| 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.
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