天麻对帕金森病大鼠神经元保护及机制的研究

【作者】 徐瑰翎；

【导师】 白丽敏； 袁红；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2007， 硕士

【摘要】 帕金森病(Parkinson’s disease,PD)是一种常见的中老年中枢神经系统退行性疾病,随着人口的老龄化,PD已成为威胁人类健康的主要疾病之一。PD主要病理变化是黑质致密部多巴胺神经元(dopaminergic neuron)脱失,导致了患者出现运动减少、僵直、震颤等临床症状。由于PD的发病原因至今不十分清楚,因此也无理想的防治措施。神经保护性治疗已成为当前PD研究的热点之一。现代药理证明天麻及其制剂具有调节免疫、抗炎、抗凋亡等功效,对神经细胞具有保护作用。本研究使用高效液相、免疫组织化学等技术研究探讨了天麻对PD大鼠行为学、形态学、与PD有关的神经递质、细胞凋亡等因素的影响,将有助于揭示PD的发病机理及天麻防治PD的作用机制,为临床用药提供理论依据,具有重要的理论意义和实用价值。实验选用SD大鼠60只,应用6-OHDA复制PD模型50只。动物随机分为六组:①正常对照组;②模型对照组;③西药对照组(美多巴组);④天麻大剂量组(简称大剂量组);⑤天麻中剂量组(简称中剂量组);⑥天麻小剂量组(简称小剂量组)。动物的存活期为14天。实验分为四个部分:①天麻对PD鼠行为学、病理学变化的影响;②天麻对PD大鼠纹状体DA及其代谢产物含量的影响;③应用免疫组化技术观察PD鼠黑质(substantia nigra,SN)与腹侧被盖区(the ventral tegmental area,VTA)肿瘤坏死因子-α(TNF-α)和胶质源神经营养因子(Glial cell line-derived neurotrophic growth factor,GDFN)的表达影响;④应用免疫组化技术观察PD鼠SN、VTA区酪氨羟化酶(Tyrosine hydroxylase,TH)和Caspase-3的表达。主要结果如下:1天麻对PD大鼠行为学和形体学的影响旋转实验:各组大鼠注射阿朴吗啡(apomorphine,APO)后10Min.都出现不同程度的兴奋状态:躁动,觅食等。大鼠旋转实验结果显示:天麻可改善PD大鼠的行为异常,其中天麻小剂量组和美多巴组与模型组比较有显著性差异(p<0.05),大、中剂量组与模型组比较无统计学意义。Morris水迷宫实验:定位航行试验的结果显示小剂量组与模型组比较有统计学意义(p<0.05)。HE染色结果:天麻各剂量组大鼠SN区神经元的形态基本相似,数量未见明显减少,仅见少数神经元变性。2天麻对PD大鼠纹状体DA及其代谢产物含量的影响高压液相色谱(法)检测结果统计显示:模型组DA、DOPAC和HVA均较正常组显著降低(p<0.01)。DOPAC/DA与HVADA/比值与正常组比较都不同程度地升高。其中小剂量组的DA、DOPAC和HVA的含量与模型组比较有显著升高(p<0.05),接近正常组和美多巴组;大、中剂量组的DA及其代谢产物提高不明显没有统计学意义。3天麻对PD鼠脑内胶质细胞TNF-α、GDNF表达的影响TNF-α图像分析及统计结果显示:①模型组VTA、SN区DA能神经元胞质TNF-α表达量较正常组显著升高(p<0.01);小剂量组、美多巴组VTA和SN与正常组比较差异均不显著(p>0.05),与模型组比较TNF-α表达明显降低且差异显著(p<0.05);小剂量组与美多巴组相比差异不显著(p>0.05)。各组右侧(未损伤侧)SN和VTA间比较差异均不显著(p>0.05)。GDNF图像分析及统计结果显示:①与模型组比较,中、小剂量组VTA、SN区DA能神经元胞质GDNF表达量显著升高(p<0.05);②与正常组相比小剂量组VTA、SN区GDNF表达升高差异显著(p<0.05);③与美多巴组比较小剂量组VTA的GDNF表达亦升高(p<0.05)。4天麻对PD鼠黑质Caspase-3表达及TH阳性神经元的影响实验研究表明:与正常组比较模型组PD大鼠中脑黑质致密部酪氨酸羟化酶(TH)阳性神经元的数目减少了约45%;各天麻治疗组可部分减少黑质致密部TH阳性神经元的丢失,其丢失程度较模型组明显减低,其中小剂量组和中剂量组,减少约16%和17%,效果较好;大剂量和美多巴组,减少约22%和20%。相应的小剂量和中级量组的Caspase-3阳性神经元表达较模型组明显减少。各组Caspase-3阳性神经元的表达与TH阳性神经元的表达呈相反趋势。结论:①免疫炎症和细胞凋亡可能是PD大鼠中脑多巴胺神经元丢失的主要方式。②天麻可通过调节上述因素减少细胞凋亡,其中天麻小剂量组有较好的作用。提示天麻可不同程度地保护多巴胺神经元,减缓PD病程的进展更多还原

【Abstract】 Parkinson’s disease (PD) is a neurodegenerative disease.It is one of the frequent senile disease in central nervous system.With the increasing ageing of population,PD is one of the main disease to threaten the health of the human being.A major pathological hallmark of Parkinson’s disease is a progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and their axons,which project to the striatum.Thus,To lead to PD patient appear the clinical symptom of hypokinesia,rigidity and tremor et al.Up to now,as the disease remains poorly understood,there is still no effective treatment.It is one of the hot subject of studying to use the neuroprotective therapy to prevent and cure PD.The results of the modern pharmacological study showed that gastrodia rhizome or its products have the well efficacy as immunological regulation and anti-apoptosis,which can protect neurons.To elucidate the effect of gastrodin in the behavior,morphology,neurotransmitter , we use the technique of high pressure liquid chromatography(HPLC) and immunohistochemistry,which to help us to reveal the etiopathogenesis of PD and the mechanism of action of the gastrodin in order to prevent and cure PD.Furthermore, to provide theoretical support for clinical medication and have significant theoretical purpose.In experimental research,male SD rats were left-CPU injected with 6-OHDA(20ug)to establish PD model.The experimental animals is divided randomly into six groups:①The normal control group,②The model contral group,③Western medicine contral group,④gastrodin large dose group,⑤gastrodin middle dose group and⑥gastrodin small dose group.The animals were killed on the 14th day after injecting with 6-OHDA.This study is consisted of four parts:①To observe the changes of ethology and pathology in PD rats and the effect of gastrodin.②To study the contents of DA,DOPAC and HVA in striatum in PD rats and the effect of gastrodin by HPLC.③To investigate the expression of TNF-αand GDFN in SNpc and VTA in PD rats and the effect of gastrodin by immunohistochemistry technique.④To observe the expression of Caspase-3 and TH in SNpc and VTA in PD rats and the effect of gastrodin by immunohistochemistry technique.The main results are displayed as follows:1 Gastrodin can improve PD rats’ethologic and morphologic dysfunction. Each group rats that are injected APO after 10 minute appear different d egree motional state,restlessness and foraging,for about 30minuts then come down.The result show that the rotation number in the low dose and madopar group significantly decreased ,comparing with model group, however, large and middle does group no significant difference.Moirris water labyrinth result show that there was significant difference between low goes group and model group(p<0.05).The HE staining result show that the neuronal shape is no change,but the number is slightly decreased and a few neuron are degenerative in rats’SN among each dose gastrodin group。2 Gastrodia can increase the content of DA and its metabolic product in Striatum in PD rats.The content of DA,DOPAC and HVA in model group significantly decreased compared with normal group(p<0.01).The ratio of DA/DOPAC and DA/HVA in model group increased compared with normal group(p<0.05).The content of DA、DOPAC and HVA in low does group significantly increased compared with model group. In treatment groups,the contents of DA,DOPAC and HVA were recovered significantly and the ratio of HVA/DA decreased.3 The expression of TNF-αis decreased and the GDNF is increased in PD rats brain glial cell in each gastrodin-treated group.Image analysis and statistic result display that the expression of TNF-αis significantly increased in the cytoplasm of DA Neurons in VTA and SN in Models group compared with normal group(p<0.01).The low does and madopar group is similar to compared with normal group and declined significantly to compared with model group(p<0.05).Image analysis and statistic result show that the expression of GDNF significantly increased in the cytoplasm of DA Neurons in VTA and SN in middle, small dose group compared with model group(p<0.05).The low does group is significantly increased not only compared with normal group but also madopar group.There was no difference among the other group.4 Gastrodine may protect the loss and apoptosis of TH+ dopaminergic neurons by declining the express of Caspase-3 in SNpc in PD rats.In the experimental studying, immunohistochemistry technique is used.The TH+ dopaminergic neurons were counted in every group.The loss of TH+ neurons are partly inhibited in SNpc of each gastrodin treat group.The number of TH+ neurons in SNpc of small dose group and middle dose group is decreased by 16% and 17%;The number of TH+ neurons in SNpc of madopar group and large dose group is decreased by 22% and 20%.Correspondingly, the number of Caspase-3+ neurons in SNpc in small does and middle does group is obviously decrease d. The trend of decreasing of TH+ neurons is opposite to the change of number of Caspase-3+ neurons.Conclusion:①first,immuni-inflammation and apoptosis may be one of main way in loss of dopaminergic neurons in Parkinson’s disease.②Secondly,gastrodin may suppress the lose and apoptosis in SNpc,by adjusting the above factor.The effect of small dose group is best.It is indicated that gastrodin can moderately protect DA neuron and slow down the process of PD.更多还原