【作者】 赵卿；

【导师】 蔡定芳； 朱大年；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2010， 博士

【摘要】 帕金森病(Parkinson’s disease, PD)是最常见的神经系统变性疾病之一。迄今为止,其发病原因和病理机制仍未完全阐明,因此尽管包括左旋多巴制剂在内的各种多巴胺替代疗法能够改善病人的某些症状,但目前仍缺乏阻止病情进展的有效措施。中国医药学是一个伟大的宝库,不论是古代医籍中对“颤振”及其治疗的记载还是现代中医药临床实践,均显示出中医药治疗PD的有效性和潜在优势。肉苁蓉具有补肾阳、益精血、润肠通便之功效,在PD的治疗中是常用的一味中药。我们在临床中发现肉苁蓉为主的方剂确实能够延缓PD患者需要增加左旋多巴制剂用量的时间。体外实验数据证实,肉苁蓉抗PD活性来自其中的苯乙醇苷类化合物,其单体成分之一的松果菊苷(Echinacoside, ECH)具有很强的药理活性,能够显著抑制MPP+诱导的多巴胺能神经元损伤。为深入探讨松果菊苷在活体内的作用效果和途径,我们根据其可能的抗凋亡活性选取MPTP亚急性损伤PD小鼠作为实验模型,首先对该模型的神经行为学异常进行了系统观察,结合病理及神经化学指标的改变筛选出敏感、稳定的行为学评价指标,然后从症状改善和对黑质纹状体多巴胺系统的神经挽救作用两个角度进一步研究,证实其有效性后再对其作用靶点进行了初步的探索,揭示其可能的作用机制。本课题的研究结果如下：目的探讨MPTP亚急性损伤PD小鼠神经行为学改变及其与黑质纹状体多巴胺通路损伤的关系,为药物干预研究提供模型依据。方法本研究以多次腹腔注射(30mg/kg×5天)的MPTP亚急性损伤C57BL／6J小鼠作为PD的动物模型,等量生理盐水腹腔注射作为对照,每组9只动物,通过对多种神经行为学指标的动态观察(MPTP末次给药后ld,3d,5d,7d,14d),探讨此类模型的运动障碍表现形式：在末次MPTP给药后2周每组5只小鼠取脑制作石蜡切片,采用免疫组织化学方法了解MPTP导致的黑质致密部酪氨酸羟化酶(TH)阳性染色神经元及纹状体TH阳性染色神经纤维损伤情况；同时每组另4只小鼠采用高效液相方法检测纹状体组织中多巴胺(DA)神经递质及其代谢产物的变化。统计学处理采用两样本均数比较t检验。结果一、模型组小鼠每次MPTP注射后数分钟至半小时即出现震颤、僵直等急性损伤症状,持续约数小时,生理盐水对照组则无上述变化；与生理盐水组比较,末次给药后1周内MPTP模型组小鼠先后出现水平移动距离缩短(1d,3d,P<0.01,0.05)、胶布移除时间延长(1d,P<0.05)、爬杆实验T-turn对间延长(1d,3d,5d,P<0.05,0.05,0.05)、滚筒停留时间缩短(1d,3d,5d,7d,P<0.05,0.05,0.01,0.01)、竖立次数减少(3d,5d,7d,P<0.05,0.001,0.01)和前后肢步长缩短(前肢3d,5d,7d,P<0.05,0.01,0.01；后肢5d,7d,P<0.05),但随观察时间延长各指标有不同程度的恢复,至末次给药后14天仅表现为前后肢步长明显缩短(P<0.01)、滚筒停留时间缩短(P<0.05)和竖立次数减少(P<0.05)；二、末次给药后14天,与生理盐水对照组比较,MPTP模型组小鼠黑质致密部TH阳性染色神经元数目显著减少(P<0.001),纹状体TH阳性染色神经纤维数目明显减少(P<0.001),光密度值也显著下降(P<0.001)；三、末次给药后14天,与生理盐水对照组比较,MPTP模型组小鼠纹状体组织中DA及代谢产物含量显著减少(P<0.01,P<0.05)。结论一、MPTP亚急性损伤小鼠能够复制大部分PD行为学异常,其中,震颤、僵直出现最早,其余依次受累的是自发活动、平衡协调能力和步态,相对而言,步态异常、平衡协调能力下降、竖立次数减少出现稍晚但敏感、稳定,而其他指标出现早且有明显的自发恢复趋势,不适合造模后给药的疗效评价；二、MPTP亚急性损伤小鼠多巴胺能神经元及轴突数目减少符合PD的病理表现,并能持续至损伤结束后14天；三、MPTP亚急性PD小鼠DA神经递质及代谢产物减少进一步证实该模型能够模拟PD典型的神经化学改变,并能持续至损伤结束后2周。目的探讨ECH对MPTP亚急性损伤PD小鼠神经行为学异常及黑质纹状体多巴胺系统损伤的挽救治疗效果及可能的作用机制。方法本研究以经第一部分实验验证的多次注射的MPTP亚急性损伤C57BL／6J小鼠作为帕金森病的动物模型,在MPTP末次注射后24小时给予低(10mg/kg)中(20mg/kg)、高(30mg/kg)三个剂量的ECH或阳性对照药物司来吉兰(1.0mg/kg)灌胃治疗,模型组给予等量生理盐水灌胃治疗,正常对照组先后给予相应剂量的生理盐水腹腔注射和灌胃。共6组,每组8只动物。在治疗第7天和第14天观察ECH和对照药对MPTP亚急性损伤小鼠步态、滚筒实验和竖立次数异常的改善作用；治疗第14天行为观察结束后每组4只动物取脑制作石蜡切片,采用免疫组织化学方法了解ECH和对照药对MPTP导致的黑质致密部TH阳性染色神经元及纹状体TH阳性染色神经纤维损伤的治疗效果；采用原位末端转移酶标记凋亡(TUNEL)方法检测各组动物黑质致密部细胞凋亡情况；同时每组另4只动物采用高效液相-电化学方法检测ECH和对照药对MPTP亚急性损伤造成的纹状体DA及其代谢产物含量变化的影响；采用实时荧光定量(Real time PCR)检测各组动物腹侧中脑黑质区组织中细胞存活／死亡相关因子GDNF、BDNF、Bcl-2蛋白家族主要成员Bax、Bcl-2及内质网应激相关因子CHOP mRNA表达水平；采用免疫印迹法(Western Blot)检测各组动物腹侧中脑黑质区组织中上述分子的蛋白质表达情况。结果一、与MPTP模型组比较,ECH高剂量组小鼠在给药第7和第14天前、后肢步长显著增长(P<0.05),与正常对照组和司来吉兰阳性对照组比较无显著差异(P>0.05),中、低剂量ECH也能延长步长,但与模型组比较无统计学差异；与模型组比较,ECH各治疗组小鼠滚筒停留时间延长,竖立次数增多,但无统计学差异(P>0.05)；二、与模型组比较,高、中剂量ECH治疗14天后小鼠黑质致密部TH阳性染色神经元数目和纹状体TH阳性染色神经纤维光密度值及数目均显著增加(P<0.001,P<0.01,P<0.001),与正常对照组和司来吉兰对照组比较无显著差异,低剂量组则无上述效果；三、治疗14天后高剂量ECH治疗组纹状体DA及其代谢产物含量均高于模型组,特别是DA含量与模型组比较有显著差异(P<0.05),该作用与司来吉兰相似；四、与正常组比较,MPTP模型小鼠黑质致密部TUNEL标记的凋亡细胞数明显增加(P<0.001),Bax/Bcl-2 mRNA和蛋白表达比值显著升高(P<0.01,P<0.001),内质网应激相关凋亡分子CHOP mRNA及蛋白表达增加(P<0.01,P<0.001),而高、中剂量ECH治疗14天可以显著抑制上述改变；另外,高、中剂量ECH治疗14天能显著上调神经营养因子GDNF和BDNF mRNA和蛋白表达。以上ECH效应均表现出一定的剂量依赖关系,且与司来吉兰作用相似。结论一、高剂量ECH造模后给药治疗能够显著改善MPTP亚急性损伤小鼠的步态异常,效果与司来吉兰相似；二、高、中剂量ECH能够减轻MPTP亚急性PD小鼠多巴胺能神经元及轴突损害,抑制DA含量下降,并呈一定剂量依赖模式,与司来吉兰治疗效果相似,表现出良好的神经挽救效应及症状改善潜能；三、ECH对MPTP模型小鼠的行为改善作用和对多巴胺能神经元及轴突的挽救作用可能与其增加神经营养因子GDNF和BDNF表达及抗凋亡作用有关,后者可能涉及线粒体／细胞色素C通路及内质网通路,并可能参与调节Bcl-2蛋白家族成员表达。更多还原

【Abstract】 Parkinson’s disease (PD) is one of common neurodegenerative diseases, which is characterized by a progressive and selective loss of nigral dopaminergic neurons, resulting in a pronounced depletion of striatal dopamine. The loss of striatal dopamine and the consequent dysfunction of the nigrostriatal pathway lead to a range of motor symptoms, including resting tremor, rigidity, bradykinesia, gait disorder and loss of postural reflex. As the disease progresses, many patients also develop some cognitive dysfunction, including anxiety, depression and dementia. In contrast to other neurodegenerative disorders, there are relatively good symptomatic therapies available for the treatment of Parkinson’s disease. These therapies consist predominantly of dopamine replacement and adjuvant surgical therapy to relieve most of the motor symptoms of the disease. However, these symptomatic therapies are not themselves without problem, for example, long-term treatment with the dopamine precursor 3,4-dihydroxyl-phenylalanine (L-DOPA) often leads to the development of debilitating dyskinesias and it can not stop the progression of PD. Furthermore, at the present time, there is no disease-modifying neuroprotective or neuroresue/neurorestorative therapy available.Recently to stimulate the endogenous expression of neurotrophic factors (NTFs), especially glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) shows a petency to prevent or halt the progress in PD. According to some results of our previous researches, we hypothesized that oral administration of ECH, a monomer extracted from herbs cistanchis, to subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, commencing after impairment of the nigrstriatal system, had neurorescue effects on both behavior,pathological and neurochemistrical deficits meanwhile attenuated the factors correlated with survival or death of dompaminergic neurons, such as GDNF and BDNF, Bcl-2 family members and endoplasmic reticulum stress mediator CHOP at both mRNA and protein levels. Objective To investigate the sensitive and stable behavior tests for the subacute MPTP mouse model of PD, find further verifications by methods of pathology and neurochemistry.Methods Observe behavior changes of C57BL/6J mice before MPTP intraperitoneal injection and 1,3,5,7,14 days after the last administration of MPTP (30mg/kg/day×5 days) including open field activity, rotarod test, pole test, gait analysis, grid test and adhesive removal test. After the last observation mice were sacrificed for TH immunohistochemistry detection and dopaminergic neurotransmitter detection by HPLC-EC. Independent samples T-Test was performed for statistical purposes.A value of P<0.05 was considered statistically significant.Results 1.The MPTP-lesioned mice demonstrated obvious tremor and rigid symptoms several to 30 minutes after every intraperitoneal injection and recovered in few hours. For MPTP administrated mice the distances of horizontal movement, rearing numbers, latency to fall in rotarod test, stride length of fore-and hindlimb decreased while the time to remove adhesive tape and time to turn in pole test were both prolonged compared with the saline control mice within 7 days after the last MPTP injection. However some of index recovered spontaneously later and only stride length, rotarod behavior dysfuctions and rearing numbers kept abnormal 14 days after the last MPTP administration.2.Comparing with the saline control animal, the number of TH-ir neurons and fibers in MPTP mice decreased and the contents of DA and DOPAC reduced significantly 14 days after the last MPTP injection (P＜0.001).Conclusion 1.The subacute MPTP mouse model can mimic most of behavior impairments of PD including tremor and muscular rigid, akinesia, bradykinesia, gait abnormalities with poor postural balance.The gait analysis is the most sensitive and stable index for research of neuroresue drugs and the next choice are rotarod test and rearing numbers.2.The dopaminergic neurons and fibers’loss in subacute MPTP mouse model are in accordance with the typical pathological changes of PD and can be retained to 14 days after the end of leision.3.The reductions of DA and its metabolisms verify the model from the view of neurochemistry further and can also last to 2 weeks after the last MPTP injection.Objective To investigate the neurorescue effects of ECH in subacute MPTP mouse model of PD and the possible mechanisms involved. Methods The subacute MPTP-lesioned mice were treated with three doses of ECH (10, 20,30mg/kg/day) and selegiline (1.0mg/kg/day)for 14 days started at 24 hours after the last MPTP injection. Behavior assessments were performed at the 7th and 14th day of treatments according to the methods of part I on gait, rotarod and rearing. After the last observation of behaviors mice were sacrificed for detections of TH immunohistochemistry, dopaminergic neurotransmitters detection by HPLC and TUNEL apoptotic cell counts. The mRNA and protein levels of GDNF, BDNF, Bax, Bcl-2 and CHOP in substantia nigra of mice were quantified by Real time PCR as well as Western blot techniques. One-way ANOVA followed by post-hoc analysis of Tukey’s HSD and Student-Newman-Keuls multiple comparisons test was performed for statistical purposes. A value of P<0.05 was considered statistically significant.Results 1. At the 7th and 14th days of high-dose ECH treatment, the gait disorder displayed obvious improvement (P<0.05) meanwhile the rotarod and rearing behavior also had slight ameliorations compared with the saline treated MPTP mice, which is similar with selegiline treatment. The behaviors in other two doses of ECH treated mice were improved to some extent (P>0.05).2.The high doses of ECH suppressed the reductions of nigral dopaminergic neurons (P<0.001) and striatal fibers (P＜0.01), contents of DA (P<0.05) and its metablisms meanwhile decreased the number of apoptotic cells compared with saline treated MPTP-lesioned animals (P<0.001). There was a relative elevation in expression of GDNF and BDNF mRNA (2.94 and 3.75-fold) and proteins (184.34% and 185.93%) in high dose of ECH treated mice compared with vehicle treated MPTP-lesioned mice. In addition, the Bax/Bcl-2 ratio as well as gene and protein of CHOP in MPTP-lesioned mice significantly increased, and these effects could be prevented by ECH. The moderate dose of ECH and selegiline had similar but weaker effects.Conclusion 1.The high dose of ECH improves the gait abnormal with poor postural balance and akinesia in the subacute MPTP-lesioned mice.2.ECH can rescue the loss of dopaminergic neurons, fibers and DA after the MPTP lesions in certain dose-dependent manner then exhibits similar activites with selegiline. So ECH demonstrates a potency in the disease-modifying and symptom treatments of PD.3.ECH up-regulates pro-survival factors GDNF and BDNF, meanwhile has anti-apoptotic effects through attenuation to Bcl-2 family members and CHOP which indicates the mitochondria and endoplasmic reticulum pathway are probably involved.更多还原