清开灵有效组分阻断脑缺血炎症反应的配伍机制研究

【作者】 徐雅；

【导师】 李澎涛；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2004， 硕士

【摘要】 大量的临床和动物实验资料表明,清开灵治疗缺血性脑血管疾病的疗效是确切的。前期的实验研究表明,清开灵有效组分中胆酸、栀子苷、黄芩苷和珍珠母在治疗缺血性中风中发挥了重要作用。本文就清开灵有效组分在阻断炎症反应这一环节的作用机制及配伍特征进行深入研究,以丰富清开灵“祛邪扶正”的生物学内涵,诠释清开灵“解毒通络”作用的现代生物学基础,初步讨论中药复方的配伍特征,为研究现代化中药制剂提供实验依据。 本课题包括文献综述和实验研究两个部分,文献综述讨论前炎症细胞因子与缺血性脑血管疾病的关系以及中医药治疗缺血性脑血管疾病的研究概况。实验研究采用大鼠MCAO 模型,研究清开灵有效组分阻断炎症反应的作用机制及配伍特征。研究结果包括:1 清开灵有效组分阻抑脑缺血损伤的病理学观察 脑缺血损伤后,可见假手术组脑组织结构清晰,皮层神经细胞排列整齐。神经元胞浆丰富,淡染,胞核居中,核仁清楚。血管内皮细胞完整、连接紧密。脑实质内未见炎细胞浸润。缺血12h和24h模型组可见损伤侧皮层神经元缺失,密度降低,间质疏松呈水肿样。神经细胞胞浆浓缩红染,胞核皱缩。胶质细胞增生、水肿。血管周围间隙增大,炎细胞聚集于血管周围,内皮细胞受损,偶见血管内多形核白细胞粘附和嵌塞。脑组织内可见散在淋巴细胞浸润。各给药组大鼠脑组织病理变化均有不同程度的减轻。尤以缺血12h合方、胆酸、珍珠母的作用较显著。 神经元特异性烯醇化酶(NSE)作为特异性高的神经元损伤标志酶,可以直接反映神经元受损的程度。实验结果显示:在缺血12h珍珠母、胆酸、合方有降低NSE含量的作用,而栀子苷和黄芩苷对NSE 含量无影响,在缺血24h 各给药组对血清中NSE 的含量均无影响,说明胆酸、珍珠母及合方能降低血清中NSE,但具有时效性,即发挥作用的时间在缺血12h。结合形态学变化推断胆酸、珍珠母以及合方在缺血损伤早期(12h)能够抑制神经元坏死,对神经元具有直接或间接的保护作用。2 清开灵有效组分对前炎症细胞因子脑损伤作用的影响 目前对前炎症细胞因子的研究中,认为在缺血的早期,IL-1β与TNF-α的产生主要起损害性作用。TNF-α产生较早,可能作为启动因素引发炎症损伤的级联反应并促进损伤的进一步加重。IL-1β是具有多向性生物功能的炎症介质,对炎症和免疫具有调节作用。所以干预这些因子的合成与分泌,有望减轻脑缺血损伤,减小梗塞体积,促进神经功能的改善。 通过对清开灵各有效组分的作用比较可以看出,在缺血12h,针对TNF-α清开灵各有效组分的作用效果都不显著,针对sTNF-R1以合方和胆酸的作用最突出,胆酸体现出强的增强作用,而合方却显示出强的抑制作用。表明四者的配伍应用在这一靶点上显示了毒性作用。针对IL-1β这一靶点,栀子苷、黄芩苷和胆酸都显示较强的抑制作用,相互之间作用强度无显著统计学差异。针对sIL-1R,只有栀子苷表现出弱的促进作用。 缺血24h,针对sTNF-R1合方与胆酸都体现出抑制作用,以合方的作用为突出。针<WP=6>中文摘要 -2-对IL-1β,各有效组分都发挥了抑制作用,栀子苷、黄芩苷、珍珠母与合方的作用最突出。针对sIL-1R,各有效组分都具有促进其分泌的作用,以胆酸与黄芩苷的作用最突出。 可见缺血 12h 胆酸具有抑制 TNF-α,升高 sTNF-R1、sIL-1R 的作用。表明胆酸发挥脑保护作用的机制是通过抑制 TNF-α的活性,促进 sTNF-R1、sIL-1R 的分泌以拮抗IL-1β与TNF-α的生物作用,从而发挥脑保护作用。栀子苷的作用主要是通过抑制IL-1β的活性,促进sIL-1R的合成以限制IL-1β与膜上固定信号受体结合,从而保护脑组织免受 IL-1β的损伤。黄芩苷在缺血 12h 能显著降低脑组织中 IL-1β的含量,而对 TNF-α、sTNF-R1、sIL-1R的作用不显著。而珍珠母在缺血12h针对TNF-α、sTNF-R1、IL-1β、sIL-1R四个靶点作用不突出。合方具有抑制sTNF-R1、IL-1β活性的作用,而对TNF-α、sIL-1R的用效果不显著。 缺血24h胆酸具有抑制TNF-α、sTNF-R1、IL-1β活性,促进sIL-1R合成的作用。栀子苷具有抑制 TNF-α、IL-1β的分泌,促进 sIL-1R 合成的作用。其主要作用体现在抑制IL-1β的活性。黄芩苷具有强的抑制IL-1β和促sIL-1R合成的作用。珍珠母具有抑制TNF-α、 β的分泌,促进sIL-1R合成的作用。合方具有强的抑制TNF-α、 IL-1 sTNF-R1、IL-1β活性和促sIL-1R 合成的作用。3 清开灵有效组分对前炎症细胞因子神经保护作用的影响 IL-6 在中枢神经系统的生理功能非常复杂,具有神经营养和神经保护作用,是一种神经元死亡抑制剂。实验结果显示:缺血12h合方具有显著抑制IL-6表达的作用,胆酸、栀子苷和黄芩苷则显著促进 IL-6 的表达。缺血 24h 只有珍珠母显示出弱的增强 IL-6 表达的作用;而栀子苷、黄芩苷、珍珠母、胆酸以及合方都具有促进sIL-6R表达的作用,尤以合方、胆酸、栀子苷和珍珠母的作用突出。通过进一步的比较可以发现栀子苷、黄芩苷和胆酸针对 IL-6 和 sIL-6R 均有促进作用,而四位配伍组成的合方却表现出不同的作用,早期抑制 IL-6 的合成,而在缺血更多还原

【Abstract】 Massive clinical and animal experimental material indicated that, QINGKAILING’seffective ingredients have accurate effection on ischemic cerebrovascular disease. Thepreliminary experimental study showed that, QINGKAILING’s effective ingredients such as thecholic acid, Jasminoidin, baicalin, and nacre play a vital role on the treatment of stroke. Thisarticle researchs on the mechanism and the character of their compatibility, in order to reveal itsbiology material of “detoxification and dredging collaterals”, initially discusses the rule of theChinese native medicine compound prescription compatibility and provides evidence on theexperiment for the research modernization Chinese native medicine preparation. This topic includes two parts: the document research and the experimental study, the formeris about the relationship between proinflammatory cytokine and ischemic cerebrovasculardisease and the research survey of the Chinese medicine treating ischemic cerebrovasculardisease. The later studies the mechanism and the compatibility character of QINGKAILING’seffective ingredients blocking the inflammation response by the rat MCAO model. The resultsare as following: 1. Pathology observation of QINGKAILING’s effective ingredients blocking theinflammation response Through hematoxylin and eosin staining, it can be seen that the brain organizationalstructure is clear and integrity, the cerebral cortex nerve cells are crowded and its arrangement isneat. Neuron plasm is rich and palely dyes, nucleus is on the core and the nucleolus is clear.Blood vessel endothelial cells are integrity and connected closely. The adhesion and Impactionof cells have not been seen. The organization around blood vesse is compact. The phlogocyte’sinfiltration has not seen in the Cortex (picture 1) in the sham-operated group. After 12h and 24h of ischemia, obvious damages can be seen in the operated groups. Wecan see that neurons are deleted obviously in the damaged cerebral cortex and its density reduces,stroma is edema and rarefaction. The nerve cell plasm is concentrated and dyed red. The nucleusis shrunk. Glial cells are proliferating. The gap around the vessel increases. The phlogocytegathers to vessel. The endothelial cell suffers injury, the partner sees in the blood vessel thepolymorphonuclear white blood cell adherency and Impaction. In the brain organizationobviously disperses in the lymphocyte infiltrates (picture 2-3). The groups administered with QINGKAILING’s effective ingredients have the varyingimprovement on pathological changes respectively. The nerve cell swelling, karyopyknosis, thespongiocyte proliferation swelling, the blood pipe wall twists, pathological change and so onaccumulation has the change for the better, the neutral granular cell and the lymphocyte infiltratethe reduction, You Yique blood 12h gathers the side, the cholic acid, the mother-of-pearl functionis more remarkable (picture 5-14). The neuron specificity enolase (NSE) can"leak off" from the neuron after the cerebral<WP=9>英文摘要 -5-ischemia. It causes the content of NSE in the blood and cerebrospinal fluid rising. It had beenproved that the content of NSE in the serum has positive correlation with the infarct area aftercerebral ischemia. At ischemia 12h, nacre, cholic acid and their compatibility can reduce thecontent of NSE in the serum. Whereas Jasminoidin and baicalin have on effect on it. At ischemia24h, all groups administered with QINGKAILING’s effective ingredients have on effect onNSE. It indicated that nacre, cholic acid and their compatibility could reduce the content ofNSE.so we can conclude that nacre, cholic acid and their compatibility have neuroprotection, buttheir mechanism is still to be discussed. 2. Mechanism discussion of QINGKAILING’s effective ingredients suppressingproinflammatory cytokine harmful function In the study of pr更多还原