安宫清开灵干预自发性高血压大鼠脑出血模型炎症反应的机制研究

【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， 2007， 博士

【摘要】 出血性中风(intracerebral hemorrhage,ICH)是一种严重的中风亚型,伴有很高的死亡率和致残率,严重影响着人类的健康。而目前治疗脑出血的药物无一具有突破性的治疗效果,尤其是继发性脑损伤导致的神经功能缺失未得到根本解决。减轻出血后继发性脑损伤,营救神经元仍然是治疗的目标,而更好的理解脑出血的病理生理机制无疑是提高临床疗效的有效途径。目前临床有关脑出血的认识上,注重血肿的压迫效应,治疗上也强调血肿的清除和促进血肿的吸收。但这些治疗措施并没能有效的降低脑出血患者的死亡率和致残率。虽然现代医学研究也逐渐认识到脑出血后血管反应进而级联的炎症反应在脑微环境的破坏,进而神经细胞损伤的病理性过程中的作用,但是炎症反应并没有引起临床医生的重视,而且临床上也缺乏相应的治疗药物。因此,研制一种针对脑出血后炎症反应的药物是当务之急。本研究认为炎症反应导致继发性脑损伤发病机理与传统中医理论“毒损脑络”的病机相吻合,因此以炎症反应为切入点,探讨“解毒通络”药物对脑出血后继发性脑损伤的治疗作用具有重要的现实意义,有望能够为出血性中风的治疗提供新型的干预途径。安宫清开灵注射液是在前期973(课题号:G1999054404)研究成果的基础上,在863计划支持下,结合出血性中风的发病特点,研制的治疗出血性中风的新一代中药制剂,是按照“解毒通络”的大法组方,主要针对ICH“毒损脑络”病机而设立的,由栀子苷、黄芩苷、猪去氧胆酸、珍珠母水解液和三七的有效组分组成,具有“解毒通络”的功效。研究的目的主要是(1)观察脑出血后炎症反应与脑组织继发性病理损伤之间的关系;(2)观察安宫清开灵干预脑出血后炎症反应的作用机制。本研究采用尾状核胶原酶注射法复制SHR大鼠脑出血模型,采用放射免疫、酶联免疫吸附方法、免疫组织化学染色、HE染色、Weatern Blotting等实验手段观察了:①脑出血后TNF-α、IL-1β和ICAM-1的时程表达;②TNF-α与脑出血后炎症反应启动及IL-1β、ICAM-1生成表达和中性白细胞活化的关系;③脑出血后脑组织的病理形态学变化以及安宫清开灵的干预特征;④安宫清开灵对前炎症细胞因子TNF-α、IL-1β的作用效果;⑤安宫清开灵对黏附分子和中性白细胞活化的影响;⑥安宫清开灵对脑出血后核转录因子和热休克蛋白-70表达的影响特征;⑦安宫清开灵对细胞凋亡的影响等七个方面的内容。实验结果如下:1.脑出血后24小时、48小时、72小时和7天,脑内TNF-α、IL-1β、ICAM-1的含量显著升高。2.采用外源性抗体治疗后,脑组织和血液中的IL-1β和sICAM-1的含量显著降低。3.脑出血后48小时,基底核部位可见典型的弥漫型出血灶,并有小血肿形成。出血灶中可见广泛弥漫性结构破坏。出血灶周边呈现脑水肿、炎细胞浸润和微血管不畅等损害表现;而安宫清开灵可显著减轻该损害,以中、高剂量组最为显著。脑出血后14天血肿部位形成了胶质瘢痕,并可见空洞的形成。而安宫清开灵可显著减小胶质瘢痕的体积以及空洞形成的数量,以中、高剂量组效果最明显。4.放射免疫方法和免疫组织化学染色方法证实,ICH后48小时安宫清开灵可以显著抑制前炎症细胞因子TNF-α表达,而出血后7天这种抑制作用消失,而对前炎症细胞因子IL-1β表达的抑制作用能够从出血后48小时持续到出血后7天。5.酶联免疫吸附实验和免疫组织化学染色方法证实,ICH后48小时安宫清开灵可以显著抑制ICAM-1的表达,作用可以持续到出血后7天;而对中性白细胞活化的抑制作用以出血后48小时最显著,至出血后7天抑制作用减弱。6.免疫组织化学染色方法证实,ICH后48小时安宫清开灵可以显著抑制NF-κB的激活,而对HSP-70没有显著的促合成作用。7.免疫组织化学染色方法证实,ICH后48小时安宫清开灵可以减少血肿周围组织凋亡细胞的数量。总结以上实验结果,可以发现ICH后48小时,通过两种以上的实验方法证实,安宫清开灵低、中、高三个剂量组对前炎症细胞因子(TNF-α和IL-1β)、黏附分子(sICAM-1和ICAM-1)、白细胞活化的标志物MPO、核转录因子NF-κB和细胞死亡的数量都有显著的抑制作用,并呈现一定的剂量依赖关系,其中以中、高剂量的作用效果最突出。至出血后7天,安宫清开灵对IL-1β、sICAM-1和MPO仍有一定的抑制作用,也具有一定的剂量依赖关系,中、高剂量的效果最明显。可见安宫清开灵在出血的急性期(出血48小时)对出血侧脑组织炎症反应的启动和细胞凋亡发挥了广泛的抑制作用,并能持续到出血后的7天。分析以上实验结果,得出如下结论:1.炎症反应与脑出血后脑组织的继发性病理损伤存在密切的关系。2.安宫清开灵注射液通过有效抑制核转录因子NF-κB和前炎症细胞因子TNF-α和IL-1β和粘附分子、炎性白细胞的活化等,对炎症反应呈现显著的抑制作用。并能改善出血周围组织循环,为神经组织的再生修复提供了良好的微环境。3.脑出血的病理过程产生的前炎症细胞因子、活化的中性白细胞、内皮细胞和神经细胞表达的黏附分子等物质从病理性质上都应归属于传统医学“毒”的范畴,这些物质产生后直接或间接作用于血管内皮细胞或神经元,改变局部微环境,使血管舒缩功能障碍,血管壁通透性增加,造成局部组织水肿和能量代谢障碍,最终导致神经细胞的坏死和凋亡,这是对“毒损脑络”病机的进一步诠释。而安宫清开灵注射液可以抑制前炎症细胞因子和黏附分子的过度表达和白细胞的过度活化,能够解除这些“毒”的侵害,既可以抑制神经细胞的坏死,又可以防止细胞凋亡,起到了“解毒通络”的作用。更多还原

【Abstract】 Intracerebral hemorrhage is a devastating stroke subtype with high mortality and morbidity, with harmful effects on human’s health. There has no breakthrough medicine to the disease; especially neurological deficits caused by secondary brain injury still exist. The therapeutic object is to relieve secondary brain injury following ICH. Nevertheless, to better understanding the path-physiological mechanisms will be a good way to enhance the clinical therapeutic effect.Vascular reaction has attracted great attention of the medical science circle,which is related to pathologic lesion following ICH, and this academic trends partly coincides with the new rising hypothesis of‘toxin hurts brain collaterals’and the therapeusis of‘removing toxin and dredging collaterals’in Chinese medicine. Vascular endothelial cell damage exerted important effect on various pathological aspect of collaterals disease. A series of reaction arose as a compensative mechanisms,such as focal vasoconstriction, activation of the coagulation cascade, releasing of thrombin, erythrocatalysis,hemoglobin and its degradation products,which can lead to vascular reaction in hematoma and its surrounding. Vascular endothelial cells are activated and generate cytokine, which may affect neuron and focal microenvironment and lead to homeostasis disorder, cell surface receptor activated, and disorder of the signal transduction. Therefore, it is important to develop a formula to intervene vascular reaction following ICH, which probably enhances the therapeutic efficacy of stroke.An Gong Qing Kai Ling injection is a multicomponent injection derived from the study results of“973”and a latest Chinese drugs pharmaceutics for hemorrhagic stroke, which is developed according to the hypothesis of‘toxin hurts brain collaterals’and the therapeusis of‘removing toxin and dredging collaterals’. The components are include jasminoidin, baicalin, hyodeoxycholic acid, concha margaritifera usta digest and Radix Notoginseng. The research of“Study on functionary mechanisms of An Gong Qing Kai Ling injection in blocking inflammation following intracerebral hemorrhage”was funded by National High Technology Research Program of China (863 Program, 2002AA2Z3220).We copied experimental intracerebral hemorrhage model of spontaneous hypertensive rats using collagenase infusion in caudate nucleus method. And we used radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), immunohistochemistry stain, hematoxylin- eosine stain (HE) and Weatern Blotting to observe:①the time course of tumor necrosis factor-alpha(TNF-α), interleukin-1 beta(IL-1β) and intercellular adhesion molecule-1(ICAM-1) following intracerebral hemorrhage;②the relationship between tumor necrosis factor-alpha and inflammatory reaction, expression of interleukin-1 beta(IL-1β) and intercellular adhesion molecule-1(ICAM-1) and activation of neutrophil;③the pathomorphism changes in brain following ICH and the effect of An Gong Qing Kai Ling Injection;④the effection of An Gong Qing Kai Ling Injection on the expression of proinflamatory cytokine--TNF-αand IL-1β;⑤the effection of An Gong Qing Kai Ling Injection on the intercellular adhesion molecule-1 and activation of neutrophil;⑥the effection of An Gong Qing Kai Ling Injection on the expression of nuclear factor-κB and heat shock protein 70;⑦the effection of An Gong Qing Kai Ling Injection on the number of apoptotic cells. Results are as follows:1. The contents of TNF-α、IL-1βand ICAM-1 are significant elevated at 24 hours,48 hours, 72 hours and 7days following ICH.2. The concentration of IL-1βand sICAM-1 in brain or blood degrades after treated by exogenous anti- TNF-αmonoclonal antibody.3. Typical widespread hemorrhagic focus is formed at 48 hours after infusion, with small hematoma formation. The hemorrhagic focus is charactered by erythrocyte and inflammatory cell (neutrophil) infiltration, edema, nuclear structure of neurons are obscure, denaturation and necrosis, and sometimes neuronophagia can be seen. The tissues around hemorrhagic focus are loose, accompanied by infiltration of lymphocyte, cellular edema in neurons and glia cell, vascular endothelial cell swelling, cell aggregation leading to capillary blocked. An Gong Qing Kai Ling Injection can relieve the degree of looseness, The same as the inflammatory cell (neutrophil) infiltration, edema and vascular endothelial cell damage, without accumulation of blood cells, especially in middle and high dose groups. At 14 days following intracerebral hemorrhage, the hematoma was absorbed and its volume became small, and glial scar was formed with lots of microglial cells, vascular proliferation. Sometimes cavity was seen. There were microglial cells around hematoma which had been swallowing hemosiderin. The volume of glial scar and the number of cavity decreased in An Gong Qing Kai Ling Injection treated groups and control group, especially in middle and high dose groups.4. It has been confirmed that An Gong Qing Kai Ling Injection can prevent the expression of TNF-αat 48hours following ICH by RIA and immunohistochemistry stain, however the preventive effect diminished at 7days after ICH. The inhibitive effect of An Gong Qing Kai Ling Injection on the expression of IL-1βcan last from 48hours to 7 days following ICH.5. It has been found that An Gong Qing Kai Ling Injection can prevent the expression of ICAM-1 at 48 hours following ICH by ELISA and immunohistochemistry stain, and the effect can last from 48 hours to 7 days following ICH. The inhibitive effect of An Gong Qing Kai Ling Injection on the activation of neutrophil was significant at 48 hours following ICH, which weakened at 7 days.6. It has been found that An Gong Qing Kai Ling Injection can prevent the expression of NF-κB at 48 hours following ICH by immunohistochemistry stain. However it had no effect on the generation of HSP-70.7. It has been found that An Gong Qing Kai Ling Injection can decrease the number of apoptotic cells at 48 hours following ICH by immunohistochemistry stain.In summary, An Gong Qing Kai Ling Injection (low dose, middle dose and high dose) can prevent the expression of TNF-α, IL-1β, sICAM-1, MPO, NF-κB and decrease the number of apoptotic cells with dose-dependent manner at 48 hours and 7 days .following ICH. It is thus clear that An Gong Qing Kai Ling Injection can extensively suppress inflammatory reaction and decrease the number of apoptotic cells in acute stage.We can make such conclusion as follows:1. The content of cytokine was elevated and its peak between 24 hours and 72 hours following ICH, soon after descending and rising again ay 7days after ICH. Therefore, we select 48 hours and 7 days as the time window to administration.2. The content of IL-1β, sICAM-1 and MPO decreased after treated by TNF-αmonoclone antibody. It is thus clear that neutralization TNF-αin circulation can effectively suppress the expression of proinflamatary cytokine and adhesion molecule, as well as the activation of neutrophil.3. An Gong Qing Kai Ling Injection can effectively interfere the generation of TNF-αand IL-1β, thus can prevent the trigger of inflammatory reaction. Its action mechanism may be related with its prevention on the NF-κB.4. An Gong Qing Kai Ling Injection also can surppress the activation of neutrophil and the expression of ICAM-1. That is to say, it possess dual effect on cell adhersion, it can not only interfere VEC, neuron and glial cell in expression ICAM-1, but also suppress the actication of neutrophil, accordingly can decrease the neutrophil infiltration in hemorrhagic focus and exert neurprotective effect.5. An Gong Qing Kai Ling Injection also can decrease the number of apoptotic cells and prevent neuronal cell death, which can improve the outcome in clinic. Its mechanism may be related to its suppression on TNF-α, IL-1β, HSP-70 and NF-κB.更多还原