药物均可抑制内皮细胞PECAM-1的表达,以栀子苷作用最著,合方次之,黄芩苷在缺血早期作用显著,至后期开始减弱,珍珠母也有作用,在缺血后不同时段有效组分发挥了较一致的阻抑作用,抑制白细胞穿越内皮细胞进入脑组织。清开灵有效组分干预缺血性中风细胞粘附机制的研究三、清开灵有效组分对脑缺血后微血管挛缩状态的干预作用 脑缺血1 Zh血浆ET一1的水平已明显上升,至24h仍保持高水平,而有效组分中只有桅子营及合方在缺血后24h才显示了阻抑其表达的作用,且合方的抑制作用优于单用桅子昔,可以认为除桅子着外其它组分单用在此环节虽无明显治疗作用,但多种有效组分可协同作用而抑制血浆ET一1的表达;脑缺血1 Zh,除合方外各有效组分均表现为不同程度的抑制血浆TxBZ表达作用,其中胆酸和黄答普还有升高血浆6一ket。一PGF,Q趋势,从而在此时段表现为除珍珠母外各组分能显著阳氏TxBZ/6一keto一PGF:Q比值,发挥较理想的舒张微血管、保护内皮的作用;至缺血24h,黄答昔、合方仍能抑制血浆TXBZ高表达及降低TxBZ/6一ket。一PGF,a比值,而珍珠母及胆酸则有升高血浆6一keto一PGF:Q趋势。从以上两个时段三个紧密相关指标的变化规律及有效组分的干预作用可以看出,有效组分对TxBZ/6一ket。一PGF,Q比值影响与对血浆TXBZ影响趋势大体一致,提示血浆TxBZ的升高是引发微血管痉挛的主要因素,药物主要通过抑制血浆TxBZ高表达从而降低TXBZ/6一ket。一GFIQ比值,使微血管痉挛状态得以改善。四、清开灵有效组分对脑缺血后血小板活化的干预作用 脑缺血不同时段毗A0大鼠血清P一5 electin水平均显著升高,桅子普、胆酸、合方在缺血1 Zh时段可以降低血清可溶性P一selectin的浓度,至缺血24h,桅子昔、胆酸作用不著,而珍珠母、黄答昔开始显效,合方则仍继续阳氏血清可溶性P一se lectin浓度,从而抑制血小板活化;脑缺血1 Zh,模型组血浆TX残含量高于正常组,除合方外的有效组分能阻抑缺血1 Zh毗AO大鼠血浆TxBZ高表达,其中珍珠母作用尤为显著;缺血24h,黄答昔、合方仍可阻抑其表达。各有效组分通过阻抑血小板的活化及其内容物的释放来减轻缺血后内皮细胞损伤、缺血程度及炎症反应。五、清开灵有效组分对脑缺血损伤的干预作用 脑?更多还原

【Abstract】 More than 70% cerebrovascular diseases are caused by ischemia in clinic. The incident rate, death rate and mutilate rate of cerebral arterial thrombosis are very high. It has been one of mainly cause for death. Central nervous system, according to traditional concept, is except from immune system for the protection of blood brain barrier. With progresses in mechanism of brain damage, more accumulating evidences show that cytokines, adhesion molecules exist in brain tissues and cause inflammation. Inflammatory reaction plays a key role in brain damage. A new hot spot is coming in this field and more and more scholars pay attentions to it. The precondition of inflammation is abnormally adhesion between leucocytes and vascular endothelia cells in circulation.QINGKAILING parenteral solution is the improved dose derived from Angongniuhuang pills. The effects of QINGKAILING include removing fever, detoxication, expectorant and purging aperture. In the early days, it widespread in treating hyperpyrexia, infection and epidemic diseases, and it was extensively used to treat acute stroke since 1980s. This therapy is successful in clinic for the main mechanism partly coinciding with the new rising hypothesis of ’toxin damage brain veins’. The mechanism, the target points of QINGKAILING parenteral solution are not clear and more evidences are required to verify its effects in treating cerebrovascular diseases caused by ischemia. Thus, it is necessary to explore the molecular evens of ’toxin’, ’meridian’ and ’toxin damage brain veins’.This study was supported by a grant(G1999054404) from The National Program For Key Bisic Reasearch Projects(973)-’The effective components and mechanisms of QingKaiLing in depress ing cascade reaction of ischemic stroke’. Two time points, 12 hours and 24hours, are defined according to previous researches. For these progresses,this study focuses on the process of adhesion between endothelia cells and leucocytes. The level of neuronal and endothelia cell injury are evaluated and the expression levels of cytokines and adhesion molecules are detected after the cerebral ischemia model rat treated with QINGKAILING Our studies are performed as follows in five parts:Part one : The intervention effects of effective components of QINGKAILING on NE- κB - the initiation factor of vascular endothelia injury.The expression level of NF— κB are depressed by cholalic acid on endothelia after 12h ischemia. Jasminoidin obviously depress the expression level of NF—κB on both neur and endothelia after 12h and more stronger after 24h on neur. The depressed level of NF—κB was observed on endothelia in different periods in baicalin treating rats. The same effects on NF—κB are observed on endothelia, neur and glial cells when rats are treated with HeFang. In a word, these components can depress inflammation reaction by decreasing the expression of NF —κB. Part two : The intervention effects of effective components of QINGKAILING on expression of adhesion molecules in different period.Although the depress level is different in different time points in this part, the results show usthat the effective components of QINGKAILING can obviously decrease the level of E-selectin in cerebral tissues of MCAO rats except nacre. Nacre, HeFang and baicalin significantly reduced the expression of ICAM-1 after ischemia 12 hrs. After ischemia 24hrs, jasminoidin and cholalic acid begin to show their effects on depressing the expression of ICAM-1. The same tendency can be seen in the results of immunochemistry. These mechanisms contribute to the effects on depressing the primary and substance adhesion between leukocytes and endothelia.The results of immunochemistry show us that the expression levels of P-selectin are obviously decreased by nacre, jasminoidin, baicalin and HeFang on endothelia, and in a way, cholalic acid depress too. All components except cholalic acid depress the expression of PECAM-1 on endothelia, the strongest one is jasminoidin and HeFang takes the second place. Baicalin is more effective in the early period and更多还原