冬虫夏草对缺血性中风后炎症反应的影响及神经保护作用的研究

【作者】 赵丹；

【导师】 刘振权；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2013， 硕士

【摘要】 缺血性中风是发病率、致残率和死亡率均较高的疾病,发病机制错综复杂,目前尚未完全阐明,严重威胁着人类的生命安全和生存质量。中医和中药是中国防治脑血管病的特色。冬虫夏草作为一味传统药材,成分丰富,具有人体所需的多种氨基酸、蛋白质、矿物质、微量元素等,对多种疾病疗效显著。现代医学研究已证明冬虫夏草具有调控免疫反应、治疗盗汗、调节胰岛活力、降低血脂水平、抗感染、抗肿瘤、抗衰老、保护肾脏及改善生殖功能、改善心血管疾病等广泛的药理学作用。并且,关于冬虫夏草提取物对大脑中动脉闭塞性脑缺血大鼠模型的神经保护及抗炎作用,课题组的初步研究已经显示较好的医药学研究价值。因此,本试验旨在前期探索基础上,研究冬虫夏草的药理学作用及其抗炎作用机制,以期为防治缺血性中风提供新的药物选择和科学依据。目的：本研究通过考察动物模型脑缺血后行为学变化、冬虫夏草的神经保护作用与缺血后损伤级联反应中的炎症反应等,探讨脑缺血后的炎症变化及冬虫夏草的抗炎作用机制。另外,由于野生冬虫夏草比较稀珍,并且是首次探索性研究,从保护资源和动物福利的科学性等方面考虑,本实验将采用冷水负重游泳小鼠模型模拟中医“中风痴呆病”气虚血瘀证入手进行验证研究冬虫夏草对脑缺血缺氧的保护作用。1.考察冬虫夏草对冷水负重游泳模型小鼠的脑保护作用；2.考察冬虫夏草对线栓法脑缺血模型大鼠的神经保护作用；3.考察脑缺血后炎症介质TNF-α与IL-1β及黏附因子ICAM-1与VCAM-1的变化及冬虫夏草对其影响。方法：1.观察并测定小鼠断头张口呼吸时间,紫外分光光度法测定模型小鼠脑组织中乳酸含量、钠钾-ATP酶活力；2.根据神经功能评分、TTC染色法求脑梗死灶范围及测定脑水肿程度等行为学方法观察冬虫夏草对线栓法脑缺血模型大鼠的神经保护作用；3.应用酶联免疫吸附测定法(ELISA法)观察脑缺血后模型大鼠脑组织中的炎症介质TNF-a与IL-1β及黏附因子ICAM-1与VCAM-1的变化及冬虫夏草的抗炎作用机制。结果：1.与模型组相比,冬虫夏草中剂量和小剂量,均不同程度延长了冷水负重游泳模型小鼠的断头张口呼吸时间(P<0.05)。乳酸结果测定显示与模型组相比,冬虫夏草大剂量组乳酸含量明显下降(P<0.01); Na+K+-ATPase活力测定结果显示仅阳性对照组较模型组明显增加(P<0.05)。2.神经功能评分结果显示,给药后相对于模型组,冬虫夏草大剂量和中剂量均有促进神经保护作用,且作用比较显著(P<0.01)。TTC(2,3,5-氯化三苯基四氮唑)染色法结果表明,与模型组对比,各给药组的梗塞灶范围均显著性减小(P<0.01)。此外,将脑缺血大鼠患侧与健侧脑含水量相比较,患侧脑含水量显著增加(P<0.001),脑水肿严重,进一步证明了模型的成功性；并且,与假手术组相比,模型组患侧脑含水量增加显著,脑水肿明显(P<0.001),给药组对于减轻脑缺血后脑水肿程度的作用明显,其中冬虫夏草大剂量组和小剂量组作用特别明显(P<0.01)。3. ELISA法测定的脑缺血损伤后脑组织中TNF-α、IL-1β、ICAM-1、VCAM-1含量的变化,结果表明,模型大鼠脑组织中TNF-α、 IL-1β、ICAM-1、VCAM-1含量增加,其中IL-1β、ICAM-1及VCAM-1含量增加显著,而给药组可明显减少模型大鼠脑组织IL-1p及VCAM-1的含量(P<0.05),从而抑制炎症的发生与发展,减轻脑损伤。结论：1.冬虫夏草在模型小鼠断头后,脑供血中断,延长了小鼠张口喘气时间,从而提升了其存活能力,可能与降低乳酸含量、调节生化指标有关。2.冬虫夏草可明显改善脑缺血损伤后大鼠的神经功能症状,减少梗死灶范围,减轻脑水肿程度,具有明显的神经保护作用。3.冬虫夏草对于脑缺血症状的神经保护作用机制,可能是通过抑制TNF-α、IL-1β. ICAM-1、VCAM-1等炎症因子的表达,减少其在脑组织中的含量,从而起到一定的抗炎效应而产生脑保护作用。更多还原

【Abstract】 Cerebral ischemia is a disease with high risk of incidence, morbidity and mortality. Pathogenesis of ischemia stroke is very complicated and has not been fully elucidated. At present, ideal drug has not been found to prevent and treat the ischemia stroke, which affected the living and life safety.The traditional Chinese medicine (TCM) plays a unique role in preventing the brain disease. Cordyceps sinesis, a typical traditional medicine, contains rich ingredients, such as amino acids, protein, minerals, etc. And modern medical research shows that C.S. has the function of treating many diseases.The extractives from C.S. has been found its neuro-protective effects and anti-inflammatory effects in middle cerebral artery occlusion cerebral ischemia models. Therefore, the target of this study is to further explore the pharmacological effects and anti-inflammatory mechanism of C.S..Objective:In this study, the behavior of animal models suffering cerebral ischemia, neuroprotective effects of C.S. and anti-inflammation effect cascading with ischemic damage cascade of were examined and observed, which were also used to elucidate the neuroprotective mechanism and anti-inflammation effect of C.S.. Moreover, the swimming mouse model with weight-bearing in cold water was used to perform qi asthenia and blood stasis of "disease of stroke and dementia" for validation study of protective effect about C.S.,1. To investigate the brain protection effect of C.S. on swimming mouse model with weight-bearing in cold water;2. To investigate the neuroprotective effect of C.S. on cerebral ischemia model in rats suture method;3. To study the variation of inflammatory mediators of TNF-alpha and IL-lbeta and adhesion factor of ICAM-1and VCAM-1changes in cerebral ischemia rat models; and to elucidate C.S. influence on these index.Methods: 1. Mouth breathing time of mouse after decollation was observed and determined;Content of lactic acid, sodium-potassium-ATP enzyme activity in brain tissue were measured by UV spectrophotometric method.2. The neuroprotective effects of C.S. on cerebral ischemia rats model was investigated by behavioral methods, such as the neurological score, TTC staining for determining the scope of in fare t and the degree of brain edema, etc.3. By using ELISA. transformation of TNF-α,IL-1β,ICAM-1,VCAM-1after cerebral ischemia were observed, which can be used for explain the anti-inflammatory mechanism of C.S..Results:1. Compared with model group, the mouth breathing time of mouse after decapitation with middle and small dosage of C.S. group showed the significance of difference(P<0.05). The content of lactic acid for the C.S. group with large dosage decreased(P<0.05); Na+K-ATP ase dynamic results showed only the positive control group was significantly increased (P<0.05).2. Neurological score results showed that the C.S., both large and middle dosage showed significant effects (P<0.01). The in fare t range of treated groups were significantly reduced (P<0.01). In addition, a significant increase of brain water content showed up in injured brain instead of the other, which confirmed the success of models; and vs models, the groups with drug show an clear function to alleviate the degree of cerebral edema, and both high and low dosage of C.S. showed significant (P<0.01).3. The content of TNF-α, IL-1β，ICAM-1, VCAM-1after cerebral ischemia was found to be increased by ELISA method. Drug administration clearly reduced the content of IL-1β and VCAM-1(P<0.05), which was the pathway for inhibiting the happening and developing the inflammation, and then relieve the damnification of brain.Conclusion:1. C.S. extended the gasping time for mouse and prolonged their survival time.2. As to the rats suffering cerebral ischemia, C.S. significantly improved their neurological symptoms, reduced infarct size and cerebral edema, also showed obvious protective effect of nerve system.3. The regulation of expression of inflammatory factors, TNF-α, IL-1β,ICAM-1, VCAM-1, and successively reduction of the content in brain tissue, was the potential mechanism routines of C.S. effect, which play a brain protection role by certain anti-inflammatory effect.更多还原