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蒙医温针疗法治疗脑缺血模型大鼠早期脑损伤的作用及机制研究
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摘要
缺血性脑卒中为卒中的主要类型,其高发病率、高死亡率、高致残率使其成为世界范围危害极大、急待攻克的一种疾病,其研究和防治已成了基础医学和临床医学的重要课题。该疾病属蒙医萨病范畴,蒙医有自己的认识和理论体系,根据长期实践经验,蒙医治疗该病症具有丰富的经验和独具特色的治疗方法,且具有一定的优势。蒙医温针疗法具有改善气血运行、抑制赫依巴达干和改善脑供血等功效,是治疗脑供血不足引起的症候群和缺血性脑梗死的行之有效的治疗力法之一,且有迅速、简便、易行等优点。本课题提出蒙医温针可能使缺血性脑卒中发生后减轻脑损伤、促进神经功能修复,这些可能通过清除自由基、抗氧化损伤、抑制炎性因子释放以及缺血时保持脑细胞的代谢起到脑保护作用的假设,采用蒙医温针治疗脑缺血再灌注模型(MCAO-R)大鼠的研究方法,阐明蒙医温针对缺血性脑卒中的早期抗脑损伤的机制,为该疾病的防治和研究提供有效、简便、易行的应用性广泛的方法和新研究思路。
     本文中概括了两个内容,其一、试述蒙医对缺血性脑血管病的认识,总结了近20年来蒙医关于脑缺血疾病的治疗情况和实验研究进展的综述;其二、蒙医温针治疗大鼠脑缺血冉灌注脑损伤急性期的治疗作用及其机制研究的实验报告。实验报告如下:
     目的:观察蒙医温针疗法对大鼠脑缺血再灌注后24h脑损伤的影响,探讨蒙医温针疗法治疗脑缺血急性期的脑水肿、神经功能损伤作用及抗脑损伤的作用及机制。
     方法:将SD雄性大鼠(体重280±20g),随机分成5组:模型组(M)、假手术组(F)蒙医温针组(W)、常规针刺组(C)、药物对照组(D)。采用改良的线栓法制备大鼠大脑中动脉缺血再灌注模型(MCAO),脑缺血90分钟后实施再灌注。治疗方法:大鼠清醒后1h进行治疗。W组,选取顶会穴、赫依穴,银针刺入5mm,连接电热针治疗仪,留针30分钟;C组,选取大椎、气海、命门穴位,分别毫针直刺5mm、2mm、4mm,留针30min,留针期间每隔10min行针1次;D组,按照尼莫地平片10.8mg/kg·d剂量,灌胃给药一次。再灌注24小时后,取材进行实验观察:(1)观察各组大鼠神经功能评分的变化;(2)将脑切片,TTC染色、固定后,采用成像系统软件处理,测定脑梗死体积;(3)将大鼠脑组织HE染色,观察脑组织形态学改变;(4)ELISA方法检测大鼠血清MDA、SOD、GSH、IL-1β、IL-8、TNF-a、NO、NOS、ET-1含量的变化。(5)ELISA方法检测大鼠海马组织GFAP、NSE含量的变化。
     结果:F组与M组之间,除了特别说明P>0.05以外,均有显著性差异,这能够表明模型组观察指标与假手术组有显著性差异。(1)神经功能评分:与M组比较,Pw,Pt<0.05,与C组比较,Pw<0.05。w、C组可明显改善神经功能,且w组更有显著性,明显优于C组。(2)脑梗死体积:与M组比较,Pr<0.05;Pw<0.01,与Y组比较,Pw,Pr<0.05。w、C组脑梗死体积明显小于组M、D组。(3)观察HE染色病理切片显示:与M组比较,w组、C组、D组神经细胞肿胀和间质水肿减轻,并w组细胞稀疏不明显,神经元数量较多。(4)MDA:与F组比较,PM,Pt,Pb>0.05;各组之间血清MDA含量无显著性差异,但均值柱状图显示:W组均值低于M组,有降低MDA趋势。SOD:与M组比较,Pb,Pw<0.01;P.<0.05;w、Y、Z组血清SOD含量明显高于M组。GSH:与M组比较,Pw,Pr<0.05;w、C组血清GSH含量明显高于M组。IL-8,TNF-a:与M组比较,Pw,Pbn<0.05;w、D组血清IL-8和TNF-a含量明显低于M组。N0,NOS:与M组比较,Pb,<0.05;Pw,P。<0.01;w、D、C组血清N0,NOS含量明显低于M组,且w,C组更有‘显著性。ET-1:与M组比较,Pb,<0.01:Pw,Pr<0.05。D、W、C组血清ET-1含量明显低于M组,且D组更有显著性。IL-1β:与M组比较,Pw,P.,Pb>0.05;与F组比较,PM,Pr,Pb>0.05;各组之间血清IL-1β禽量无显著性差异。(5)海马组织NSE:与M组比较,Pw<0.01;w组海马组织NSE的含量明显低于M组。GFAP:与M红比较,Pw,Pr,Pb,>0.05;与F组比较PM<0.05。D、w、C组之间海马组织GFAP的含量无显著性差异;F组与M组之间有显著性差异。
     结论:(1)蒙医温针疗法能够明显改善脑缺血冉灌注早期神经功能缺损,从神经功能改善角度揭示蒙医温针疗法的脑保护作用。(2)通过观察TTC染色脑切片和HE病理切片,蒙医温针疗法有缩小脑梗塞体积,减轻脑水肿作用,因此在脑缺血再灌注早期可能起到抑制半暗带的继续损伤及神经细胞凋亡,从而能够改善脑缺血冉灌注早期神经功能缺损,亦能够提高脑梗塞预后康复程度。(3)通过观察测定脑缺血再灌注早期血清MDA、SOD、GSH、NO、NOS、ET-1等指标,可总结出蒙医温针疗法对脑缺血再灌注损伤早期有抗氧化、抗自山基损伤的趋势,其机制可能是脑缺血再灌注早期能够降低血清NO、NOS、ET-1,提高SOD和GSH含量而有效的拮抗自山基损伤,提高脑组织抗氧化能力来实现的。(4)通过观察测定脑缺血再灌注早期血清TNF-a、IL-lβ、IL-8等指标,可总结出蒙医温针疗法对脑缺血再灌注损伤早期有抗炎作用,其机制可能是脑缺血再灌注早期能够降低血清TNF-a、IL-8,提高SOD和GSH含量而有效的拮抗炎症因子来实现脑保护作用的。(5)NSE是在脑缺血早期能反映脑损伤程度,并可作为评定恢复期神经功能康复程度的客观生化指标之—,本次研究结果初步表明蒙医温针疗法对脑缺血再灌注损伤早期有降低海马组织(?)NSE(?)勺含量,这有可能成为蒙医温针能够起到挽救和保护缺血区神经元的依据之一。
Cerebral Ischemic Stroke is one of the main types of Stroke. It has the characteristics of high incidence rate, high death rate and high disabled rate. Because of the characteristics mentioned above, Ischemic Stroke has become the most dangerous disease that has to be conquered. The study of Ischemic Stroke has also become an important subject in the basic medical and clinical medical aspects. This kind of disease belongs to the category of Hemiplegia. Mongolian medicine has its own recognition and theoretical system about this kind of disease, and according to long term practical experience, it has plenty of experience and unique way to treat the disease of Ischemic Cerebrovascular. Mongolian Medical Warm Acupuncture has the functions of improving blood-going, protecting Hectar in Sparta and improving blood-supplying to the brain. Thus It has become one of the treating methods to the syndrome and the brain infarctions caused by the lack of brain blood-supplying, and it has also the advantages of rapid, easy and easy-doing characteristics. This dissertation puts forward that Mongolian Medical Warm Acupuncture can reduce brain damage after Ischemic Stroke, promote the reparation of neural function, assume the brain protection to the metabolic of brain cells by clearing free-radicals, anti-oxide damage, and by protecting the release of Inflammatory cytokines. The dissertation also uses the research method of MACO-R to narrate the mechanism of Mongolian Medical Warm Acupuncture to the early anti-brain damage by Cerebral Ischemia. This is for better clinical guidance, and also for an effective, convenient and easy-going method and research thought of such kind of disease
     This Dissertation summarizes the following two contents:one is to narrate the recognition of Mongolian medicine to Ischemic Cerebrovascular, to summarize the therapy condition of Mongolian medicine about Brain-blood diseases, and the literature review of the experiment research in the past20years; The other is the experiment report on the functions and mechanisms of Medical Warm Acupuncture for the Early Brain Damage by the24h Cerebral Ischemia Reperfusion Injury of the Rat. The experiment reports are as follows:
     Aim:observe the effect of Mongolian Medical Warm Acupuncture for Brain Damage after24hours by the Cerebral Ischemia Reperfusion Injury of the Rat, and discuss the function mechanisms of Mongolian Warm Acupuncture for the urgent Brain edema as well as the damage of the neural function.
     Method:Divide the SD male rat (weight280±20g) into the following five groups randomly:Model Group (M). False Operation Group (F), Warm Acupuncture Group (W). Conventional Acupuncture Group (C). Drug Contrastive Group (D). Use MCAO with the improved line plug method and go on reperfusion90min after the Ischemia. Treating method:treat1hour after the rat woke. Group W: choose Oroil Beqir, Hein Beqir and pierce the silver needle into them, them connect the electronic needle Therapeutic Apparatus, and keep the needle for30minutes; Group C, choose the acupuncture points of the big spine, air sea and Aminsodar. The Filoform needle pierces directly into them5mm,2mm and4mm, and it also needs keeping the needle30minutes. During the needle keeping, pierce the needle once in10minutes. Group D. fill the stomach once in accordance with the amount of10.8mg/ke of Nimodipine. Do the perfusion24hours after the Cerebral Ischemia of the rat, and choose the materials to do further observation to the experiment:(1) observe the score changes of the neural function evaluation for all the rats;(2) do the brain slice TTC staining, and after its firmation, deal with the brain slices of the rat with the Imaging System Software, and determinate the infarction volume of left brain damage;(3) do HE staining of the rat's brain organization and observe the morphology change of the brain organization;(4) examine the rat's serum and the contents change of MDA、 SOD、GSH、IL-1β、 IL-8、TNF-a、NO、NOS and ET-1with the method of ELISA;(5) Examine the content change of Hippocampus GFAP, NSE with the method of ELISA.
     Results:Among Group F and Group M, except for the special explanation of P>0.05, both have evident difference. This indicates that the contents between Ischemia and Non-Ischemia have big differences.(1) Neural function scores: compared with Group M, Pw, Pc<0.05; compared with Group C, Pw<0.05. Group W and Group C improve the neural function evidently, and Group W is far better than Group C.(2) Infarction volume of the brain: compared with Group M, Pc <0.05, Pw<0.01; Compared with Group D, Pw, Pc<0.05. The Infarction volume of the brain of Group W and Group C is clearly smaller than that of Group M and Group D.(3) Observation of the pathological section of HE staining:compared with Group M, the swelling and edema of the Nerve Cells of Group W, Group C and Group D are reduced, the cells of Group W does not sparse evidently and the neurons amount are increased.(4) MDA: compared with Group F, Pm, Pc, Pd>0.05, The content of serum MDA among all groups has no significant difference. But the mean value bar graph shows that the mean value of Group W is lower than that of Group M. SOD:compared with Group M, Pd, Pw<0.01. Pc<0.05; The content of serum SOD of Group W, Group D and Group C is higher than that of Group M. GSH:compared with Group M, Pw, Pc<0.05. The content of serum GSH of Group W and Group C is clearly higher than that of Group M. IL-8:compared with Group M, Pw, Pd<0.05. The content of serum IL-8and TNF-a of Group M and Group D is clearly less than that of Group M. NO, NOS:compared with Group M, Pd<0.05, Pw, Pc<0.01; The content of serum NO, NOS of Group W, Group D and Group C is clearly less than that of Group M, and Group W and Group C are far more significant. ET-1: compared with Group M, Pd<0.01, Pw, Pc<0.05. The content of serum ET-1of Group D, Group and Group C is evidently less than that of Group M, and Group D is the most significant. IL-1β, compared with Group M, Pw, Pc, Pd>0.05; Compared with Group F, Pm, Pc, Pd>0.05; The content of serum IL-1β among all Groups has no significant difference.(5) Organization of Hippocampus NSE:compared with Group M, Pw<0.01; The content of Hippocampus NSE is clearly less than that of Group M. GFAP:compared with Group M, Pw, Pc, Pd>0.05; Compared with Group F, Pm<0.05. The content of Hippocampus GFAP of Group D, Group W and Group C has no significant difference, but Group F and Group M have clear difference.
     Conclusion:(1) Mongolian Medical Warm Acupuncture can reduce evidently the defect score of the rat's neural function by Cerebral Ischemic Reperfusion, and can improve the neural function and reveal the protective function of the Mongolian Medical Warm Acupuncture therapy from the aspect of neuroethology.(2) Mongolian Medical Warm Acupuncture has the function to reduce the volume of brain infarction and brain edema by observing TTC staining slice and HE pathological section, thus it can protect the continuous damage of the half dark bands in the early times of Cerebral Ischemic Reperfusion by reducing brain edema and narrowing the volume of brain infarction. It can also save and protect the injured neurons, and improve the treating role of the neural function.(3) By observing and evaluating the content of serum MDA, SOD, GSH, NO, NOS, ET-1in the early times of Cerebral Ischemic Reperfusion, it can be concluded that Mongolian Medical Warm Acupuncture has the tendency of anti-oxidize and anti-free radical damage to Cerebral Ischemic Reperfusion in its early period. Its mechanism maybe lower serum NO, NOS and ET-1, and increase serum SOD and GSH in the early period of Cerebral Ischemic Reperfusion. In this way, it can protect the free radical damage effectively and increase the anti-oxidize ability of brain tissue.(4) By observing and evaluating the content of serum TNF-a. IL-1β and IL-8, it can be concluded that Mongolian Medical Warm Acupuncture has the function of anti-inflammatory to Cerebral Ischemic Reperfusion in its early period. Its mechanism maybe reduce serum TNF-a and IL-8, and increase serum SOD and GSH in the early period of Cerebral Ischemic Reperfusion to achieve the role of anti-inflammatory and brain-protection effectively.(5) NSE can reflect the degree of brain damage in the early period of Cerebral Ischemic and act as one of the objective Biochemical indicators to the recovery degree of neural function recovery period. The research results indicate initially that Mongolian Medical Acupuncture has the effect of reducing the content of Hippocampus NSE in the early period of Cerebral Ischemic Reperfusion. This may become one of the reasons for why Mongolian Medical Acupuncture can save and protect the Ischemic Neurons.
引文
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