解毒通络复方对缺血性脑损伤后突触再生的作用

【作者】 张万辉；

【导师】 潘彦舒；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2012， 硕士

【摘要】 脑血管病是导致人类死亡的常见疾病之一,其中缺血性中风的病死率和致残率高,严重威胁人类健康,影响患者生活质量。但当前对于该疾病的治疗仍无突破性进展,特别是对中风恢复期的治疗仍然是临床上棘手问题之一。本课题组认为,中风属于“络病”范畴,其核心病机为“毒损脑络”。解毒通络复方正是以这一创新性病机理论假说为指导,在结合缺血性中风现代病理生理学和中药生物效应以及国内外最新研究成果的基础上,研制出的由中药栀子、丹参、黄芪、天麻的有效组分配伍而成的中药新配方。基于此,本实验旨在研究缺血性脑损伤后突触再生的机制,以及解毒通络复方对其的影响,探讨中医药在解毒通络、调和营卫、凉血解毒、和血通络等综合功效下,有效地实现对脑缺血炎症级联反应病理过程的阻断作用,改善大脑微环境,诱导神经突触的结构和功能再建,发挥其对神经元保护作用的机制,为研究中医药治疗缺血性脑血管病的作用途径与靶点提供有意义的思路。目的：本研究从突触结构以及突触生长微环境变化两方面来研究缺血性脑损伤后突触再生的机制及解毒通络复方对其的影响。1验证解毒通络复方对大鼠缺血性脑损伤后神经功能及形态的影响2观察解毒通络复方对大鼠缺血性脑损伤后突触结构的影响3观察解毒通络复方对大鼠缺血性脑损伤后突触生长微环境中的活性因子的影响方法：1.应用步态分析和神经功能功能评分观察神经功能恢复的情况。2.应用HE染色法光镜下观察脑组织一般形态。3.应用镀银染色、透射电镜的方法来观察神经突触一般和超微结构的变化。4.应用免疫组织化学法观察突触素、肌动蛋白的表达,来揭示大鼠脑缺血损伤后突触再生的情况。5.应用酶联免疫(ELISA)法和免疫组织化学法观察突触生长微环境中生物活性因子脑源性神经生长因子(BDNF)、碱性成纤维细胞生长因子(BFGF)和血管内皮生长因子(VEGF)的含量和表达变化。结果：1.在步态分析结果显示,解毒通络复方组与模型组相比明显增大,有显著的差异。神经功能功能评分结果显示,解毒通络复方组与模型组相比的评分明显降低,具有明显差异。以上结果初步验证了：解毒通络复方对缺血后受损后神经功能的恢复起到了一定的促进作用。2.HE染色光镜下观察结果：可见大鼠缺血性脑损伤4周和8周时,皮层顶叶区神经元大量丢失,皮层区神经元和星形胶质细胞出现明显病理变化,给予解毒通络复方后梗死灶面积与模型组相比明显减小。神经元和星形胶质细胞损伤程度均有所减轻。3.镀银染色观察结果：正常组神经元形态结构完整,分布密集,树突数量多,轴突长。各个突触间连接紧密。可见轴突-树突式突触、轴突-胞体式突触等突触类型。模型组神经元坏死严重,数量明显减少。残存神经元突触数量少。与模型组相比,解毒通络复方组神经元数量增多,形态结构基本完整,突触数量增多,有见部分突触间相互连接。电镜观察结果：正常组神经元突触较密集,结构清晰,可见突触前膜、突触后膜、突触间隙以及突触前部囊泡等。突触后致密区染色深,密度大。模型组神经元突触数量明显减少,突触前、后膜结构模糊不清,突触后致密区密度低。与模型组相比,解毒通络复方组神经元突触数量增加,可见新生的穿孔突触,突触前膜、突触后膜以及突触前囊泡清晰可见,突触后致密区密度大。4.用免疫组化法观察大鼠局灶性脑缺血性损伤后脑组织皮质区突触素SYN的表达,结果显示：模型4周组SYN的表达与正常组相比明显降低,有显著性差异(P<0.01)。解毒通络复方4周组与模型组相比明显增高,有显著性差异(P<0.001)。模型8周组SYN的表达与正常组相比有明显降低,具有显著性差异(P>0.001)。解毒通络复方8周组与模型组相比SYN的表达呈升高趋势,有显著性差异(P<0.05)。免疫组化法观察大鼠局灶性脑缺血性损伤后脑组织皮质区肌动蛋白ACTIN的表达,结果显示：模型4周组ACTIN的表达与正常组相比略有降低。解毒通络复方4周组与模型组相比有升高趋势。模型8周组ACTIN的表达与正常组相比有明显降低,具有极显著性差异(P<0.001)。解毒通络复方8周组与模型组相比显著增高,有极显著性差异(P<0.001)。初步验证了：解毒通络复方在对大鼠脑缺血性损伤后神经元和突触的再生方面有一定的促进作用。5.通过酶联免疫(ELISA)法和免疫组织化学法观察突触生长微环境中生物活性因子脑源性神经生长因子(BDNF)、碱性成纤维细胞生长因子(BFGF)和血管内皮生长因子(VEGF)的含量和表达变化。结果显示：用ELISA法检测大鼠局灶性脑缺血性损伤后各组血清和患侧(左侧)脑匀浆中BDNF、VEGF、BFGF含量1) BDNF模型4周组组与正常组相比降低。解毒通络复方8周组与模型组有明显升高趋势,但无显著性差异。,模型8周组与正常组相比明显降低,有显著性差异(P<0.05)。解毒通络复方8周组与模型组相比明显增高。有显著性差异(P<0.05).2) BFGF模型4周组与正常组相比明显增高,有极显著性差异(P<0.001)。解毒通络复方4周组与模型组相比明显降低。有极显著性差异(P<0.001)。模型8周组与正常组相比明显增高,有极显著性差异(P<0.001)。解毒通络复方8周组与模型组相比明显降低。有极显著性差异(P<0.001)。3) VEGF模型4周组与正常组相比明显增高,有显著性差异(P<0.01)。解毒通络复方4周组与模型组相比明显降低,有显著性差异(血清中P<0.01)、(脑匀浆中P<0.05)。模型8周组与正常组相比明显增高,有极显著性差异(血清中P<0.001)、(脑匀浆中P<0.05)。解毒通络复方8周组与模型组相比明显降低,有极显著性差异(血清中P<0.01)、(脑匀浆中P<0.001)。免疫组化结果与ELISA结果基本呈一致性。初步验证了：解毒通络复方可能是通过调节突触生长微环境中的活性因子的变化来促进突触再生,进而达到神经保护和神经功能恢复的目的。结论：1.大鼠脑缺血性损伤后神经元明显受损,解毒通络复方对缺血后损伤神经功能的恢复起到了一定的促进作用。2解毒通络复方在对大鼠脑缺血性损伤后神经元和突触的再生方面有一定的促进作用。3.解毒通络复方可能是通过调节突触生长微环境中的活性因子的变化来促进突触再生,进而达到神经保护和神经功能恢复的目的。更多还原

【Abstract】 Cerebrovascular disease is one of the three diseases inducing human beings’death, and ischemic stroke which belongs to cerebrovascular disease has high mortality and disability rates, seriously threatens mankind’s health and effects their qualities of life. But, until now there haven’t been dramatic breakthroughs in the treatment of the disease, especially in the aspect of treating the patients in the recovery period of stroke.Based on our previous research, stroke belongs to collateral disease in traditional chinese medicine TCM, and its major mechanism in TCM is "the damage of toxic to cerebral collaterals". Based on the above mechanism in TCM and the latest achievements of both modern pathological physiology and bioeffects of Chinese herbs in the research of ischemic stroke, JieDuTongLuo Compound consisting of Gardenia、Salvia,、Astragalus、 Gastrodia has been developed. Therefore, this study is to research the mechanism of synaptic regeneration following cerebral ischemic injury and the beneficial effect of JieDuTongLuo Compound on this injury; to explore the effect of Chinese medicine on Cerebral ischemia inflammation levels even reaction, the improvement of brain microenvironment, reconstruction of structures and functions in synapses and the protection of neurons in order to provide a meaningful way of thinking for the acting way and targets in the treatment of ischemic cerebrovascular disease with Chinese medicine.Objective:This research demonstrate the mechanism of synapse regeneration after ischemic cerebral damage and the impact of JieDuTongLuo compound via two aspects, the structure of synapse and changes of its microenvironment.1. To verify the effect of JieDuTongLuo compound to recovery of nerve function after ischemic cerebral damage;2. To observe the changes of synapse structure after ischemic cerebral damage and the effect of JieDuTongLuo compound;3. To observe the changes of microenvironment around synapse after ischemic cerebral damage on rat and the impact of JieDuTongLuo compound.Methods:1. Prove the effect of JieDuTongLuo compound to the recovery of nerve function from ethology and morphology, including gait analysis meter, nerve function score, HE staining; 2. Observe changes of general synapse structure and its ultra microstructure via plating silver staining and transmission electron microscope TEM, and test the amount of SYP and actin via IHC;3.Observe the amount and changes of BDNF, BFGF, VEGF around microenvironment of synapse via ELISA and IHC.Results:1.To verify the effect of JieDuTongLuo compound to recovery of nerve function after ischemic cerebral damage;1) HE staining:By optical microscope we found that neurons of parietal cortex area significantly decreased4weeks and8weeks after ischemic cerebral damage on rats, and there is obvious pathological change on neurons and astroglias of cortex area, the infarct area significant decreased by effect of JieDuTongLuo compound. At high magnification lens we can see that neurons and astroglias are slightly damaged with newborn vessel, neuron and synapse.2) On the aspect of intensity of pressure in gait analysis, JieDuTongLuo compound group is significantly higher than control group.3) On the aspect of nerve function loss scale, JieDuTongLuo compound group is much lower than control group.2. Observe changes of general synapse structure and its ultra microstructure via plating silver staining and transmission electron microscope TEM, and test the amount of SYP and actin via IHC;1) Results of silver plating staining:the neuron in normal group was equably stained, had unabridged structure, many dendrite and long axon, also intensively distributed. Each synapse was tightly connected. We can see axon-dendrite synapse, axon-soma synapse, etc. However, neurons in model group was severely decreased, unevenly stained, badly pyknosis, few dendrite and hardly no axon. JieDuTongLuo compound group had more neurons, fairly equably stained, full structure, more axon and dendrite than model group, also had connect between synapse.2) Result of electron microscope:normal group:neuron had full structure, normal shape, clearly two layer of karyotheca, chromatin was evenly distributed in nucleus, organoid had normal structure. A dense synaptic with clear presynaptic membrane, postsynaptic membrane, synaptic cleft and anterior synaptic capsule. And there is also a deep staining and a large density in postsynaptic dense zone, model group:unclear structures in neuron, their membranes and nuclear membranes, chromatin was distributed mainly in the lower part of nucleus, organelles in cytoplasm were decreased with nuclear structures and mitochondria swelling. A significant reduction in the number of synaptic with nuclear presynaptic membrane and postsynaptic membrane, and a low density in postsynaptic dense zone. JieDuTongLuo compound group:a basic integrity of structure and an almost normal shape in neurons with clear nuclear membranes and well-distributed chromatin could be found. In addition, an increase in the number of some organelles such as Golgi complex, ribosome and rough endoplasmic reticulum, a slight swelling in some mitochondria could be seen. A large density in the distribution of synaptic and postsynaptic dense zone could be found. The connection between synaptic, presynaptic membrane, postsynaptic membrane and anterior synaptic capsule could also be observed clearly.3) Analysis the amount of SYN in cortex area after focal cerebral damage by IHC, we found that amount of SYN in model group was significantly lower than that in normal group after the administration for4weeksP<0.01. That of JieDuTongLuo compound group was obviously higher than model groupP<0.001. After8weeks the amount of SYN in model group is significantly lower than normal group P <0.001. SYN in JieDuTongLuo compound group showed an obvious increasing trendP<0.05, and was significantly higher than that of4weeks P<0.001. After analysing the amount of actin of cortex area after focal cerebral damage on rats via IHC, we found that after4weeks, amount of actin in model group was slightly lower than normal group. That in JieDuTongLuo compound group was higher than model group. After8weeks, the amount of actin in model group is significantly lower than normal group P<0.001. That of JieDuTongLuo compound was significantly higher than model group P<0.001, and was higher than that of4weeksP<0.05.3.Observe the amount and changes of BDNF, BFGF, VEGF around microenvironment of synapse via ELISA and IHC.The results from the content of BDNF, VEGF and BFGF both in serums and tissues of injury brains following the local brain injury in rats with the method of ELISA:1BDBF:the content of BDNF in model group decreased compared with that in control group, and showed an increasing trend compared with that in other groups without statistical significance following the administration of JieDuTongLuo compound for4weeks. After the administration of JieDuTongLuo compound for8weeks, there was an obvious decrease in the content of BDNF in model group compared with that in control groupP<0.05and an obvious increase of BDNF in JieDuTongLuo compound group compared with that in model groupP<0.05.2BFGF:the content of BFGF in model group decreased compared with that in control group, and increased compared with that in other groups with most statistical significant difference following the administration of JieDuTongLuo compound for4weeksP<0.001. After the administration of JieDuTongLuo compound for8weeks, there was an obvious decrease in the content of BFGF in model group compared with that in control groupP<0.001and an obvious increase of BFGF in JieDuTongLuo compound group compared with that in model groupP<0.001.3VEGF:the content of VEGF in model group decreased compared with that in control group, and increased compared with that in other groups with statistical significant difference following the administration of JieDuTongLuo compound for4weeks(in serum:P<0.01; in the brain homogenate:P<0.05), After the administration of JieDuTongLuo compound for8weeks, there was an obvious decrease in the content of VEGF in model group compared with that in control groupin serum:P<0.001; in the brain homogenate:P<0.05and an obvious increase of VEGF in JieDuTongLuo compound group compared with that in model groupin serum:P<0.01; in the brain homogenate:P<0.001. And these results were consistent with those from IHC.Conclusions:1. Neurons were damaged severely following the local cerebral ischemia in rat brain, and JieDuTongLuo compound had a beneficial effect on the recovery of nervous functions induced by ischemia.2. JieDuTongLuo compound could effectively promote the regeneration of neurons and synaptic after their damage mediated by local cerebral ischemia in rat brain.3JieDuTongLuo compound might promote the regeneration of synaptic via regulating the changes of some active factors in synaptic microenvironment in order to exert its effects on the protection of neurons and the recovery of nervous functions.更多还原