【作者】 文晓东；

【导师】 雒晓东；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2012， 博士

【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。更多还原

【Abstract】 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 DiseaseObject iveTo objectively evaluate the clinical efficacy of Pabing II Formula, a Chinese medicine compound, in treating Parkinson’s disease (PD) at early stage.MethodsRandomized, 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.Results1.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.ConelusionThe 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 RatsObjectiveTo explore neuroprotective effect of Pabing II Formula on the nigros triatal dopaminergic neurons of Parkinson’s disease ratsMethodsPD 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.Results1. 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.ConelusionThe 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 RatsObjectiveTo study effect of Parbin Ⅱ Formula on the oxidation stress response of PD rats. MethodsPD 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)ResultsCompared 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.ConclusionsPabing Ⅱ Formula can significantly enhance antioxidation ability and eliminate free radicals of PD rats, and its effect is in a dose dependent manner.更多还原