培补肝肾中药治疗帕金森病的机制研究
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摘要
帕金森病(Parkinson's disease，PD)是一种常见于中老年的神经系统变性疾病，其发病率在65岁以上人群高达1％。其病理特征是中脑黑质纹状体系统多巴胺(Dopamine，DA)能神经元变性死亡，当DA能神经元死亡超过50％时，导致脑内神经递质DA明显减少，出现静止性震颤、肌僵直和运动迟缓等临床表现。其病因不甚明确，可能与遗传、环境、老龄等因素有关。目前认为自由基生成、氧化反应增强、谷胱甘肽含量降低以及线粒体功能异常、内源性和外源性毒物是可能导致黑质DA能神经细胞变性的原因。该病的内科治疗以左旋多巴制剂为主，疗效较好，但这种制剂的长期应用，会出现明显的疗效减退及较严重的副作用，包括运动症状波动，开关现象等。临床以培补肝肾中药为主治疗PD有较好的疗效，本研究旨在从抗氧化作用和抗黑质细胞凋亡两方面来探讨培补肝肾中药为主治疗PD可能的作用机制。本研究拟通过动物实验研究证实：1．培补肝肾复方对PD小鼠模型黑质组织匀浆活性氧、MDA、GSH、GSH-Px、和SOD等的影响；2．培补肝肾复方及肉苁蓉对PC12细胞活性和细胞凋亡的影响。3．MPP+作用于体外培养的正常SD胎鼠黑质DA能神经元细胞，建立细胞凋亡模型，并观察培补肝肾中药肉苁蓉对DA能神经元的活性和凋亡的影响；实验内容如下：
实验一：目的观察MPTP对PD小鼠模型黑质抗氧化系统的作用及培补肝
第四军医人学硕卜学位论文
肾复方对其影响。方法采用MPTP腹腔注射小鼠造成PD模型，并用培补肝
肾中药进行治疗，同时设立正常对照组、模型组、治疗组共3组，各组分
别为10、20、20只。观察培补肝肾复方对氧化应激的影响。结果模型组
活性氧和丙二醛(MDA)分别为403士7 IU .mg一，，10.3士3.2 nmol .mg一，与
培补肝肾组(201士34)U .mg一，和(5士2) nmol .mg一，相比较明显降低，统
计学差异显著(P(0.05);GSH、GSH一Px、和SOD含量模型组为(4.6士1.3)
mg .9一，，(2.4士0.3)nU .9一，，(136士5)nU .9一，，与中药治疗组相比(6.4
士2.3)mg .9一，，(3.2士0.3) nU .9一‘和(168士8) nU .mg一，明显增高，统
计学差异显著(P<0 .05)。结论培补肝肾中药可能通过增加MPTP诱导的PD
模型小鼠的抗氧化能力，清除自由基，而减低自由基对黑质的损伤。
实验二:目的探讨培补肝肾复方对MPP‘所致PC12细胞损伤模型的保护作
用。方法采用血清药理学的方法，以肾上腺嗜铬细胞瘤克隆化细胞株PC12
细胞为材料，制备中药含药血清，利用M尸P+建立经神经生长因子(NG曰诱导
前后的PC12细胞的损伤模型，观察给药不同的培补肝肾中药剂量血清对Pc12
细胞的不同作用。结果形态学检查发现等效剂量培补肝肾中药复方血清对
诱导前后的PC12细胞具有保护作用，可显著提高 PC12细胞的存活数，减低
细胞损伤程度。结论培补肝肾复方中药血清可能通过对抗淤P+引起的细胞
损伤，对体外培养的PC12细胞具有一定的保护作用。
实验三:目的观察中药肉从蓉含药血清对PC12细胞凋亡的影响。方法采
用l一甲基一4一苯基毗陡离子(1一methyl一4一phenylpyridinium，MpP+)建立
PC12细胞凋亡模型，应用血清药理学方法，结合光镜荧光染色核形分析、
透射电镜、流式细胞仪等检测手段，研究含中药兔血清对PC12细胞凋亡模
型的保护作用。结果含等效剂量组中药兔血清(灌胃剂量为肉从蓉生药
1.6眺g)、2倍等效剂量组中药兔血清(灌胃剂量为肉从蓉生药3.2叭g)‘
处理的PC12细胞经MPP+作用后，凋亡率分别为1 0.2土0.60%，2名士0.21%;
第四军医大学硕士学位论文
与空白兔血清组(18.6士0.67%)相比有显著统计学差异(尸<0.05)。结论肉
从蓉可能具有拮抗MPP+导致的细胞凋亡作用。
实验四:目的建立MPP+诱导的原代培养新生鼠中脑DA能神经细胞凋亡
模型，并探讨肉从蓉对中脑DA能神经细胞的保护作用和机制.方法采用
血清药理学的方法，肉从蓉含药血清培养DA能神经细胞，观察培补肝肾
复方含药血清对MPP+损伤DA能神经元细胞存活时间、存活率的影响、
及细胞形态学的变化。经MPP+处理后，用流式细胞仪检测，观察肉从蓉
对凋亡细胞的保护作用。结果浓度为加0阿。比的侧田P+使DA能神经细
胞发生典型的凋亡，等效剂量培补肝肾中药复方含药血清(灌胃剂量为3
眺g)可延长MPP+损伤DA神经细胞的存活时间，提高细胞的存活率。流
式细胞仪结果显示等效剂量肉从蓉含药血清(动物灌胃浓度生药1 .6glkg)
培养液组DA神经细胞凋亡率为9.80士 0.51%，空白模型组的凋亡率为24.20
士1.09%，光镜下计数每视野凋亡阳性细胞数显示:含等效剂t肉从蓉含药
血清及2倍等效剂量肉从蓉含药血清培养液组明显低于模型组，经统计学
分析差异显著(尸<0.05)。结论肉从蓉药能有效减轻MPP+对体外培养损
伤的DA能神经细胞的损害作用，其机制与抗凋亡作用有关。
PD is a neurodegenerative progressive disorder with a prevalence of 1% in the population above 65 years of age. It is characterized by degeneration and dysfunction of dopaminergic neurons in the mid-brain substantial nigra,neurons that project to the caudate putamen.the clinical symptoms are caused by the loss of dopaminergic neurons that exceed 50% of all and the consequent dysfunction of the nigrostriatal pathway. The disease is detected as a mild resting tremor of one or more limbs, muscular rigidity, postural abnormalities, and bradykinesia-akinesia. The mechanism by which dopaminergic (DA) neurons degenerate in PD is still unknown. Yet, several lines of evidence suggest that oxidative stress, excitotoxic mechanisms and altered protein catabolism participate in the cascade of events leading to neuronal degeneration in this disease. These cellular and molecular alterations ultimately lead to apoptosis. Although nearly four decades have passed since its introduction into the clinical practice , replacement
of deficient dopamine with its direct precursor, -3,4-dihydroxyphenylalanine ( L -DOPA), is still the most effective and commonly prescribed treatment for PD. However, long-term treatment with
-DOPA is associated with numerous, well-known complications that are extremely discomforting for the patient. These include "on-off" fluctuations, freezing episodes, "wearing off" and, above all, abnormal, uncontrollable movements, known as dyskinesias .we had found that pei bu gan shen herbs had effection on the parkinson's diseases patients,so we investgated the possible mechnism of pei bu gan shen herbs in the therapyment of Parkinson disease.first,we observe the effects of pei bu gan shen herbs on the content of Reactive Oxygen Species,MDA,GSH,GSH-Px,and SOD of nigra in the rat models of Parkinson's disease. Next we study the effects of pei bu gan shen herbs and Cistanche on the apoptosis of PC 12 cells;last,we investigate the effects of Cistanche on the primary culture dopaminergic neurons apoptosis cell induced by MPP+.
Experiment 1
Objective: To investigate the effects of PEI BU GAN SHEN Chinese herbs on the substantial nigra antioxidant system in the MPTP model of parkinson's rat.Methods: rat models of PD were induced by injecting MPTP into abdomen and treated with Chinese herbs in three groups: normal group,model group,and treatment group( 10 , 20 , 20 rats respectively),and the effect of the Chinese herbs on oxidation stress response was observed. Results: compared with the model group (403 71 U . mg-1. 10.3 3.2 nmol mg-1) , the active oxygen and MDA contents in herbal treatment group were obviously reduced (201 34)U - mg-1and (5 2) nmol . mg-1(P<0.01); whileGSH,GSH-Px and SOD contents of the model group (4.6 1.3) mg . g-1 , (2.4 0.3) nU . g-1 , (136 5) nU - g-1), were obviously increased, respectively (6.4 2.3') mg . g-1, (3.2 0.3)nU g-1 and (168 8) nU . mg-1 (P<0.05) after treatment of the herbs Conclusion: the -PEI B U GAN SHEN Chinese herbs can
-
enhance anti-oxidation ability and eliminate free radicals in the substantial nigra, preventing the neurons from the lesion of free radicals.
Experiment 2
Objective: To investigate the protective effects of PEI BU GAN SHEN Chinese materia medica on MPP+ injured PC12 Cell.Methods: PEI BU GAN SHEN herbs is composed of DAYUN, GOUQIZI, SHOUWU.To prepare the rabbite serum comprised of theses Chinese herbs employs the method of serum pharmacology.Then assay the Chinese materia medica effect on PC12 Cell from MPP+ injured models, by using morphological, MTT staining and LDH measurement. Results: the equivalent dosage of the serum of PEI BU GAN SHEN herbs possessed obvious protective effects on PC 12 Cell, the serum of PEI BU GA SHEN herbs could increase the number of life cells and decrease LDH activity significantly.Conciusion: the serum of PEI B U GAN SHEN herbs protected PC 12 Cells from injured models in vitro by MPP+. Experiment 3
Objective: To investigate the effect of Cistanche on MPP+-induced PC 12 Cells apoptosis. Methods: PC12 cells w
实验一：目的观察MPTP对PD小鼠模型黑质抗氧化系统的作用及培补肝
第四军医人学硕卜学位论文
肾复方对其影响。方法采用MPTP腹腔注射小鼠造成PD模型，并用培补肝
肾中药进行治疗，同时设立正常对照组、模型组、治疗组共3组，各组分
别为10、20、20只。观察培补肝肾复方对氧化应激的影响。结果模型组
活性氧和丙二醛(MDA)分别为403士7 IU .mg一，，10.3士3.2 nmol .mg一，与
培补肝肾组(201士34)U .mg一，和(5士2) nmol .mg一，相比较明显降低，统
计学差异显著(P(0.05);GSH、GSH一Px、和SOD含量模型组为(4.6士1.3)
mg .9一，，(2.4士0.3)nU .9一，，(136士5)nU .9一，，与中药治疗组相比(6.4
士2.3)mg .9一，，(3.2士0.3) nU .9一‘和(168士8) nU .mg一，明显增高，统
计学差异显著(P<0 .05)。结论培补肝肾中药可能通过增加MPTP诱导的PD
模型小鼠的抗氧化能力，清除自由基，而减低自由基对黑质的损伤。
实验二:目的探讨培补肝肾复方对MPP‘所致PC12细胞损伤模型的保护作
用。方法采用血清药理学的方法，以肾上腺嗜铬细胞瘤克隆化细胞株PC12
细胞为材料，制备中药含药血清，利用M尸P+建立经神经生长因子(NG曰诱导
前后的PC12细胞的损伤模型，观察给药不同的培补肝肾中药剂量血清对Pc12
细胞的不同作用。结果形态学检查发现等效剂量培补肝肾中药复方血清对
诱导前后的PC12细胞具有保护作用，可显著提高 PC12细胞的存活数，减低
细胞损伤程度。结论培补肝肾复方中药血清可能通过对抗淤P+引起的细胞
损伤，对体外培养的PC12细胞具有一定的保护作用。
实验三:目的观察中药肉从蓉含药血清对PC12细胞凋亡的影响。方法采
用l一甲基一4一苯基毗陡离子(1一methyl一4一phenylpyridinium，MpP+)建立
PC12细胞凋亡模型，应用血清药理学方法，结合光镜荧光染色核形分析、
透射电镜、流式细胞仪等检测手段，研究含中药兔血清对PC12细胞凋亡模
型的保护作用。结果含等效剂量组中药兔血清(灌胃剂量为肉从蓉生药
1.6眺g)、2倍等效剂量组中药兔血清(灌胃剂量为肉从蓉生药3.2叭g)‘
处理的PC12细胞经MPP+作用后，凋亡率分别为1 0.2土0.60%，2名士0.21%;
第四军医大学硕士学位论文
与空白兔血清组(18.6士0.67%)相比有显著统计学差异(尸<0.05)。结论肉
从蓉可能具有拮抗MPP+导致的细胞凋亡作用。
实验四:目的建立MPP+诱导的原代培养新生鼠中脑DA能神经细胞凋亡
模型，并探讨肉从蓉对中脑DA能神经细胞的保护作用和机制.方法采用
血清药理学的方法，肉从蓉含药血清培养DA能神经细胞，观察培补肝肾
复方含药血清对MPP+损伤DA能神经元细胞存活时间、存活率的影响、
及细胞形态学的变化。经MPP+处理后，用流式细胞仪检测，观察肉从蓉
对凋亡细胞的保护作用。结果浓度为加0阿。比的侧田P+使DA能神经细
胞发生典型的凋亡，等效剂量培补肝肾中药复方含药血清(灌胃剂量为3
眺g)可延长MPP+损伤DA神经细胞的存活时间，提高细胞的存活率。流
式细胞仪结果显示等效剂量肉从蓉含药血清(动物灌胃浓度生药1 .6glkg)
培养液组DA神经细胞凋亡率为9.80士 0.51%，空白模型组的凋亡率为24.20
士1.09%，光镜下计数每视野凋亡阳性细胞数显示:含等效剂t肉从蓉含药
血清及2倍等效剂量肉从蓉含药血清培养液组明显低于模型组，经统计学
分析差异显著(尸<0.05)。结论肉从蓉药能有效减轻MPP+对体外培养损
伤的DA能神经细胞的损害作用，其机制与抗凋亡作用有关。
PD is a neurodegenerative progressive disorder with a prevalence of 1% in the population above 65 years of age. It is characterized by degeneration and dysfunction of dopaminergic neurons in the mid-brain substantial nigra,neurons that project to the caudate putamen.the clinical symptoms are caused by the loss of dopaminergic neurons that exceed 50% of all and the consequent dysfunction of the nigrostriatal pathway. The disease is detected as a mild resting tremor of one or more limbs, muscular rigidity, postural abnormalities, and bradykinesia-akinesia. The mechanism by which dopaminergic (DA) neurons degenerate in PD is still unknown. Yet, several lines of evidence suggest that oxidative stress, excitotoxic mechanisms and altered protein catabolism participate in the cascade of events leading to neuronal degeneration in this disease. These cellular and molecular alterations ultimately lead to apoptosis. Although nearly four decades have passed since its introduction into the clinical practice , replacement
of deficient dopamine with its direct precursor, -3,4-dihydroxyphenylalanine ( L -DOPA), is still the most effective and commonly prescribed treatment for PD. However, long-term treatment with
-DOPA is associated with numerous, well-known complications that are extremely discomforting for the patient. These include "on-off" fluctuations, freezing episodes, "wearing off" and, above all, abnormal, uncontrollable movements, known as dyskinesias .we had found that pei bu gan shen herbs had effection on the parkinson's diseases patients,so we investgated the possible mechnism of pei bu gan shen herbs in the therapyment of Parkinson disease.first,we observe the effects of pei bu gan shen herbs on the content of Reactive Oxygen Species,MDA,GSH,GSH-Px,and SOD of nigra in the rat models of Parkinson's disease. Next we study the effects of pei bu gan shen herbs and Cistanche on the apoptosis of PC 12 cells;last,we investigate the effects of Cistanche on the primary culture dopaminergic neurons apoptosis cell induced by MPP+.
Experiment 1
Objective: To investigate the effects of PEI BU GAN SHEN Chinese herbs on the substantial nigra antioxidant system in the MPTP model of parkinson's rat.Methods: rat models of PD were induced by injecting MPTP into abdomen and treated with Chinese herbs in three groups: normal group,model group,and treatment group( 10 , 20 , 20 rats respectively),and the effect of the Chinese herbs on oxidation stress response was observed. Results: compared with the model group (403 71 U . mg-1. 10.3 3.2 nmol mg-1) , the active oxygen and MDA contents in herbal treatment group were obviously reduced (201 34)U - mg-1and (5 2) nmol . mg-1(P<0.01); whileGSH,GSH-Px and SOD contents of the model group (4.6 1.3) mg . g-1 , (2.4 0.3) nU . g-1 , (136 5) nU - g-1), were obviously increased, respectively (6.4 2.3') mg . g-1, (3.2 0.3)nU g-1 and (168 8) nU . mg-1 (P<0.05) after treatment of the herbs Conclusion: the -PEI B U GAN SHEN Chinese herbs can
-
enhance anti-oxidation ability and eliminate free radicals in the substantial nigra, preventing the neurons from the lesion of free radicals.
Experiment 2
Objective: To investigate the protective effects of PEI BU GAN SHEN Chinese materia medica on MPP+ injured PC12 Cell.Methods: PEI BU GAN SHEN herbs is composed of DAYUN, GOUQIZI, SHOUWU.To prepare the rabbite serum comprised of theses Chinese herbs employs the method of serum pharmacology.Then assay the Chinese materia medica effect on PC12 Cell from MPP+ injured models, by using morphological, MTT staining and LDH measurement. Results: the equivalent dosage of the serum of PEI BU GAN SHEN herbs possessed obvious protective effects on PC 12 Cell, the serum of PEI BU GA SHEN herbs could increase the number of life cells and decrease LDH activity significantly.Conciusion: the serum of PEI B U GAN SHEN herbs protected PC 12 Cells from injured models in vitro by MPP+. Experiment 3
Objective: To investigate the effect of Cistanche on MPP+-induced PC 12 Cells apoptosis. Methods: PC12 cells w
引文
1．陶国枢．帕金森病流行特征及其预防对策．解放军保健医学杂志．2002；4(3)：145-147．
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