帕病2号方治疗早期帕金森病的临床观察及药效学研究
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摘要
帕金森病(Parkinson's diseaes, PD)是影响中老年人身体健康的主要神经系统变性疾病,其病因及发病机制仍然尚未完全清楚。复方左旋多巴制剂仍然是目前药物治疗的“金标准”,因其长期治疗存在严重的副作用及并发症,人们普遍看重PD的神经保护治疗。许多研究证实中药治疗PD的效果是确切的,具有神经保护作用,但目前从细胞、分子水平深入研究中药治疗PD的具体作用机理则相对较少。导师雒晓东教授总结出治疗PD的经验方帕病2号方,在临床上初步观察到该方能改善临床症状,延缓病情进展,提高患者的生活质量。
本课题临床研究部分观察帕病2号方对早期PD患者的运动功能、中医证候、生存质量及睡眠质量的改善情况,客观评价中药复方帕病2号方治疗早期PD的临床疗效。实验研究部分用6-羟基多巴(6-OHDA)纹状体两点注射单侧毁损法制作PD大鼠模型,通过病理形态学、免疫组化、生化检测等方法,从抗氧化应激、抑制神经细胞凋亡等方面对帕病2号方治疗PD的作用机理进行了系统研究。旨在探讨帕病2号方的药效学作用及相关作用机制。本课题分为三部分,摘要如下：
第一部分帕病2号方治疗早期帕金森病的临床疗效观察
目的：
客观评价中药帕病2号方治疗早期帕金森病的临床疗效。
方法：
采用随机、对照研究方法进行临床试验。将60例早期帕金森病患者随机分为2组：帕病2号方组、美多巴组,每组各30例。帕病2号方组采用中药帕病2号方(由乌梅、白芍、当归、熟地黄、何首乌、葛根、天麻、龟板等组成)治疗,美多巴组给予美多巴治疗,疗程3个月。3个月疗程结束后进行疗效评价,疗效评价指标为统一帕金森病量表(UPDRS)第三部分(运动功能)、改良Webster量表评分、Hoehn&Yahr分级、(自拟)肝肾不足型帕金森病中医证候积分量表、帕金森病生存质量量表(PDQ-39)、睡眠量表(PDSS)评分相对于基线的变化,记录完成相关运动试验时间(双手1min记时运动)。同时观察血压、脉搏、血尿常规、肝肾功能、心电图及不良反应等
结果：
1.在改善运动功能方面：帕病2号方组29例患者,显效13.79%(4/29),有效65.52%(19/29),总有效率79.31%；美多巴组28例患者,显效17.86%(5/28),有效64.29%(18/28),总有效率82.14%,两组疗效比较差异无统计学意义(P>0.05)。帕病2号方组的UPDRSⅢ、改良Webster量表评分等指标治疗前后有明显改善(P<0.05),Hoehn-Yahr分级、双手1min记时运动试验治疗前后差异无统计学意义(P>0.05)；美多巴组的UPDRSIII、改良Webster量表等指标治疗前后有明显改善(P<0.05),Hoehn-Yahr分级、双手1min记时运动试验治疗前后差异无统计学意义(P>0.05)；两组组间比较无差异性(P>0.05)。提示在改善早期PD患者运动功能方面,中药复方帕病2号方与美多巴疗效相当。
2.患者中医证候改善情况：帕病2号方组29例患者,显效6例,有效20例,总有效率为89.66%,美多巴组28例患者,显效0例,有效5例,总有效率为17.86%,两组中医证候疗效比较有显著差异(P＜0.01)。帕病2号方组对肝肾不足证患者常见症状如震颤、肢体拘挛、运动迟缓、头晕、失眠多梦、腰膝酸软、便秘等症状均有明显改善(P<0.05),美多巴组仅有肢体拘挛、运动迟缓症状有明显改善(P<0.05)。
3.对早期PD患者生存质量的影响
帕病2号方组治疗前后生活质量问卷分类评分组内比较,运动、日常生活活动、情感、耻辱感、认知力、躯体不适等方面评分差异有统计学意义(P<0.05),美多巴组仅运动、日常生活活动积分差异有统计学意义(P<0.05)。两组情感、耻辱感、认知力、躯体不适等分类评分治疗前后差值组间比较,差异有统计学意义(P<0.05)。在改善睡眠质量方面,帕病2号方组PDSS积分治疗后明显改善(P<0.05),美多巴组治疗前后组内比较,差异无统计学意义(P>0.05),两组组间比较具有差异性(P<0.05)。
4.安全性评价
帕病2号方及美多巴治疗前后血尿常规、肝肾功能检查结果皆在正常范围内,心率、血压和心电图未见明显的变化,未发现有严重不良反应。
结论：
中药帕病2号方对改善早期帕金森病患者的运动功能有效,无明显不良反应,疗效与美多巴相当,在改善中医证候、患者生存质量及睡眠质量方面明显优于美多巴。这将为治疗早期帕金森病增加一可供选择的有效药物。
第二部分帕病2号方对帕金森病大鼠模型多巴胺能神经元保护作用的研究目的：
研究帕病2号方对PD大鼠模型黑质多巴胺能神经元的保护作用。方法：
用6-羟基多巴胺(6-OHDA)纹状体左侧两点注射法制作PD大鼠模型,阿朴吗啡(APO)腹腔注射诱导并诊断大鼠旋转行为,将造模成功大鼠随机分为帕病2号方高剂量组(32.0g生药/kg)、帕病2号方中剂量组(16.0g生药/kg)、帕病2号方低剂量组(8.0g生药/kg)、美多巴组(美多巴0.075g/kg)、模型组,同时设立空白对照组,每组8只,空白对照组和模型组予等容积蒸馏水灌胃,连续灌胃4周,观察阿朴吗啡诱导的行为学改变,应用TUNEL免疫组化染色检测多巴胺能神经元凋亡的情况,TH免疫组化染色检测大鼠黑质TH阳性神经元数目,HE染色观察多巴胺能神经元的病理形态学改变。
结果：
1.行为学方面,模型组大鼠治疗前后旋转圈数无显著差异,帕病2号方高、中剂量对阿朴吗啡诱导的旋转行为有明显改善,旋转圈数与模型组相比较明显减少(P<0.05),且治疗前后组内比较,差异均有统计学意义(P<0.05)；而帕病2号方低剂量组治疗前后组内比较及与模型组同期组间比较,差异均无统计学意义(P>0.05)。美多巴组大鼠旋转圈数与模型组相比亦明显减少(P<0.05)。
2.组织形态学结果表明,空白对照组黑质区组织细胞正常。模型组大鼠黑质神经元有明显损伤,表现为该区神经元数量减少,部分发生固缩,胶质细胞浸润,而帕病2号方高、中剂量组尽管黑质内也存在神经元减少的现象,但程度明显较轻。帕病2号方低剂量组及美多巴组神经元减少程度与模型组相似。说明帕病2号方可对抗6-OHDA所致的神经元损伤。
3.TH免疫组化结果：空白对照组大鼠黑质多巴胺神经元数量较多,PD大鼠模型毁损侧黑质部位多巴胺神经元显著减少,与模型组相比,帕病2号方高、中剂量组大鼠毁损侧黑质多巴胺神经元数量明显增多(P＜0.05),高剂量组细胞数量增加尤为显著(P<0.01),而帕病2号方低剂量组及美多巴组毁损侧多巴胺神经元数量与模型组相比无统计学差异(P>0.05)。说明帕病2号方能保护黑质多巴胺能神经元。
4.原位凋亡(TUNEL)检测结果显示,空白对照组未见凋亡细胞；模型组大鼠毁损侧黑质区凋亡细胞数量较多并且出现凋亡小体。与模型组比较,帕病2号方高、中剂量组黑质区凋亡细胞数量显著减少(P<0.01或P<0.05)；而帕病2号方低剂量组、美多巴黑质区凋亡细胞数量与模型组比较无显著改变(P>0.05)。说明帕病2号方可减少6-OHDA致黑质区神经细胞的凋亡结论：
帕病2号方对帕金森病大鼠模型黑质多巴胺能神经元有保护作用,能抑制神经细胞的凋亡,增加黑质内TH的表达。
第三部分帕病2号方对帕金森病大鼠模型纹状体GSH-Px、SOD和MDA含量的影响目的：
观察帕病2号方对PD大鼠氧化应激的影响。
方法：
模型制作、分组及治疗同第二部分,分6组,每组7只大鼠。麻醉状态下迅速取毁损侧纹状体,用冰生理盐水制成10%的组织匀浆,取上清液,用化学比色法检测纹状体部位GSH-Px、SOD和MDA的含量。
结果：
与空白对照组相比,模型组大鼠毁损侧纹状体内GSH-Px、SOD活性显著降低,MDA
含量则明显增高(P<0.05)；与模型组相比,帕病2号方高剂量组GSH-Px、SOD活性明显升高(P<0.05或P＜0.01),MDA含量则显著下降(P<0.05)；帕病2号方中剂量组可提高GSH-Px活性并降低MDA含量(P<0.05),而SOD活性则无明显变化(P>0.05)；而帕病2号方低剂量组仅GSH-Px活性增加(P<0.05),MDA、SOD含量则无显著改变(P>0.05)。美多巴组上述指标均无显著改善(P>0.05),且MDA呈“恶化趋势”
结论：
帕病2号方能提高机体抗氧化和清除自由基的能力,且抗氧化作用呈一定的量效关系。
Parkinson's disease (PD) is a common degenerative disease of the central nervous system, which affects the health of middle aged and elderly patients. Up to date, its etiology and pathogenesis is still unknown. Levodopa has been the mainstay of Parkinson's disease (PD) therapy for over40years, however it has obvious side-effects and complications in long-term treatment. Therefore, people pay close attention to the neuroprotective therapy. Plenty of experiments have confirmed that the Chinese medicine had obvious therapeutic effect on PD, which is regarded as one of the neuroprotective therapy. However, there is few study on the mechanism of Chinese medicine's therapeutic effect on PD on the cellular and molecular level. Pabing Ⅱ Formula, which is the experiential prescription of professor Luoxiaodong, We have observed that Pabing Ⅱ Formula can meliorate symptoms of early PD, improve the quality of life of paitients and modify the progression of PD. To objectively evaluate the clinical efficacy of Chinese medicine Pabing Ⅱ Formula, we observe the motor signs, TCM symptoms, quality of life inclding quality of sleep in early-stage PD patients in three-month treatment. In order to explore the pharmacodynamic of Pabing Ⅱ Formula, we establish the hemilateral rat model of PD by6-hydroxydopamine (6-OHDA) twice stereotaxi-cally into the left side of striatum, pathomorphology, immunohistochemical staining and biochemical detection are proformed in order to probe into the therapeutic effect of Pabing Ⅱ Formula, and identify its mechanism of antioxidation and anti-apoptosis. The study is divided into three parts as follows:
The first part Therapeutic Effect Observation of Pabing Ⅱ Formula in Pa tients with Early-stage Parkinson's Disease
Object ive
To objectively evaluate the clinical efficacy of Pabing II Formula, a Chinese medicine compound, in treating Parkinson's disease (PD) at early stage.
Methods
Randomized, parallel-group clinical trial. Sixty subjects with early P D, not requiring dopaminergic therapy, were divided into two groups:Pabi ng II Formula treatment group and madopar control group, each group had t hirty cases. Subjects in Pabing Ⅱ Formula group were administerd with tr aditional Chinese medicine Pabing II Formula decoction (mainly compose of Dark Plum Fruit, White Paeony Root, Radix Angelicae Sinensis, prepared r hizome of Rehmannia, Polygonum Multiflorum, Radix Puerariae, Gastrodia El ata, Carapax Testudinis,et al.), while subjects in madopar group received madopar,150-750mg/d. The treatment course was3months for all. Efficacy was evaluated with the unified Parkinson Disease rating scale (UPDRS)III (motor function section), modified Webster scale, Hoehn&Yahr scale, symp toms of Parkinson's disease of liver-kidney Yin deficiency rating scale,P arkinson's Disease Questionnaire(PDQ-39) and Parkinson disease sleep sea le(PDSS). One-minute bimanual timed motor test was also involved. The base line scores were assessed before treatment and compared with the scores r ecorded at the end of treatment. Meanwhile, the blood pressure, pulse rat e, blood and urine routine, liver and renal functions, electrocardiogram(E CG) and adverse reactions were monitored as the indices for safety superv lse.
Results
1.In improving motor signs:After treatment, there were4patients (13.79%) in Pabing II Formula treatment group markedly improved and19patien ts(65.52%) improved, the total effective rate was79.31%; while in madopar group, the corresponding outcomes were5(17.86%) and18(64.29%), respecti vely, the total effective rate was82.14%; showing insignificant differen ce between the two groups(P>0.05). As far as motor signs were concerned, parameters such as UPDRSIII scores and modified Webster scale scores were all significantly decreased in Pabing II Formula treatment group after tr eatment. And in madopar group, significant lowering also showed in terms of UPDRSIII and modified Webster scale (P<0.05). However, the above parame ters showed no statistical significant difference between groups(P>0.05). When comparing Hoehn and Yahr Scale, there was no significant difference within or between groups after treatment (P>0.05). Comparing the times of one-minute bimanual timed motor test on both hands before and after the t reatment, there existed no significant changes within the Pabing II Formu la treatment group and Madopar group (P>0.05).
2. Changes of TCM symptoms:There were6patients in Pabing Ⅱ Formula treatment group markedly improved and20patients improved, the total effe ctive rate was89.66%; while in madopar group, the corresponding outcomes were0and5, respectively, the total effective rate was17.86%; showing significant difference between the two groups(P<0.01). The clinical syrapto ms such as tremor, rigidity, bradykinesia, dizziness, insomnia, waist achin g and limp, constipation of patients in Pabing II Formula treatment group made good clinical efficacy, while the madopar group showed no significan t difference in improving the clinical symptoms except rigidity and brady kinesia.
3. Effects on quality of life:The scores of some aspects of PDQ-39su ch as emotion, sense of disgrace, cognition and psychophysiologic disorde rs in Pabing II Formula treatment group showed statistical significant di fference compared to the control group (P<0.05).The items of PDSS scores in Pabing II Formula treatment group were significantly increased after t reatment (P<0.05), while changes had no statistical significance(P>0.05) in the control group.
4. Adverse reaction:Both the two therapy were considered safety and t he adverse effects were mild. It showed no changes either in biochemical e xaminations of routine blood and urine,liver and renal function tests, or cardiovascular examinations including heart rhythm, blood pressure and E CG.Severe side-effects had not been observed during the clinical trial.
Conelusion
The results of clinical trial showed the Chinese medicine Pabing II F ormula therapy can ameliorate motor signs of early-stage PD patients and had no marked adverse effects, the curative effect was similar to madopar. TCM symptoms of PD patients improved markedly in Pabing II Formula group. In some aspects of quality of life including sleep quality, Pabing Ⅱ For mula was superior to madopar. And it may become one of the choices for the treatment of early-stage Parkinson's disease.
The second part Involvement in Neuroprotective Effect of Pabing II Formu la on the Dopaminergic Neurons of Parkinson's disease Rats
Objective
To explore neuroprotective effect of Pabing II Formula on the nigros triatal dopaminergic neurons of Parkinson's disease rats
Methods
PD rats were induced by6-hydroxydopamine(6-OHDA) twice stereotaxical ly into the left side of striatum, rotation test was diagnosed by injecti ng apomorphine (APO) and the hemilateral model of PD were obtained. PD ra ts were radomly divided into five groups:model group, Parbin II Formula high dose group(32.0g·kg-1), Parbin Ⅱ Formula media dose group(16.0g·kg), Parbin II Formula low dose group(8.0g·kg-1) and madopar group(0.075g·kg-1), at the same time, the normal control group was established, each group had8rats. The rats in Parbin II Formula high, media, low dose grou p were administered with Chinese medicine Parbin II Formula decoction(32.0,16.0,8.0g#kg-1), respectively. The rats in madopar group were treated with madopar (0.075g·kg-1), The normal group and model group were treated with distilled water. The treatment lasted for4weeks and only one time a day. All animals rotational behavior were detected by recording the num ber of apomorphine-induced turns. Tyrosine hydroxylase(TH) immunohistoche mical staining was used to investigate the numbers of dopaminergic neuron s in substantia nigra compacta(SNc). The TUNEL staining methods were adop ted to observe apoptosis changes of nigrostriatal dopaminergic neurons. H E staining was used to observe the pathomorphological changes of SNc.
Results
1. In rotational behavior:There was no significant difference in the rotation behaviour in model group before and after treatment; Compared to model group, the rotation behaviour of PD rats was significantly amelior ated in Parbin Ⅱ Formula high and media dose group(P<0.05), Madopar can also obviously change the rotation behaviour of PD rats (P<0.05). However, Parbin Ⅱ Formula low dose group showed no statistical significant diffe rence compared with modelgroup(P>0.05).
2. Histomorphological observation showed the neurons in SNc zone of mo del rats were obviously damaged,which were manifested by severely decrea se of neurons, condensed cellular nucleus and partly infiltrated glial ce11s. Parbin Ⅱ Formula decoction (32.0,16.0g·kg-1) administration can pa rtly reverse the changes. However, the values of neurons in Formula low d ose (8.0g·kg-1) and madopar group had no obvious changes compared to those of model rats. It means that Parbin II Formula protected neurons against the neurotoxicity of6-OHDA.
3. TH immunohistochemical staining results suggested TH positive neuro ns in the impaired side of SNc of the PD model rats singificnatly decreas e compared to normal control group(P<0.01). After being treated with Parbi n II Formula decoction (32.0,16.0g·kg-1), they could partly be restored, high dose group(32.0g·kg-1) showed the prominent TH expression in SNc. H owever, TH positive neurons in the Parbin II Formula low dose (8.0g·kg-1) and madopar group had no statistical difference compared to6-OHDA-induce d model group (P>0.05). It means that Parbin II Formula treatment can el evate TH expression, the marker of dopaminergic neurons(P<0.05).
4.TUNEL assay showed the normal control rats had none of apoptotic ce11s, while apoptotic body and neurons apoptosis were obvious in model gro up. Parbin Ⅱ Formula decoction (32.0,16.0g·kg-1) treatment can obviously decrease apoptotic body and neurons apoptosis(P<0.01or P<0.05). Compare dwith model group, the numbers of apoptosis nigral cells in Parbin II For mula low dose (8.0g·kg-1) and madopar group had no evident difference (P>0.05). It means Parbin II Formula may mitigate the injury of SNc evidentl y by inhibiting6-OHDA-induced neurons apoptosis.
Conelusion
The present study demonstrated that Parbin II Formula treatment showe d its neuroprotective effects, and its possible mechanism maybe involved i n anti-apoptotic pathway and elevate TH expression in SNc.
The third part The Influence of Pabing II Formula on Contents of GSH-Px, SOD and MDA in Striatum of PD Rats
Objective
To study effect of Parbin Ⅱ Formula on the oxidation stress response of PD rats.
Methods
PD rats producing, dividing into groups and medication intervention, all the three steps were the same as the second part,each group had7rat s. In deeply anesthetized condition, the injured side of striatum was take n out, made10%tissue homogenate, the colorimetric assays were used to de tect the activities of glutathione peroxidase(GSH-Px), superoxidase dismu tase (SOD) and the levels of malonaldehyde (MDA)
Results
Compared with the normal control group, MDA contents were obviously i ncreased and the activities of GSH-Px and SOD were obviously reduced in m odel group (P<0.05). However, the above indicators were improved in Parbi n Ⅱ Formula high dose group (32.0g·kg-1) and had statistical significant difference (P<0.05or P<0.01). Parbin Ⅱ Formula media dose group(16.0g·kg ') enhanced the activities of GSH-Px and dereased MDA contents and had th e significant difference (P<0.05), SOD activities showed no statistical s ignificant difference. Parbin Ⅱ Formula low dose group(8.0g·kg-1) can onl y remove the activities of GSH-Px and had no change in MDA and SOD conten ts. However, there was no significant difference in the the above indicat ors in madopar group (P>0.05) and the content of MDA seemed to be worsen.
Conclusions
Pabing Ⅱ Formula can significantly enhance antioxidation ability and eliminate free radicals of PD rats, and its effect is in a dose dependent manner.
本课题临床研究部分观察帕病2号方对早期PD患者的运动功能、中医证候、生存质量及睡眠质量的改善情况,客观评价中药复方帕病2号方治疗早期PD的临床疗效。实验研究部分用6-羟基多巴(6-OHDA)纹状体两点注射单侧毁损法制作PD大鼠模型,通过病理形态学、免疫组化、生化检测等方法,从抗氧化应激、抑制神经细胞凋亡等方面对帕病2号方治疗PD的作用机理进行了系统研究。旨在探讨帕病2号方的药效学作用及相关作用机制。本课题分为三部分,摘要如下：
第一部分帕病2号方治疗早期帕金森病的临床疗效观察
目的：
客观评价中药帕病2号方治疗早期帕金森病的临床疗效。
方法：
采用随机、对照研究方法进行临床试验。将60例早期帕金森病患者随机分为2组：帕病2号方组、美多巴组,每组各30例。帕病2号方组采用中药帕病2号方(由乌梅、白芍、当归、熟地黄、何首乌、葛根、天麻、龟板等组成)治疗,美多巴组给予美多巴治疗,疗程3个月。3个月疗程结束后进行疗效评价,疗效评价指标为统一帕金森病量表(UPDRS)第三部分(运动功能)、改良Webster量表评分、Hoehn&Yahr分级、(自拟)肝肾不足型帕金森病中医证候积分量表、帕金森病生存质量量表(PDQ-39)、睡眠量表(PDSS)评分相对于基线的变化,记录完成相关运动试验时间(双手1min记时运动)。同时观察血压、脉搏、血尿常规、肝肾功能、心电图及不良反应等
结果：
1.在改善运动功能方面：帕病2号方组29例患者,显效13.79%(4/29),有效65.52%(19/29),总有效率79.31%；美多巴组28例患者,显效17.86%(5/28),有效64.29%(18/28),总有效率82.14%,两组疗效比较差异无统计学意义(P>0.05)。帕病2号方组的UPDRSⅢ、改良Webster量表评分等指标治疗前后有明显改善(P<0.05),Hoehn-Yahr分级、双手1min记时运动试验治疗前后差异无统计学意义(P>0.05)；美多巴组的UPDRSIII、改良Webster量表等指标治疗前后有明显改善(P<0.05),Hoehn-Yahr分级、双手1min记时运动试验治疗前后差异无统计学意义(P>0.05)；两组组间比较无差异性(P>0.05)。提示在改善早期PD患者运动功能方面,中药复方帕病2号方与美多巴疗效相当。
2.患者中医证候改善情况：帕病2号方组29例患者,显效6例,有效20例,总有效率为89.66%,美多巴组28例患者,显效0例,有效5例,总有效率为17.86%,两组中医证候疗效比较有显著差异(P＜0.01)。帕病2号方组对肝肾不足证患者常见症状如震颤、肢体拘挛、运动迟缓、头晕、失眠多梦、腰膝酸软、便秘等症状均有明显改善(P<0.05),美多巴组仅有肢体拘挛、运动迟缓症状有明显改善(P<0.05)。
3.对早期PD患者生存质量的影响
帕病2号方组治疗前后生活质量问卷分类评分组内比较,运动、日常生活活动、情感、耻辱感、认知力、躯体不适等方面评分差异有统计学意义(P<0.05),美多巴组仅运动、日常生活活动积分差异有统计学意义(P<0.05)。两组情感、耻辱感、认知力、躯体不适等分类评分治疗前后差值组间比较,差异有统计学意义(P<0.05)。在改善睡眠质量方面,帕病2号方组PDSS积分治疗后明显改善(P<0.05),美多巴组治疗前后组内比较,差异无统计学意义(P>0.05),两组组间比较具有差异性(P<0.05)。
4.安全性评价
帕病2号方及美多巴治疗前后血尿常规、肝肾功能检查结果皆在正常范围内,心率、血压和心电图未见明显的变化,未发现有严重不良反应。
结论：
中药帕病2号方对改善早期帕金森病患者的运动功能有效,无明显不良反应,疗效与美多巴相当,在改善中医证候、患者生存质量及睡眠质量方面明显优于美多巴。这将为治疗早期帕金森病增加一可供选择的有效药物。
第二部分帕病2号方对帕金森病大鼠模型多巴胺能神经元保护作用的研究目的：
研究帕病2号方对PD大鼠模型黑质多巴胺能神经元的保护作用。方法：
用6-羟基多巴胺(6-OHDA)纹状体左侧两点注射法制作PD大鼠模型,阿朴吗啡(APO)腹腔注射诱导并诊断大鼠旋转行为,将造模成功大鼠随机分为帕病2号方高剂量组(32.0g生药/kg)、帕病2号方中剂量组(16.0g生药/kg)、帕病2号方低剂量组(8.0g生药/kg)、美多巴组(美多巴0.075g/kg)、模型组,同时设立空白对照组,每组8只,空白对照组和模型组予等容积蒸馏水灌胃,连续灌胃4周,观察阿朴吗啡诱导的行为学改变,应用TUNEL免疫组化染色检测多巴胺能神经元凋亡的情况,TH免疫组化染色检测大鼠黑质TH阳性神经元数目,HE染色观察多巴胺能神经元的病理形态学改变。
结果：
1.行为学方面,模型组大鼠治疗前后旋转圈数无显著差异,帕病2号方高、中剂量对阿朴吗啡诱导的旋转行为有明显改善,旋转圈数与模型组相比较明显减少(P<0.05),且治疗前后组内比较,差异均有统计学意义(P<0.05)；而帕病2号方低剂量组治疗前后组内比较及与模型组同期组间比较,差异均无统计学意义(P>0.05)。美多巴组大鼠旋转圈数与模型组相比亦明显减少(P<0.05)。
2.组织形态学结果表明,空白对照组黑质区组织细胞正常。模型组大鼠黑质神经元有明显损伤,表现为该区神经元数量减少,部分发生固缩,胶质细胞浸润,而帕病2号方高、中剂量组尽管黑质内也存在神经元减少的现象,但程度明显较轻。帕病2号方低剂量组及美多巴组神经元减少程度与模型组相似。说明帕病2号方可对抗6-OHDA所致的神经元损伤。
3.TH免疫组化结果：空白对照组大鼠黑质多巴胺神经元数量较多,PD大鼠模型毁损侧黑质部位多巴胺神经元显著减少,与模型组相比,帕病2号方高、中剂量组大鼠毁损侧黑质多巴胺神经元数量明显增多(P＜0.05),高剂量组细胞数量增加尤为显著(P<0.01),而帕病2号方低剂量组及美多巴组毁损侧多巴胺神经元数量与模型组相比无统计学差异(P>0.05)。说明帕病2号方能保护黑质多巴胺能神经元。
4.原位凋亡(TUNEL)检测结果显示,空白对照组未见凋亡细胞；模型组大鼠毁损侧黑质区凋亡细胞数量较多并且出现凋亡小体。与模型组比较,帕病2号方高、中剂量组黑质区凋亡细胞数量显著减少(P<0.01或P<0.05)；而帕病2号方低剂量组、美多巴黑质区凋亡细胞数量与模型组比较无显著改变(P>0.05)。说明帕病2号方可减少6-OHDA致黑质区神经细胞的凋亡结论：
帕病2号方对帕金森病大鼠模型黑质多巴胺能神经元有保护作用,能抑制神经细胞的凋亡,增加黑质内TH的表达。
第三部分帕病2号方对帕金森病大鼠模型纹状体GSH-Px、SOD和MDA含量的影响目的：
观察帕病2号方对PD大鼠氧化应激的影响。
方法：
模型制作、分组及治疗同第二部分,分6组,每组7只大鼠。麻醉状态下迅速取毁损侧纹状体,用冰生理盐水制成10%的组织匀浆,取上清液,用化学比色法检测纹状体部位GSH-Px、SOD和MDA的含量。
结果：
与空白对照组相比,模型组大鼠毁损侧纹状体内GSH-Px、SOD活性显著降低,MDA
含量则明显增高(P<0.05)；与模型组相比,帕病2号方高剂量组GSH-Px、SOD活性明显升高(P<0.05或P＜0.01),MDA含量则显著下降(P<0.05)；帕病2号方中剂量组可提高GSH-Px活性并降低MDA含量(P<0.05),而SOD活性则无明显变化(P>0.05)；而帕病2号方低剂量组仅GSH-Px活性增加(P<0.05),MDA、SOD含量则无显著改变(P>0.05)。美多巴组上述指标均无显著改善(P>0.05),且MDA呈“恶化趋势”
结论：
帕病2号方能提高机体抗氧化和清除自由基的能力,且抗氧化作用呈一定的量效关系。
Parkinson's disease (PD) is a common degenerative disease of the central nervous system, which affects the health of middle aged and elderly patients. Up to date, its etiology and pathogenesis is still unknown. Levodopa has been the mainstay of Parkinson's disease (PD) therapy for over40years, however it has obvious side-effects and complications in long-term treatment. Therefore, people pay close attention to the neuroprotective therapy. Plenty of experiments have confirmed that the Chinese medicine had obvious therapeutic effect on PD, which is regarded as one of the neuroprotective therapy. However, there is few study on the mechanism of Chinese medicine's therapeutic effect on PD on the cellular and molecular level. Pabing Ⅱ Formula, which is the experiential prescription of professor Luoxiaodong, We have observed that Pabing Ⅱ Formula can meliorate symptoms of early PD, improve the quality of life of paitients and modify the progression of PD. To objectively evaluate the clinical efficacy of Chinese medicine Pabing Ⅱ Formula, we observe the motor signs, TCM symptoms, quality of life inclding quality of sleep in early-stage PD patients in three-month treatment. In order to explore the pharmacodynamic of Pabing Ⅱ Formula, we establish the hemilateral rat model of PD by6-hydroxydopamine (6-OHDA) twice stereotaxi-cally into the left side of striatum, pathomorphology, immunohistochemical staining and biochemical detection are proformed in order to probe into the therapeutic effect of Pabing Ⅱ Formula, and identify its mechanism of antioxidation and anti-apoptosis. The study is divided into three parts as follows:
The first part Therapeutic Effect Observation of Pabing Ⅱ Formula in Pa tients with Early-stage Parkinson's Disease
Object ive
To objectively evaluate the clinical efficacy of Pabing II Formula, a Chinese medicine compound, in treating Parkinson's disease (PD) at early stage.
Methods
Randomized, parallel-group clinical trial. Sixty subjects with early P D, not requiring dopaminergic therapy, were divided into two groups:Pabi ng II Formula treatment group and madopar control group, each group had t hirty cases. Subjects in Pabing Ⅱ Formula group were administerd with tr aditional Chinese medicine Pabing II Formula decoction (mainly compose of Dark Plum Fruit, White Paeony Root, Radix Angelicae Sinensis, prepared r hizome of Rehmannia, Polygonum Multiflorum, Radix Puerariae, Gastrodia El ata, Carapax Testudinis,et al.), while subjects in madopar group received madopar,150-750mg/d. The treatment course was3months for all. Efficacy was evaluated with the unified Parkinson Disease rating scale (UPDRS)III (motor function section), modified Webster scale, Hoehn&Yahr scale, symp toms of Parkinson's disease of liver-kidney Yin deficiency rating scale,P arkinson's Disease Questionnaire(PDQ-39) and Parkinson disease sleep sea le(PDSS). One-minute bimanual timed motor test was also involved. The base line scores were assessed before treatment and compared with the scores r ecorded at the end of treatment. Meanwhile, the blood pressure, pulse rat e, blood and urine routine, liver and renal functions, electrocardiogram(E CG) and adverse reactions were monitored as the indices for safety superv lse.
Results
1.In improving motor signs:After treatment, there were4patients (13.79%) in Pabing II Formula treatment group markedly improved and19patien ts(65.52%) improved, the total effective rate was79.31%; while in madopar group, the corresponding outcomes were5(17.86%) and18(64.29%), respecti vely, the total effective rate was82.14%; showing insignificant differen ce between the two groups(P>0.05). As far as motor signs were concerned, parameters such as UPDRSIII scores and modified Webster scale scores were all significantly decreased in Pabing II Formula treatment group after tr eatment. And in madopar group, significant lowering also showed in terms of UPDRSIII and modified Webster scale (P<0.05). However, the above parame ters showed no statistical significant difference between groups(P>0.05). When comparing Hoehn and Yahr Scale, there was no significant difference within or between groups after treatment (P>0.05). Comparing the times of one-minute bimanual timed motor test on both hands before and after the t reatment, there existed no significant changes within the Pabing II Formu la treatment group and Madopar group (P>0.05).
2. Changes of TCM symptoms:There were6patients in Pabing Ⅱ Formula treatment group markedly improved and20patients improved, the total effe ctive rate was89.66%; while in madopar group, the corresponding outcomes were0and5, respectively, the total effective rate was17.86%; showing significant difference between the two groups(P<0.01). The clinical syrapto ms such as tremor, rigidity, bradykinesia, dizziness, insomnia, waist achin g and limp, constipation of patients in Pabing II Formula treatment group made good clinical efficacy, while the madopar group showed no significan t difference in improving the clinical symptoms except rigidity and brady kinesia.
3. Effects on quality of life:The scores of some aspects of PDQ-39su ch as emotion, sense of disgrace, cognition and psychophysiologic disorde rs in Pabing II Formula treatment group showed statistical significant di fference compared to the control group (P<0.05).The items of PDSS scores in Pabing II Formula treatment group were significantly increased after t reatment (P<0.05), while changes had no statistical significance(P>0.05) in the control group.
4. Adverse reaction:Both the two therapy were considered safety and t he adverse effects were mild. It showed no changes either in biochemical e xaminations of routine blood and urine,liver and renal function tests, or cardiovascular examinations including heart rhythm, blood pressure and E CG.Severe side-effects had not been observed during the clinical trial.
Conelusion
The results of clinical trial showed the Chinese medicine Pabing II F ormula therapy can ameliorate motor signs of early-stage PD patients and had no marked adverse effects, the curative effect was similar to madopar. TCM symptoms of PD patients improved markedly in Pabing II Formula group. In some aspects of quality of life including sleep quality, Pabing Ⅱ For mula was superior to madopar. And it may become one of the choices for the treatment of early-stage Parkinson's disease.
The second part Involvement in Neuroprotective Effect of Pabing II Formu la on the Dopaminergic Neurons of Parkinson's disease Rats
Objective
To explore neuroprotective effect of Pabing II Formula on the nigros triatal dopaminergic neurons of Parkinson's disease rats
Methods
PD rats were induced by6-hydroxydopamine(6-OHDA) twice stereotaxical ly into the left side of striatum, rotation test was diagnosed by injecti ng apomorphine (APO) and the hemilateral model of PD were obtained. PD ra ts were radomly divided into five groups:model group, Parbin II Formula high dose group(32.0g·kg-1), Parbin Ⅱ Formula media dose group(16.0g·kg), Parbin II Formula low dose group(8.0g·kg-1) and madopar group(0.075g·kg-1), at the same time, the normal control group was established, each group had8rats. The rats in Parbin II Formula high, media, low dose grou p were administered with Chinese medicine Parbin II Formula decoction(32.0,16.0,8.0g#kg-1), respectively. The rats in madopar group were treated with madopar (0.075g·kg-1), The normal group and model group were treated with distilled water. The treatment lasted for4weeks and only one time a day. All animals rotational behavior were detected by recording the num ber of apomorphine-induced turns. Tyrosine hydroxylase(TH) immunohistoche mical staining was used to investigate the numbers of dopaminergic neuron s in substantia nigra compacta(SNc). The TUNEL staining methods were adop ted to observe apoptosis changes of nigrostriatal dopaminergic neurons. H E staining was used to observe the pathomorphological changes of SNc.
Results
1. In rotational behavior:There was no significant difference in the rotation behaviour in model group before and after treatment; Compared to model group, the rotation behaviour of PD rats was significantly amelior ated in Parbin Ⅱ Formula high and media dose group(P<0.05), Madopar can also obviously change the rotation behaviour of PD rats (P<0.05). However, Parbin Ⅱ Formula low dose group showed no statistical significant diffe rence compared with modelgroup(P>0.05).
2. Histomorphological observation showed the neurons in SNc zone of mo del rats were obviously damaged,which were manifested by severely decrea se of neurons, condensed cellular nucleus and partly infiltrated glial ce11s. Parbin Ⅱ Formula decoction (32.0,16.0g·kg-1) administration can pa rtly reverse the changes. However, the values of neurons in Formula low d ose (8.0g·kg-1) and madopar group had no obvious changes compared to those of model rats. It means that Parbin II Formula protected neurons against the neurotoxicity of6-OHDA.
3. TH immunohistochemical staining results suggested TH positive neuro ns in the impaired side of SNc of the PD model rats singificnatly decreas e compared to normal control group(P<0.01). After being treated with Parbi n II Formula decoction (32.0,16.0g·kg-1), they could partly be restored, high dose group(32.0g·kg-1) showed the prominent TH expression in SNc. H owever, TH positive neurons in the Parbin II Formula low dose (8.0g·kg-1) and madopar group had no statistical difference compared to6-OHDA-induce d model group (P>0.05). It means that Parbin II Formula treatment can el evate TH expression, the marker of dopaminergic neurons(P<0.05).
4.TUNEL assay showed the normal control rats had none of apoptotic ce11s, while apoptotic body and neurons apoptosis were obvious in model gro up. Parbin Ⅱ Formula decoction (32.0,16.0g·kg-1) treatment can obviously decrease apoptotic body and neurons apoptosis(P<0.01or P<0.05). Compare dwith model group, the numbers of apoptosis nigral cells in Parbin II For mula low dose (8.0g·kg-1) and madopar group had no evident difference (P>0.05). It means Parbin II Formula may mitigate the injury of SNc evidentl y by inhibiting6-OHDA-induced neurons apoptosis.
Conelusion
The present study demonstrated that Parbin II Formula treatment showe d its neuroprotective effects, and its possible mechanism maybe involved i n anti-apoptotic pathway and elevate TH expression in SNc.
The third part The Influence of Pabing II Formula on Contents of GSH-Px, SOD and MDA in Striatum of PD Rats
Objective
To study effect of Parbin Ⅱ Formula on the oxidation stress response of PD rats.
Methods
PD rats producing, dividing into groups and medication intervention, all the three steps were the same as the second part,each group had7rat s. In deeply anesthetized condition, the injured side of striatum was take n out, made10%tissue homogenate, the colorimetric assays were used to de tect the activities of glutathione peroxidase(GSH-Px), superoxidase dismu tase (SOD) and the levels of malonaldehyde (MDA)
Results
Compared with the normal control group, MDA contents were obviously i ncreased and the activities of GSH-Px and SOD were obviously reduced in m odel group (P<0.05). However, the above indicators were improved in Parbi n Ⅱ Formula high dose group (32.0g·kg-1) and had statistical significant difference (P<0.05or P<0.01). Parbin Ⅱ Formula media dose group(16.0g·kg ') enhanced the activities of GSH-Px and dereased MDA contents and had th e significant difference (P<0.05), SOD activities showed no statistical s ignificant difference. Parbin Ⅱ Formula low dose group(8.0g·kg-1) can onl y remove the activities of GSH-Px and had no change in MDA and SOD conten ts. However, there was no significant difference in the the above indicat ors in madopar group (P>0.05) and the content of MDA seemed to be worsen.
Conclusions
Pabing Ⅱ Formula can significantly enhance antioxidation ability and eliminate free radicals of PD rats, and its effect is in a dose dependent manner.
引文
[1]Ravina BM, Fagan SC, Hart MD, et al. Neuroprotective agents for clinical trials in Parkinson's disease[J]. Neurology,2003,60(8):1234.
[2]王慧新,刘毅,李如奎.止颤汤联合美多巴片治疗帕金森病30例临床研究[J].中医杂志,2010,51(6)(增刊)：146-147.
[3]汪美霞,杨文明,汪瀚,等.滋养肝肾育阴熄风法治疗帕金森病临床疗效观察[J].辽宁中医药大学学报,2011,13(6)：174-175.
[4]赵国华,孟庆刚,于向东,等.龟羚帕安胶囊治疗帕金森病多中心、随机、双盲、对照临床研究[J].中国中西医结合杂志,2009,29(7)：590-594.
[5]袁灿兴,支惠萍,陈顺中,等.熟地平颤汤结合西医常规疗法治疗帕金森病的临床多中心随机对照研究[J].上海中医药杂志,2010,44(6)：3-6.
[6]袁红,刘平,许建阳,等.天麻对帕金森病模型大鼠行为学、生物化学的影响[J].武汉医学,2008,19(3)：239-242.
[7]王振华,何建成,张春燕.复方地黄方对帕金森病大鼠细胞凋亡的影响[J].新中医,2010,42(4)：86-88.
[8]王文武,何建成,丁宏娟.天麻钩藤饮对帕金森病大鼠神经行为学及氧化应激反应的影响[J].中国老年学杂志,2010,30(12)：1657-1659.
[9]祝维峰,罗荣敬,周丽萍,等.震颤宁治疗帕金森病的实验研究[J]。广州中医药大学学报,2004,21(4)：284-287.
[10]袁灿兴,叶青.滋补肝肾、通络解毒中药对异动症大鼠行为学及纹状体多巴胺D2受体活性的影响[J].中西医结合学报,2008,6(10)：1024-1028.
[11]Abeliovich A, Flint Beal M. Parkinsonism genes:culprits and clues [J]. J Neurochem, 2006,99 (4):1062-1072.
[12]McNaught KS, Olanow CW. Protein aggregation in the pathogenesis of familial and sporadic Parkinson's disease [J]. Neurobiol Aging,2006,27(4):530.
[13]Reeve AK, Park TK, Jaros E, et al. Relationship Between Mitochondria and a-Synuclein: A Study of Single Substantia Nigra Neurons [J].Arch Neurol,2012,69(3):385-393.
[14]王涛,梁直厚,侯晓妹,等.帕金森病患者parkin基因多态性位点的研究[J].卒中与神经疾病,2003,10(2)：87-89.
[15]Lucking CB, Durr A, Bonifati V, et al. Association between early-onset Parkinson's disease and mutations in the parkin gene. French Parkinson's Disease Genetics Study Group[J]. N Engl J Med,2000,342(11):1560-1567.
[16]肖颖,张本恕.UCH-L1基因多态性与帕金森病的关联研究[J].中华医学遗传学杂志,2008, 25(5)：586-587.
[17]熊慧,张雅坤.帕金森病致病基因DJ-1的研究[J].内科理论与实践,2010,5(5)：390-392.
[18]杜青青,杨学超,吴娟娟,等.PD相关基因LRRK2表达调控的初探[J].南通大学学报(医学版)：2011,31(6)：409-413.
[19]廖冰,郭纪锋,唐北沙PINK1基因与帕金森病[J].中华神经科杂志,2007,40(2)：136-138.
[20]Ramirez A, Heimbach A, Grttndemann J, et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5-type ATPase[J]. Nature Genet,2006,38(10):1184-1191.
[21]梁卉,郑文,邓昊.帕金森病与ATP13A2基因 [J].基础医学与临床,2011,31(10)：1161-1164.
[22]赵燕燕,林兴建,刘卫国,等.中国汉族人群PARK16基因多态性与帕金森病易患性的关联[J].中华神经科杂志,2011,44(5)：343-346.
[23]Li S, Pu XP. Neuroprotective effect of kaempferol against a l-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease[J]. Biol Pharm Bull,2011,34(8):1291-1296.
[24]Xiong N, Huang J, Zhang Z, et al. Stereotaxical infusion of rotenone:a reliable rodent model for Parkinson's disease[J]. PLoS One,2009,4(11):e7878.
[25]Guilarte TR. Manganese and Parkinson's disease:a critical review and new findings [J].Cien Saude Colet,2011,16(11):4549-4566.
[26]Pastorin G, Marchesan S, Hoebeke J, et al. Design and activity of cationic fullerence derivatives as inhibitors of acetylcholinesterase[J]. Org Biomol Chem,2006,4(13): 2556-2562.
[27]潘静,陈生弟.氧化应激与神经退行性疾病[J].国际神经病学神经外科学杂志,2008,35(2)：143-144.
[28]Chakrabarti S, Munshi S, Baneriee K, et al. Mitochondrial Dysfunction during Bra in Aging:Role of oxidative stress and modulation by antioxidant supplementation[J]. Ag ing Dis,2011,2(3):242-256.
[29]Hong YUAN, J ing-Chen ZHENG, Ping LIU, Pathogenesis of Parkinson's disease:oxidative stress, environmental impact factors and inflammatory processes[J]. Neuroscience Bul letin,2007,23(2):125-130.
[30]袁红,梁立武,刘平,等.氧化应激与帕金森病多巴胺神经元死亡[J].武警医学,2007,18(6)：448-450.
[31]Przedborski S, Ischiropoulos H. Reactive oxygen and nitrogen species:weapons of neuronal destruction in models of Parkinsons disease [J]. Antioxid Redox Signal,2005,7 (5-6):685-693.
[32]Sehapira AH, Cooper JM, Dexter D, et al. Mitochondrial complex I deficiency in Parkinson's disease[J]. Lancet,1989,1(8649):1269.
[33]解洪荣,张玉花,李雪松,等.MPTP/MPP诱导的帕金森病实验模型的研究进展[J].医学综述,2011,17(5)：669-672.
[34]Karunakaran S, Di wakar L, Saeed U, et al. Activation of apoptosis signal regulating kinasel (ASK1) and translocation of death-associated protein, Daxx, in substantia nigra pars compacta in a mouse model of Parkinson's disease:protection by alphal ipoic acid [J]. FASEB J,2007,21(9):2226-2236.
[35]刘军,肖颂华,周道友,等.6-羟多巴胺诱致大鼠早期黑质多巴胺能神经元凋亡的实验研究[J].中华老年医学杂志,2003,22(11)：681-683.
[36]曹非,骆芳,段国安.大鼠帕金森病发病过程中黑质细胞凋亡及其相关基因表达[J].中国老年学杂志,2007(27)：2369-2370.
[37]曹非,孙圣刚,童萼塘,等.Bcl-2、Bax蛋白表达与帕金森病大鼠黑质细胞凋亡相关性的实验研究[J].中华老年医学杂志,2003,22(10)：625-626.
[38]Hartmann A, Hunot S, Michel P P, et al. Caspase-3:a vulnerability factor and final effector in apoptosis death of dopaminergic neurons in Parkinson's disease[J]. Pore Natl Acad Sci USA,2000,97(16):2875-2880.
[39]Cheng E H Y, Kirsch D G, Clem R G, et al. Conversion of Bcl-2 to a Bax-like death effector by Caspase[J]. Science,1997,278(12):1966-1968.
[40]Bogdanov M, Matson WR, Wang L, et al. Metabolomic profiling to develop blood bio markers for Parkinson's disease[J]. Brain,2008,131(2):389-396.
[41]LeWitt P, Schultz L, Auinger P, et al. CSF xanthine, homovanillic acid, and their ratio as biomarkers of Parkinson's disease[J]. Brain Res,2011,1408 (11):88-97.
[42]Bogdanov M, Matson WR, Wang L, et al. Mctaboklmic profiling to develop blood biomarkers for Parkinson's disease [J]. Brain,2008,131(2):389-396.
[43]Dorsey ER, Holloway RG, Ravina BM. Biomarkers in Parkinson's disease[J]. Expert Rev Neuother,2006,6(6):823-831.
[44]Mascalchi M, Vella A, Ceravolo R. Movement disorders:role of imaging in diagnosis[J]. J Magn Reson imaging,2012,35(2):239-256.
[45]Wang J, Zuo CT, Jiang YP, et al.18F-FP-CIT PET imaging and SPM analysis of dopamine transporters in Parkinson's disease in various Hoehn&Yahr stages [J]. J Neurol,2007,254 (3):185-190.
[46]Dagher A, Nagano-Saito A. Functional and anantomical maganetic imaging in Parkinson's disese[J]. Mol Imaging Biol.2007,9 (6):234-242.
[47]Nanhagopal R, McKeown M J,Stoessl A J, et al. Functional imaging in Parkinson's disease[J]. Neurology,2008,70 (4):1478-1488.
[48]卢琦,黄建军,张兰,等.磁共振波谱分析在帕金森病早期诊断中的应用研究[J].中国CT和MRI杂志,2009,7(5)：1-4.
[49]Naruse S, Kizu 0, Furuya S. The role of magnetic resonance spec.troscopic imaging in patients with Pakinson's disease[J]. Nippon Rinsho,1997,55(1):262-264.
[50]张莉,孔岩,徐耑,等.豆状核质子磁共振波谱对帕金森病的诊断价值[J].临床荟萃,2009,24(13)：1115一1117.
[51]王大力,刘昊,王坚,等.质子磁共振波谱对帕金森病的诊断价值[J].临床神经病学杂志,2002,15(4)：213-214.
[52]Lu YW. Tan EK. Molecular biology changes associated with LRRK2 mutations in Parkins on's disease [J]. J Neurosci Res,2008,86(9):1895-1901.
[53]田华,田有勇,孙圣刚.帕金森病基因治疗研究现状[J].临床内科杂志,2007,24(6)：375-378.
[54]陈生弟,周海燕.帕金森病发病机制的研究进展[J].中国实用内科杂志,2006,26(4)：246-248.
[55]Duyckaerts C, Sazdovitch V, Seilhean D. Update on the pathophysiology of Parkin-son'sdisease[J]. Bull Acad Natl Med,2010,194(7):1287-1303.
[56]Ravina BM, Fagan SC, Hart MD, et al. Neuroprotective agents for clinical trials in Parkinson's disease[J]. Neurology,2003,60(8):1234.
[57]Boll MC, Alcaraz-ZubeldiaM, Rios C. Medical management of Parkinson's disease:focus on neuroprotection[J]. Curr Neuropharmacol,2011,9(2):350-359.
[58]Ybot-Gorrin I, Vivoncos-Matellano F, Chacon-Pena JR, et al, Assessment of Parkinson disease:what do we need to show neuroprotection? [J]. Nerologist,2011,17(6 Suppl 1): S21-9.
[59]陈生弟,汪锡金.帕金森病的神经保护治疗一现状与分析[J].中国神经免疫学和神经病学杂志,2010,17(3)：159-161.
[60]Li C, Guo Y, Xie W, et al. Neuroprotection of pramipexole in UPS impairment induced animal model of Parkinson's disease[J]. Neurochem Res,2010,35(10):1546-1556.
[61]Li C, Biswas S, Li X, et al. Novel D3 dopamine receptor-preferring Agonist D-264: Evidence of neuroprotective property.in Parkinson's disease animal models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and lactacystin[J]. J Neurosci Res,2010, 88(11):2513-2523.
[62]Olanow CW, Stern MB, Sethi K. The scientific and clinical basis for the treatment of Parkinson's disease [J]. Neurology,2009,72(suppl 4):S1-S136.
[63]Rascol 0, Dubois B, Caldas AC, et al. Early piribedil monotherapy of Parkinson's disease:a planned seven-month report of the REGAIN study[J]. Mov Disord,2008,21(12): 2110-2115.
[64]曲颖,罗晓光,任艳.普拉克索治疗早期帕金森病疗效分析[J].中国误诊学杂志,2009,9(13)：3084-3085.
[65]胥巧凤.盐酸思来吉兰治疗帕金森病患者疗效观察[J].现代医药卫生,2008,24(14)：2151.
[66]Chetsawang B, Kooncumchoo P, Govitrapong P, et al. 1-Methyl-4-phenyl-pyridiniumion-induced oxidative stress, c-Jun phosphorylation and DNA fragmentation factor-45 cleav age in SK-N-SH cells are averted by selegiline[J]. Neurochem Int,2008,53(6-8):283-288.
[67]BIGLAN KM, SCHWID S, EBERLY S, et al. Rasagiline Improves Quality of Life In Patients with Parkinson's Disease [J]. Mov Disord(S0885-3185),2008,21(5):616-623.
[68]Reichmann H, Emre M. Optimizing levodopa therapy to treat wearing-off symptoms in Parkinson's disease:focus on levodopa/carbidopa/entacapone[J]. Expert Rev Neurother, 2012,12(2):119-131.
[69]Lei Z, Jiang Y, Li T, et al. Signaling of glial cell line-derived Neurotrophic factor and its receptor GFR α 1 induce Nurrl and Pitx3 to promote survival of grafted midbrain-derived neural stem cells in a rat model of Parkinson disease [J]. J Neuropathol Exp Neur ol,2011,70(9):736-747.
[70]Sullivan AM, Toulouse A. Neurotrophic factors for the treatment of Parkinson's disea se[J]. Cytokine Growth Factor Rev,2011,2(3):157-165.
[71]Beck KD, Valverde J, Alexi T, et al. Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult rat[J]. Nature,1995,373(6512) :339-341.
[72]Furukawa K, Ohmi Y, Ohkawa Y, et al. Regulatory mechanisms of nervous systems with glycosphingolipids[J]. Neurochem Res,2011,36(9):1578-1586.
[73]Zhang H, Ding J, Tian W, et al. Ganglioside GM1 binding the N-terminus of amyloid precursor protein[J]. Neurobiol Aging,2009,30(8):1245-1253.
[74]CHOI J S, KIM J A, J00 CK. Activation of MAPK and CREB by GM1 induces survival of RGCs in the axotomized nerve[J]. Invest Ophthalmol Vis Sci,2003,44(4):1747-1752.
[75]Lindvall 0, Bjorklund A. Cell therapeutics in Parkinson's disease [J]. Neurotherapeu tics,2011,8(4):539-548.
[76]李海斌,石炳毅.帕金森病的干细胞治疗进展[J].中国组织工程研究与临床康复,2009,13(1)：173-176.
[77]Espay AJ, Fasano A, Morgante F. The six gaps in the search of neuroprotection for Parkinson's disease[J].Expert Rev Neurother,2012,12(2):111-113.
[78]张华,雒晓东.中医六经厥阴辨证治疗早中期帕金森病的临床研究[J].中医药学报,2008,36(6)：34-36.
[79]李军艳,杨明会,赵冠英.试论肾虚血瘀是帕金森病的基本病机[J].中华中医药杂志, 2008,23(9)：768-771.
[80]杨明会,窦永起,刘毅.抑颤汤对帕金森病模型大鼠行为学及脑黑质细胞形态学的作用[J].中国中西医结合杂志,2002,22(11)：841-843.
[81]何建成.帕金森病中医治疗的再思考—兼谈“滋肾平肝,化痰活血,解毒散结是帕金森病的基本治疗法则”[J].中医药通报,2005,4(1)：12-14.
[82]陈晓春,周宜灿,陈滢,等.人参皂甙Rgl保护多巴胺能神经元的实验研究[J].医学研究通讯,2005,34(6)：28.
[83]陈滢,陈晓春.Bc1-2家族是人参皂甙Rgl抗黑质神经元凋亡的重要调控蛋白[J].解剖学报,2002,33(5)：496-499.
[84]Li N, Liu B, Du Z D, et al. Protective effects of ginsenoside Rg2 against glutamate induced neurotoxicity in PC 12 cells[J].J Ethnopharmacol,2007,111(3):458.
[85]Bo J, Ming BY, Gang LZ, et al. Protection by puerarin against MPP+-induced neuro-toxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase--3-like Activation[J]. Neurosci Res,2005,53(2):183.
[86]李雪莉,孙圣刚.葛根素对帕金森病保护作用的实验研究[J].脑与神经疾病杂志,2002,10(1)：7-9.
[87]蒲小平,李晓蓉,李慧浓,等.肉苁蓉成分campneosidell对神经毒素MPP+诱发细胞凋亡的保护作用[J].北京大学学报(医学版),2001,33(3)：217-220.
[88]曹非,孙圣刚,曹学兵,等.银杏提取物防止帕金森病大鼠黑质细胞损伤的实验研究[J].中国老年学杂志,2002,22(5)：217.
[89]奚淑芳,陈建宗,王百忍,等.银杏叶提取物对MPP+诱导的中脑多巴胺能神经细胞损伤的保护作用[J].第四军医大学学报,2006,27(1)：73-76.
[90]李永梅,李霞,周兆丽.何首乌提取物对百草枯引起的小鼠多巴胺能神经元损伤的保护作用[J].中国临床药理学杂志,2007,23(5)：395-396.
[91]黄凌,朱毅,邝少轶.益智仁挥发油抗帕金森模型小鼠黑质神经元凋亡的作用研究[J].中国药房,2011,22(47)：4430-4433.
[92]吕娥,吕宁宁,付文玉,等.益精养血方对帕金森病大鼠多巴胺能神经元凋亡的影响[J].解剖学杂志,2007,30(5)：589-592.
[93]杨明全,窦永起,刘毅,等.抑颤汤对PD大鼠行为学及脑黑质细胞形态学的作用[J].中国中西医结合杂志,2002,22(11)：84.
[94]孟海伟,刘树伟,许继平,等.中药颤复宁血清对6-羟基多巴诱导的PCI2细胞凋亡的抑制作用[J].山东大学学报(医学版),2005,43(10)：916-919.
[95]李海龙,栗艳,厚荣荣,等.天麻素对百草枯和代森锰诱导的小鼠脑黑质多巴胺能神经元损伤的保护作用[J].成都中医药大学学报,2010,33(1)：57-59.
[96]巴晓红,刘英.银杏叶提取物及银杏总内酯对帕金森病大鼠黑质神经元损伤的保护作用[J]. 中国临床康复,2006,10(11)：39-41.
[97]何建成,袁灿兴,李亚明,等.滋补肝。肾通络解毒中药对PD大鼠模型氧化应激反应的影响[J].中国新药与临床,2003,22(3)：160.
[98]陆征宇,赵虹,汪涛,等.补肾养肝熄风方药对帕金森病小鼠纹状体抗氧化酶和细胞凋亡的影响[J].江苏中医药,2011,43(3)：85-87.
[99]鲍远程,汪瀚,张波,等.抗震止痉胶囊对PD作用机制实验研究[J].中国中西医结合杂志,2001,21(3)：193.
[100]袁崇刚,白桦,阳飞昆,等.首乌制剂对MPTP引起的小鼠PD的防治[J].华东师范大学学报(自然科学版),2002,3：95-98.
[101]陈建宗,李刚,谢永宏,等.培补肝肾中药对PD小鼠模型纹状体某些单胺类递质的影响[J].成都中医药大学学报,2002,25(4)：34.
[102]隆呈祥,邹忆怀,韩晓军,等.中药颤震平治疗老年颤证临床和实验研究[J].北京中医药大学学报,1999,22(6)：57.
[103]和欣,高誉珊,孙红梅,等.大补阴丸、牵正散及合方对帕金森小鼠脑线粒体酶复合物活性作用的实验研究[J].江苏中医药,2010,42(9)：72-73.
[104]伦学庆.兴奋性氨基酸及受体与帕金森病[J].功能性和立体定向神经外科杂志,1997,10(1)：40-42.
[105]罗瑞静,何建成.兴奋性氨基酸与帕金森病研究进展[J].医学研究杂志,2010,39(10)：13-15.
[106]马丽炎,肖培根,梁发权,等.三七总皂苷对神经细胞的保护作用[J].中国药学杂志,1998,10(3)：37.
[107]金雪红,包仕尧.粉防己碱联合还原型谷胱甘肽对帕金森病大鼠纹状体兴奋性氨基酸的影响[J].中国实用神经疾病杂志,2010,13(7)：1-3.
[108]何其华,周慧芳,薛冰,等.雷公藤单体T,o对谷氨酸所致PCI2细胞损伤的保护作用及机制研究[J].北京大学学报(医学版),2003,35(3)：252-255.
[109]袁惠莉,汪璇,张丽娟,等.中药在防治帕金森病中的作用及研究进展[J].中国药理学通报,26(7)：850-853.
[110]吴斌,赵书志,王修敏,等.中医药对帕金森病动物模型的治疗机制,中国中药杂志,2011,36(18)：2588-2591.
[111]刘国卿.神经元保护剂的研究进展[J].药学学报,2002,37(8)：657-662.
[112]Holloway RG, Shoulson I, Fahn S, et al. Pramipexole vs levodopa as initial treatment for Parkinson disease:a 4-year randomized controlled trial[J]. Arch Neurol,2004,61(9): 1044-1053.
[113]中华医学会神经病学分会运动障碍及帕金森病学组.帕金森病治疗指南[J].中华神经科杂志,2006,39(6)：409-412.
[114]张振馨.帕金森病的诊断[J].中华神经科杂志,2006,39(6)：408-409.
[115]中华全国中医学会老年医学会.中医老年颤证诊断和疗效评定标准[J].北京中医学院学报,1992,15(4)：39-40.
[116]王忆勤.中医诊断学[M].北京：中国中医药出版社,2004,135-136.
[117]中华医学会神经病学分会帕金森病及运动障碍学组,中国帕金森病治疗指南(第二版)[J].中华神经科杂志,2009,42(5)：332-335.
[118]陈海波.统一帕金森病评定量表[J].中华老年医学杂志,1999,18(1)：61-62.
[119]王志英,叶放,周学平,等.周仲瑛教授临证思辨特点概要[J].南京中医药大学学报,2007,23(1)：45.
[120]安红梅,胡兵,张学文.从肾阴虚入手证病结合治疗帕金森病思路探讨[J].中国中医急症,2004,13(2)：818-819.
[121]王文武,何建成.基于肝肾同源理论的帕金森病病因病机及治法研究[J].辽宁中医杂志,2009,36(8)：1252-1254.
[122]刘垣,陈彪.帕金森病患者中医证候调查[J].中医杂志,2009,50(8)：728-729.
[123]罗瑞静,何建成.帕金森病中医证候规范化研究[J].中华中医药学刊,2011,29(11)：2445-2447.
[124]陈婉珉,郑春叶,连新福.100例帕金森病患者中医证候要素及证型分布规律[J].中医杂志,2011,52(3)：214-217.
[125]汤湘江,雒晓东,黄培新.从《伤寒论》的六经辨证看帕金森病的中医药治疗[J].深圳中西医结合杂志,2005,15(4)：214-215.
[126]范宇鹏,曾亮,孙玉芝,等.帕病2号方治疗震颤型帕金森病的临床研究[J].天津中医药,2010,27(3)：190-191.
[127]Martinez MP, Gil LA, Gracia LM, et al. Unified Parkinson's Disease Rating Scale characteristics and structure[J].Mov Disord,1994,9(1):76-83.
[128]庄海,徐武华,邹海强.帕金森病非运动症状临床诊治的新进展[J].中华神经医学杂志,2011,10(12)：1294-1296.
[129]Tsang KL, Chi I, Ho SL, et al. Translation and validation of the standard Chinese version of PDQ-39:A quality-of-life measure for patientswith Parkinson's disease[J]. Mov Disord,2002,17(5):1036-1040.
[130]Leentjens AF. Depression in Parkinson's disease:conceptual issues and clinical challenges[J]. J Geriatr Psychiatry Neurol,2004,17(3):120-126.
[131]董青,王智樱,李焰生.帕金森病患者健康相关生活质量的影响因素研究[J].中国神经精神疾病杂志,2008,34(10)：598-600.
[132]Chaudhuri KR, Naidu Y. Early Parkinson's disease and nonmotor issues[J].J Neur ol,2008,255 (Supp15):33-38.
[133]刘文彦,刘军,丁健青,等.帕金森病患者的睡眠障碍[J].中华神经科杂志,2011,44(9)：627-629.
[134]王玉虹,徐良洲.名老中医王淑玲从肝论治失眠的经验[J].陕西中医,2008,29(10)：1356-1357.
[135]George Paxinos, Charles Waton大鼠脑立体定位图谱[M].北京：人民卫生出版社(第三版),2005,18-21.
[136]Sanchez-lglesias S, Rey P, Mendez-Alvarez E, et al. Time course of brain oxidative damage caused by in trastriatal administration of 6-hydroxydopamine in a rat model of Parkinson's disease[J]. Neurochem Ras,2007,32(1):99-105.
[137]王尧,杜子威.神经生物化学与分子生物学[M].北京：人民卫生出版社,1997,131-132.
[138]David B, Sakina T, Nathalie L, et al. Molecular pathway involved in the neurotoxic ity of 6-OHDA, dopamine and MPTP:contribution to the apoptotic theory in Parkinson's disease[J]. Prog Neurobiol,2001,65(11):35.
[139]Thiele SL, Warre R, Nash JE. Development of a Unilaterally-lesioned 6-OHDA Model of Parkinson's Disease[J]. J Vis Exp.2012,60(14):3234.
[140]Valastro B, Dekundy A, Danysz W, et al. Oral creatin supplementation attenuates L-DOPA induced dyskinesia in 6-hydroxydopamine-lesioned rats [J]. Behav Brain Res,2009, 197(1):90-96.
[141]Hudson JL, Van Horne CG, Stromberg I, et al. Correlation of apomarphine and amphe tamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopa mine lesioned rats[J]. Brain Res,1993,626(12):167.
[142]Iancu R, Mohapel P,Brundin P,et al. Behavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson's disease in mice [J]. Behav Brain Res,2005,162 (1): 1-10.
[143]唐涌.细胞凋亡在帕金森病发病中的作用[J].徐州医学院学报,2004,24(1)：87-90.
[144]He Y, Lee T, Leong SK.6-hydroxydopamine induced apoptosis of dopaminergic cells in the rat substantia nigra[J].Brain Res,2000,858(21):163-166.
[145]Migheli A, Caralla P, Marino S, et al.A study of apoptosis in normal and pathologic nerous tissue after in situ end-labeling of DNA strand breaks[J]. J Neuropath Exp Neurol, 1994,53(8):606-616.
[146]HAAVIK J, TOSKA K. Tyrosine hydroxylase and Parkinson's disease[J]. Mol Neurobiol, 1998,16(3):285-309.
[147]曹非,骆芳,段国安.左旋多巴加速帕金森病大鼠黑质细胞凋亡的实验研究[J].临床神经电生理学杂志,2007,16(2)：67-70.
[148]朱美娥,舒丹,陈江帆,等.川芎嗪对MPTP所致小鼠多巴胺能神经元损伤的保护作用及机制 [J].中风与神经疾病杂志,2009,26(3)：333-335.
[149]盛阳,张捷,熊玉波,等.当归注射液对6-羟基多巴胺诱导PC12：细胞帕金森病模型SIRT1表达及细胞凋亡的影响[J].武汉大学学报(医学版),2011,32(4)：436-439.
[150]苗明三,方晓艳.制何首乌多糖对衰老模型小鼠抗氧化作用的研究[J].中药药理与临床,2002,18(5)：23-24.
[151]李吴,杜小平,杨期东,等.何首乌对海人藻酸致大鼠脑AChE神经元及纤维损伤的保护作用[J].卒中与神经疾病,2002,9：299-302.
[152]邓汝东,李伊为,陈东风,等.龟板抗帕金森病大鼠多巴胺能神经元凋亡[J].神经解剖学杂志,2008,24(3)：301-306.
[153]Chun HS, Gibson GE, DeGiorgio LA, et al. Dopaminergic cell death induced by MPP, oxidant and specifiec neurotoxins shares the common molecular mechanism[J]. Jounal of neurochemistry,2001,76(40):1010-1021.
[154]Przedborski S, Ischiropoulos H. Reactive oxygen and nitrogen species:weapons of neuronal destruction in models of Parkinsons disease[J]. Antioxid Redox Signal,2005, 7(5-6):685-693.
[155]Mazzio E, Remas RR, Soliman KF, et al. The role of oxidative stress, impaired glycolysis and mitochondrial respiratory redox failure in the cytotoxic effects of 6-hydroxydpomaine in virto. Brain Research[J].2004,1004(1-2):29-44.
[156]孙晓芳,王丹巧,吴兆恩,等.首乌方对帕金森病模型大鼠血液和纹状体细胞外液左旋多巴药代动力学影响的研究[J].中国实验方剂学杂志,2011,17(11)：114-115
[157]张飞,李劲松.乌梅的研究进展[J].海峡药学,2006,18(4)：21-23.
[158]刘玮,吴华璞,祝晓光,等.白芍总苷对全脑缺血再灌损伤的保护作用[J].中国药理学通报,2004,20(2)：211-213.
[159]孙晓芳,王巍,王丹巧,等.天麻及其制剂神经保护机制的研究进展[J].中国中药杂志,2004,29(4)：292.
[160]周宜灿,陈晓春,朱元贵,等.人参皂苷Rgl可能通过抗氧化作用来保护帕金森病鼠黑质神经元[J].中国临床药理学与治疗学,2003,8(3)：273-277.
[2]王慧新,刘毅,李如奎.止颤汤联合美多巴片治疗帕金森病30例临床研究[J].中医杂志,2010,51(6)(增刊)：146-147.
[3]汪美霞,杨文明,汪瀚,等.滋养肝肾育阴熄风法治疗帕金森病临床疗效观察[J].辽宁中医药大学学报,2011,13(6)：174-175.
[4]赵国华,孟庆刚,于向东,等.龟羚帕安胶囊治疗帕金森病多中心、随机、双盲、对照临床研究[J].中国中西医结合杂志,2009,29(7)：590-594.
[5]袁灿兴,支惠萍,陈顺中,等.熟地平颤汤结合西医常规疗法治疗帕金森病的临床多中心随机对照研究[J].上海中医药杂志,2010,44(6)：3-6.
[6]袁红,刘平,许建阳,等.天麻对帕金森病模型大鼠行为学、生物化学的影响[J].武汉医学,2008,19(3)：239-242.
[7]王振华,何建成,张春燕.复方地黄方对帕金森病大鼠细胞凋亡的影响[J].新中医,2010,42(4)：86-88.
[8]王文武,何建成,丁宏娟.天麻钩藤饮对帕金森病大鼠神经行为学及氧化应激反应的影响[J].中国老年学杂志,2010,30(12)：1657-1659.
[9]祝维峰,罗荣敬,周丽萍,等.震颤宁治疗帕金森病的实验研究[J]。广州中医药大学学报,2004,21(4)：284-287.
[10]袁灿兴,叶青.滋补肝肾、通络解毒中药对异动症大鼠行为学及纹状体多巴胺D2受体活性的影响[J].中西医结合学报,2008,6(10)：1024-1028.
[11]Abeliovich A, Flint Beal M. Parkinsonism genes:culprits and clues [J]. J Neurochem, 2006,99 (4):1062-1072.
[12]McNaught KS, Olanow CW. Protein aggregation in the pathogenesis of familial and sporadic Parkinson's disease [J]. Neurobiol Aging,2006,27(4):530.
[13]Reeve AK, Park TK, Jaros E, et al. Relationship Between Mitochondria and a-Synuclein: A Study of Single Substantia Nigra Neurons [J].Arch Neurol,2012,69(3):385-393.
[14]王涛,梁直厚,侯晓妹,等.帕金森病患者parkin基因多态性位点的研究[J].卒中与神经疾病,2003,10(2)：87-89.
[15]Lucking CB, Durr A, Bonifati V, et al. Association between early-onset Parkinson's disease and mutations in the parkin gene. French Parkinson's Disease Genetics Study Group[J]. N Engl J Med,2000,342(11):1560-1567.
[16]肖颖,张本恕.UCH-L1基因多态性与帕金森病的关联研究[J].中华医学遗传学杂志,2008, 25(5)：586-587.
[17]熊慧,张雅坤.帕金森病致病基因DJ-1的研究[J].内科理论与实践,2010,5(5)：390-392.
[18]杜青青,杨学超,吴娟娟,等.PD相关基因LRRK2表达调控的初探[J].南通大学学报(医学版)：2011,31(6)：409-413.
[19]廖冰,郭纪锋,唐北沙PINK1基因与帕金森病[J].中华神经科杂志,2007,40(2)：136-138.
[20]Ramirez A, Heimbach A, Grttndemann J, et al. Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5-type ATPase[J]. Nature Genet,2006,38(10):1184-1191.
[21]梁卉,郑文,邓昊.帕金森病与ATP13A2基因 [J].基础医学与临床,2011,31(10)：1161-1164.
[22]赵燕燕,林兴建,刘卫国,等.中国汉族人群PARK16基因多态性与帕金森病易患性的关联[J].中华神经科杂志,2011,44(5)：343-346.
[23]Li S, Pu XP. Neuroprotective effect of kaempferol against a l-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease[J]. Biol Pharm Bull,2011,34(8):1291-1296.
[24]Xiong N, Huang J, Zhang Z, et al. Stereotaxical infusion of rotenone:a reliable rodent model for Parkinson's disease[J]. PLoS One,2009,4(11):e7878.
[25]Guilarte TR. Manganese and Parkinson's disease:a critical review and new findings [J].Cien Saude Colet,2011,16(11):4549-4566.
[26]Pastorin G, Marchesan S, Hoebeke J, et al. Design and activity of cationic fullerence derivatives as inhibitors of acetylcholinesterase[J]. Org Biomol Chem,2006,4(13): 2556-2562.
[27]潘静,陈生弟.氧化应激与神经退行性疾病[J].国际神经病学神经外科学杂志,2008,35(2)：143-144.
[28]Chakrabarti S, Munshi S, Baneriee K, et al. Mitochondrial Dysfunction during Bra in Aging:Role of oxidative stress and modulation by antioxidant supplementation[J]. Ag ing Dis,2011,2(3):242-256.
[29]Hong YUAN, J ing-Chen ZHENG, Ping LIU, Pathogenesis of Parkinson's disease:oxidative stress, environmental impact factors and inflammatory processes[J]. Neuroscience Bul letin,2007,23(2):125-130.
[30]袁红,梁立武,刘平,等.氧化应激与帕金森病多巴胺神经元死亡[J].武警医学,2007,18(6)：448-450.
[31]Przedborski S, Ischiropoulos H. Reactive oxygen and nitrogen species:weapons of neuronal destruction in models of Parkinsons disease [J]. Antioxid Redox Signal,2005,7 (5-6):685-693.
[32]Sehapira AH, Cooper JM, Dexter D, et al. Mitochondrial complex I deficiency in Parkinson's disease[J]. Lancet,1989,1(8649):1269.
[33]解洪荣,张玉花,李雪松,等.MPTP/MPP诱导的帕金森病实验模型的研究进展[J].医学综述,2011,17(5)：669-672.
[34]Karunakaran S, Di wakar L, Saeed U, et al. Activation of apoptosis signal regulating kinasel (ASK1) and translocation of death-associated protein, Daxx, in substantia nigra pars compacta in a mouse model of Parkinson's disease:protection by alphal ipoic acid [J]. FASEB J,2007,21(9):2226-2236.
[35]刘军,肖颂华,周道友,等.6-羟多巴胺诱致大鼠早期黑质多巴胺能神经元凋亡的实验研究[J].中华老年医学杂志,2003,22(11)：681-683.
[36]曹非,骆芳,段国安.大鼠帕金森病发病过程中黑质细胞凋亡及其相关基因表达[J].中国老年学杂志,2007(27)：2369-2370.
[37]曹非,孙圣刚,童萼塘,等.Bcl-2、Bax蛋白表达与帕金森病大鼠黑质细胞凋亡相关性的实验研究[J].中华老年医学杂志,2003,22(10)：625-626.
[38]Hartmann A, Hunot S, Michel P P, et al. Caspase-3:a vulnerability factor and final effector in apoptosis death of dopaminergic neurons in Parkinson's disease[J]. Pore Natl Acad Sci USA,2000,97(16):2875-2880.
[39]Cheng E H Y, Kirsch D G, Clem R G, et al. Conversion of Bcl-2 to a Bax-like death effector by Caspase[J]. Science,1997,278(12):1966-1968.
[40]Bogdanov M, Matson WR, Wang L, et al. Metabolomic profiling to develop blood bio markers for Parkinson's disease[J]. Brain,2008,131(2):389-396.
[41]LeWitt P, Schultz L, Auinger P, et al. CSF xanthine, homovanillic acid, and their ratio as biomarkers of Parkinson's disease[J]. Brain Res,2011,1408 (11):88-97.
[42]Bogdanov M, Matson WR, Wang L, et al. Mctaboklmic profiling to develop blood biomarkers for Parkinson's disease [J]. Brain,2008,131(2):389-396.
[43]Dorsey ER, Holloway RG, Ravina BM. Biomarkers in Parkinson's disease[J]. Expert Rev Neuother,2006,6(6):823-831.
[44]Mascalchi M, Vella A, Ceravolo R. Movement disorders:role of imaging in diagnosis[J]. J Magn Reson imaging,2012,35(2):239-256.
[45]Wang J, Zuo CT, Jiang YP, et al.18F-FP-CIT PET imaging and SPM analysis of dopamine transporters in Parkinson's disease in various Hoehn&Yahr stages [J]. J Neurol,2007,254 (3):185-190.
[46]Dagher A, Nagano-Saito A. Functional and anantomical maganetic imaging in Parkinson's disese[J]. Mol Imaging Biol.2007,9 (6):234-242.
[47]Nanhagopal R, McKeown M J,Stoessl A J, et al. Functional imaging in Parkinson's disease[J]. Neurology,2008,70 (4):1478-1488.
[48]卢琦,黄建军,张兰,等.磁共振波谱分析在帕金森病早期诊断中的应用研究[J].中国CT和MRI杂志,2009,7(5)：1-4.
[49]Naruse S, Kizu 0, Furuya S. The role of magnetic resonance spec.troscopic imaging in patients with Pakinson's disease[J]. Nippon Rinsho,1997,55(1):262-264.
[50]张莉,孔岩,徐耑,等.豆状核质子磁共振波谱对帕金森病的诊断价值[J].临床荟萃,2009,24(13)：1115一1117.
[51]王大力,刘昊,王坚,等.质子磁共振波谱对帕金森病的诊断价值[J].临床神经病学杂志,2002,15(4)：213-214.
[52]Lu YW. Tan EK. Molecular biology changes associated with LRRK2 mutations in Parkins on's disease [J]. J Neurosci Res,2008,86(9):1895-1901.
[53]田华,田有勇,孙圣刚.帕金森病基因治疗研究现状[J].临床内科杂志,2007,24(6)：375-378.
[54]陈生弟,周海燕.帕金森病发病机制的研究进展[J].中国实用内科杂志,2006,26(4)：246-248.
[55]Duyckaerts C, Sazdovitch V, Seilhean D. Update on the pathophysiology of Parkin-son'sdisease[J]. Bull Acad Natl Med,2010,194(7):1287-1303.
[56]Ravina BM, Fagan SC, Hart MD, et al. Neuroprotective agents for clinical trials in Parkinson's disease[J]. Neurology,2003,60(8):1234.
[57]Boll MC, Alcaraz-ZubeldiaM, Rios C. Medical management of Parkinson's disease:focus on neuroprotection[J]. Curr Neuropharmacol,2011,9(2):350-359.
[58]Ybot-Gorrin I, Vivoncos-Matellano F, Chacon-Pena JR, et al, Assessment of Parkinson disease:what do we need to show neuroprotection? [J]. Nerologist,2011,17(6 Suppl 1): S21-9.
[59]陈生弟,汪锡金.帕金森病的神经保护治疗一现状与分析[J].中国神经免疫学和神经病学杂志,2010,17(3)：159-161.
[60]Li C, Guo Y, Xie W, et al. Neuroprotection of pramipexole in UPS impairment induced animal model of Parkinson's disease[J]. Neurochem Res,2010,35(10):1546-1556.
[61]Li C, Biswas S, Li X, et al. Novel D3 dopamine receptor-preferring Agonist D-264: Evidence of neuroprotective property.in Parkinson's disease animal models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and lactacystin[J]. J Neurosci Res,2010, 88(11):2513-2523.
[62]Olanow CW, Stern MB, Sethi K. The scientific and clinical basis for the treatment of Parkinson's disease [J]. Neurology,2009,72(suppl 4):S1-S136.
[63]Rascol 0, Dubois B, Caldas AC, et al. Early piribedil monotherapy of Parkinson's disease:a planned seven-month report of the REGAIN study[J]. Mov Disord,2008,21(12): 2110-2115.
[64]曲颖,罗晓光,任艳.普拉克索治疗早期帕金森病疗效分析[J].中国误诊学杂志,2009,9(13)：3084-3085.
[65]胥巧凤.盐酸思来吉兰治疗帕金森病患者疗效观察[J].现代医药卫生,2008,24(14)：2151.
[66]Chetsawang B, Kooncumchoo P, Govitrapong P, et al. 1-Methyl-4-phenyl-pyridiniumion-induced oxidative stress, c-Jun phosphorylation and DNA fragmentation factor-45 cleav age in SK-N-SH cells are averted by selegiline[J]. Neurochem Int,2008,53(6-8):283-288.
[67]BIGLAN KM, SCHWID S, EBERLY S, et al. Rasagiline Improves Quality of Life In Patients with Parkinson's Disease [J]. Mov Disord(S0885-3185),2008,21(5):616-623.
[68]Reichmann H, Emre M. Optimizing levodopa therapy to treat wearing-off symptoms in Parkinson's disease:focus on levodopa/carbidopa/entacapone[J]. Expert Rev Neurother, 2012,12(2):119-131.
[69]Lei Z, Jiang Y, Li T, et al. Signaling of glial cell line-derived Neurotrophic factor and its receptor GFR α 1 induce Nurrl and Pitx3 to promote survival of grafted midbrain-derived neural stem cells in a rat model of Parkinson disease [J]. J Neuropathol Exp Neur ol,2011,70(9):736-747.
[70]Sullivan AM, Toulouse A. Neurotrophic factors for the treatment of Parkinson's disea se[J]. Cytokine Growth Factor Rev,2011,2(3):157-165.
[71]Beck KD, Valverde J, Alexi T, et al. Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the adult rat[J]. Nature,1995,373(6512) :339-341.
[72]Furukawa K, Ohmi Y, Ohkawa Y, et al. Regulatory mechanisms of nervous systems with glycosphingolipids[J]. Neurochem Res,2011,36(9):1578-1586.
[73]Zhang H, Ding J, Tian W, et al. Ganglioside GM1 binding the N-terminus of amyloid precursor protein[J]. Neurobiol Aging,2009,30(8):1245-1253.
[74]CHOI J S, KIM J A, J00 CK. Activation of MAPK and CREB by GM1 induces survival of RGCs in the axotomized nerve[J]. Invest Ophthalmol Vis Sci,2003,44(4):1747-1752.
[75]Lindvall 0, Bjorklund A. Cell therapeutics in Parkinson's disease [J]. Neurotherapeu tics,2011,8(4):539-548.
[76]李海斌,石炳毅.帕金森病的干细胞治疗进展[J].中国组织工程研究与临床康复,2009,13(1)：173-176.
[77]Espay AJ, Fasano A, Morgante F. The six gaps in the search of neuroprotection for Parkinson's disease[J].Expert Rev Neurother,2012,12(2):111-113.
[78]张华,雒晓东.中医六经厥阴辨证治疗早中期帕金森病的临床研究[J].中医药学报,2008,36(6)：34-36.
[79]李军艳,杨明会,赵冠英.试论肾虚血瘀是帕金森病的基本病机[J].中华中医药杂志, 2008,23(9)：768-771.
[80]杨明会,窦永起,刘毅.抑颤汤对帕金森病模型大鼠行为学及脑黑质细胞形态学的作用[J].中国中西医结合杂志,2002,22(11)：841-843.
[81]何建成.帕金森病中医治疗的再思考—兼谈“滋肾平肝,化痰活血,解毒散结是帕金森病的基本治疗法则”[J].中医药通报,2005,4(1)：12-14.
[82]陈晓春,周宜灿,陈滢,等.人参皂甙Rgl保护多巴胺能神经元的实验研究[J].医学研究通讯,2005,34(6)：28.
[83]陈滢,陈晓春.Bc1-2家族是人参皂甙Rgl抗黑质神经元凋亡的重要调控蛋白[J].解剖学报,2002,33(5)：496-499.
[84]Li N, Liu B, Du Z D, et al. Protective effects of ginsenoside Rg2 against glutamate induced neurotoxicity in PC 12 cells[J].J Ethnopharmacol,2007,111(3):458.
[85]Bo J, Ming BY, Gang LZ, et al. Protection by puerarin against MPP+-induced neuro-toxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase--3-like Activation[J]. Neurosci Res,2005,53(2):183.
[86]李雪莉,孙圣刚.葛根素对帕金森病保护作用的实验研究[J].脑与神经疾病杂志,2002,10(1)：7-9.
[87]蒲小平,李晓蓉,李慧浓,等.肉苁蓉成分campneosidell对神经毒素MPP+诱发细胞凋亡的保护作用[J].北京大学学报(医学版),2001,33(3)：217-220.
[88]曹非,孙圣刚,曹学兵,等.银杏提取物防止帕金森病大鼠黑质细胞损伤的实验研究[J].中国老年学杂志,2002,22(5)：217.
[89]奚淑芳,陈建宗,王百忍,等.银杏叶提取物对MPP+诱导的中脑多巴胺能神经细胞损伤的保护作用[J].第四军医大学学报,2006,27(1)：73-76.
[90]李永梅,李霞,周兆丽.何首乌提取物对百草枯引起的小鼠多巴胺能神经元损伤的保护作用[J].中国临床药理学杂志,2007,23(5)：395-396.
[91]黄凌,朱毅,邝少轶.益智仁挥发油抗帕金森模型小鼠黑质神经元凋亡的作用研究[J].中国药房,2011,22(47)：4430-4433.
[92]吕娥,吕宁宁,付文玉,等.益精养血方对帕金森病大鼠多巴胺能神经元凋亡的影响[J].解剖学杂志,2007,30(5)：589-592.
[93]杨明全,窦永起,刘毅,等.抑颤汤对PD大鼠行为学及脑黑质细胞形态学的作用[J].中国中西医结合杂志,2002,22(11)：84.
[94]孟海伟,刘树伟,许继平,等.中药颤复宁血清对6-羟基多巴诱导的PCI2细胞凋亡的抑制作用[J].山东大学学报(医学版),2005,43(10)：916-919.
[95]李海龙,栗艳,厚荣荣,等.天麻素对百草枯和代森锰诱导的小鼠脑黑质多巴胺能神经元损伤的保护作用[J].成都中医药大学学报,2010,33(1)：57-59.
[96]巴晓红,刘英.银杏叶提取物及银杏总内酯对帕金森病大鼠黑质神经元损伤的保护作用[J]. 中国临床康复,2006,10(11)：39-41.
[97]何建成,袁灿兴,李亚明,等.滋补肝。肾通络解毒中药对PD大鼠模型氧化应激反应的影响[J].中国新药与临床,2003,22(3)：160.
[98]陆征宇,赵虹,汪涛,等.补肾养肝熄风方药对帕金森病小鼠纹状体抗氧化酶和细胞凋亡的影响[J].江苏中医药,2011,43(3)：85-87.
[99]鲍远程,汪瀚,张波,等.抗震止痉胶囊对PD作用机制实验研究[J].中国中西医结合杂志,2001,21(3)：193.
[100]袁崇刚,白桦,阳飞昆,等.首乌制剂对MPTP引起的小鼠PD的防治[J].华东师范大学学报(自然科学版),2002,3：95-98.
[101]陈建宗,李刚,谢永宏,等.培补肝肾中药对PD小鼠模型纹状体某些单胺类递质的影响[J].成都中医药大学学报,2002,25(4)：34.
[102]隆呈祥,邹忆怀,韩晓军,等.中药颤震平治疗老年颤证临床和实验研究[J].北京中医药大学学报,1999,22(6)：57.
[103]和欣,高誉珊,孙红梅,等.大补阴丸、牵正散及合方对帕金森小鼠脑线粒体酶复合物活性作用的实验研究[J].江苏中医药,2010,42(9)：72-73.
[104]伦学庆.兴奋性氨基酸及受体与帕金森病[J].功能性和立体定向神经外科杂志,1997,10(1)：40-42.
[105]罗瑞静,何建成.兴奋性氨基酸与帕金森病研究进展[J].医学研究杂志,2010,39(10)：13-15.
[106]马丽炎,肖培根,梁发权,等.三七总皂苷对神经细胞的保护作用[J].中国药学杂志,1998,10(3)：37.
[107]金雪红,包仕尧.粉防己碱联合还原型谷胱甘肽对帕金森病大鼠纹状体兴奋性氨基酸的影响[J].中国实用神经疾病杂志,2010,13(7)：1-3.
[108]何其华,周慧芳,薛冰,等.雷公藤单体T,o对谷氨酸所致PCI2细胞损伤的保护作用及机制研究[J].北京大学学报(医学版),2003,35(3)：252-255.
[109]袁惠莉,汪璇,张丽娟,等.中药在防治帕金森病中的作用及研究进展[J].中国药理学通报,26(7)：850-853.
[110]吴斌,赵书志,王修敏,等.中医药对帕金森病动物模型的治疗机制,中国中药杂志,2011,36(18)：2588-2591.
[111]刘国卿.神经元保护剂的研究进展[J].药学学报,2002,37(8)：657-662.
[112]Holloway RG, Shoulson I, Fahn S, et al. Pramipexole vs levodopa as initial treatment for Parkinson disease:a 4-year randomized controlled trial[J]. Arch Neurol,2004,61(9): 1044-1053.
[113]中华医学会神经病学分会运动障碍及帕金森病学组.帕金森病治疗指南[J].中华神经科杂志,2006,39(6)：409-412.
[114]张振馨.帕金森病的诊断[J].中华神经科杂志,2006,39(6)：408-409.
[115]中华全国中医学会老年医学会.中医老年颤证诊断和疗效评定标准[J].北京中医学院学报,1992,15(4)：39-40.
[116]王忆勤.中医诊断学[M].北京：中国中医药出版社,2004,135-136.
[117]中华医学会神经病学分会帕金森病及运动障碍学组,中国帕金森病治疗指南(第二版)[J].中华神经科杂志,2009,42(5)：332-335.
[118]陈海波.统一帕金森病评定量表[J].中华老年医学杂志,1999,18(1)：61-62.
[119]王志英,叶放,周学平,等.周仲瑛教授临证思辨特点概要[J].南京中医药大学学报,2007,23(1)：45.
[120]安红梅,胡兵,张学文.从肾阴虚入手证病结合治疗帕金森病思路探讨[J].中国中医急症,2004,13(2)：818-819.
[121]王文武,何建成.基于肝肾同源理论的帕金森病病因病机及治法研究[J].辽宁中医杂志,2009,36(8)：1252-1254.
[122]刘垣,陈彪.帕金森病患者中医证候调查[J].中医杂志,2009,50(8)：728-729.
[123]罗瑞静,何建成.帕金森病中医证候规范化研究[J].中华中医药学刊,2011,29(11)：2445-2447.
[124]陈婉珉,郑春叶,连新福.100例帕金森病患者中医证候要素及证型分布规律[J].中医杂志,2011,52(3)：214-217.
[125]汤湘江,雒晓东,黄培新.从《伤寒论》的六经辨证看帕金森病的中医药治疗[J].深圳中西医结合杂志,2005,15(4)：214-215.
[126]范宇鹏,曾亮,孙玉芝,等.帕病2号方治疗震颤型帕金森病的临床研究[J].天津中医药,2010,27(3)：190-191.
[127]Martinez MP, Gil LA, Gracia LM, et al. Unified Parkinson's Disease Rating Scale characteristics and structure[J].Mov Disord,1994,9(1):76-83.
[128]庄海,徐武华,邹海强.帕金森病非运动症状临床诊治的新进展[J].中华神经医学杂志,2011,10(12)：1294-1296.
[129]Tsang KL, Chi I, Ho SL, et al. Translation and validation of the standard Chinese version of PDQ-39:A quality-of-life measure for patientswith Parkinson's disease[J]. Mov Disord,2002,17(5):1036-1040.
[130]Leentjens AF. Depression in Parkinson's disease:conceptual issues and clinical challenges[J]. J Geriatr Psychiatry Neurol,2004,17(3):120-126.
[131]董青,王智樱,李焰生.帕金森病患者健康相关生活质量的影响因素研究[J].中国神经精神疾病杂志,2008,34(10)：598-600.
[132]Chaudhuri KR, Naidu Y. Early Parkinson's disease and nonmotor issues[J].J Neur ol,2008,255 (Supp15):33-38.
[133]刘文彦,刘军,丁健青,等.帕金森病患者的睡眠障碍[J].中华神经科杂志,2011,44(9)：627-629.
[134]王玉虹,徐良洲.名老中医王淑玲从肝论治失眠的经验[J].陕西中医,2008,29(10)：1356-1357.
[135]George Paxinos, Charles Waton大鼠脑立体定位图谱[M].北京：人民卫生出版社(第三版),2005,18-21.
[136]Sanchez-lglesias S, Rey P, Mendez-Alvarez E, et al. Time course of brain oxidative damage caused by in trastriatal administration of 6-hydroxydopamine in a rat model of Parkinson's disease[J]. Neurochem Ras,2007,32(1):99-105.
[137]王尧,杜子威.神经生物化学与分子生物学[M].北京：人民卫生出版社,1997,131-132.
[138]David B, Sakina T, Nathalie L, et al. Molecular pathway involved in the neurotoxic ity of 6-OHDA, dopamine and MPTP:contribution to the apoptotic theory in Parkinson's disease[J]. Prog Neurobiol,2001,65(11):35.
[139]Thiele SL, Warre R, Nash JE. Development of a Unilaterally-lesioned 6-OHDA Model of Parkinson's Disease[J]. J Vis Exp.2012,60(14):3234.
[140]Valastro B, Dekundy A, Danysz W, et al. Oral creatin supplementation attenuates L-DOPA induced dyskinesia in 6-hydroxydopamine-lesioned rats [J]. Behav Brain Res,2009, 197(1):90-96.
[141]Hudson JL, Van Horne CG, Stromberg I, et al. Correlation of apomarphine and amphe tamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopa mine lesioned rats[J]. Brain Res,1993,626(12):167.
[142]Iancu R, Mohapel P,Brundin P,et al. Behavioral characterization of a unilateral 6-OHDA-lesion model of Parkinson's disease in mice [J]. Behav Brain Res,2005,162 (1): 1-10.
[143]唐涌.细胞凋亡在帕金森病发病中的作用[J].徐州医学院学报,2004,24(1)：87-90.
[144]He Y, Lee T, Leong SK.6-hydroxydopamine induced apoptosis of dopaminergic cells in the rat substantia nigra[J].Brain Res,2000,858(21):163-166.
[145]Migheli A, Caralla P, Marino S, et al.A study of apoptosis in normal and pathologic nerous tissue after in situ end-labeling of DNA strand breaks[J]. J Neuropath Exp Neurol, 1994,53(8):606-616.
[146]HAAVIK J, TOSKA K. Tyrosine hydroxylase and Parkinson's disease[J]. Mol Neurobiol, 1998,16(3):285-309.
[147]曹非,骆芳,段国安.左旋多巴加速帕金森病大鼠黑质细胞凋亡的实验研究[J].临床神经电生理学杂志,2007,16(2)：67-70.
[148]朱美娥,舒丹,陈江帆,等.川芎嗪对MPTP所致小鼠多巴胺能神经元损伤的保护作用及机制 [J].中风与神经疾病杂志,2009,26(3)：333-335.
[149]盛阳,张捷,熊玉波,等.当归注射液对6-羟基多巴胺诱导PC12：细胞帕金森病模型SIRT1表达及细胞凋亡的影响[J].武汉大学学报(医学版),2011,32(4)：436-439.
[150]苗明三,方晓艳.制何首乌多糖对衰老模型小鼠抗氧化作用的研究[J].中药药理与临床,2002,18(5)：23-24.
[151]李吴,杜小平,杨期东,等.何首乌对海人藻酸致大鼠脑AChE神经元及纤维损伤的保护作用[J].卒中与神经疾病,2002,9：299-302.
[152]邓汝东,李伊为,陈东风,等.龟板抗帕金森病大鼠多巴胺能神经元凋亡[J].神经解剖学杂志,2008,24(3)：301-306.
[153]Chun HS, Gibson GE, DeGiorgio LA, et al. Dopaminergic cell death induced by MPP, oxidant and specifiec neurotoxins shares the common molecular mechanism[J]. Jounal of neurochemistry,2001,76(40):1010-1021.
[154]Przedborski S, Ischiropoulos H. Reactive oxygen and nitrogen species:weapons of neuronal destruction in models of Parkinsons disease[J]. Antioxid Redox Signal,2005, 7(5-6):685-693.
[155]Mazzio E, Remas RR, Soliman KF, et al. The role of oxidative stress, impaired glycolysis and mitochondrial respiratory redox failure in the cytotoxic effects of 6-hydroxydpomaine in virto. Brain Research[J].2004,1004(1-2):29-44.
[156]孙晓芳,王丹巧,吴兆恩,等.首乌方对帕金森病模型大鼠血液和纹状体细胞外液左旋多巴药代动力学影响的研究[J].中国实验方剂学杂志,2011,17(11)：114-115
[157]张飞,李劲松.乌梅的研究进展[J].海峡药学,2006,18(4)：21-23.
[158]刘玮,吴华璞,祝晓光,等.白芍总苷对全脑缺血再灌损伤的保护作用[J].中国药理学通报,2004,20(2)：211-213.
[159]孙晓芳,王巍,王丹巧,等.天麻及其制剂神经保护机制的研究进展[J].中国中药杂志,2004,29(4)：292.
[160]周宜灿,陈晓春,朱元贵,等.人参皂苷Rgl可能通过抗氧化作用来保护帕金森病鼠黑质神经元[J].中国临床药理学与治疗学,2003,8(3)：273-277.