左旋多巴对帕金森病大鼠毒性作用的实验研究

目的　研究左旋多巴 (L dopa)对帕金森病 (PD)模型大鼠异常行为、黑质抗氧化系统、线粒体呼吸链功能和神经递质代谢的影响及其机制。方法　应用 6 羟基多巴胺 (6 OHDA)立体定向注射制作PD大鼠模型 ,给PD大鼠L dopa 2 5mg/ (kg·d)灌胃 ,共 4 5d。给药前后分别进行行为学测试 ,给药后测定黑质区谷胱甘肽过氧化物酶 (GSH Px)、丙二醛 (MDA)、活性氧 (ROS)及线粒体呼吸链酶复合体Ⅰ水平 ,测定尾状核头部多巴胺 (DA)、高香草酸 (HVA)、单胺氧化酶 B(MAO B)的水平。结果　 (1)L dopa组大鼠旋转速度给药前为(13.1± 1.5 )r/min ,给药后为 (7.2± 1.6 )r/min,给药前后比较差异有显著性 (P <0 .0 1) ;(2 )L dopa组GSH Px活性、呼吸链酶复合体Ⅰ水平降低 ,MDA含量、ROS活性升高 ,与对照组比较差异均有显著性 (均P <0 .0 1) ;(3)L dopa组MAO B活性、DA、HVA含量及DA/HVA比值与对照组比较均显著升高 (P <0 .0 5～ 0 .0 1)。结论L dopa能有效改善PD大鼠的旋转行为 ,但可加重黑质区氧化应激损伤 ,抑制线粒体呼吸链酶活性。     

还原型谷胱甘肽保护性治疗帕金森病的机制研究

目的观察还原型谷胱甘肽(GSH)对帕金森病(PD)大鼠模型的异常行为、黑质抗氧化系统、线粒体呼吸链功能及细胞形态学的影响。方法应用6-羟基多巴胺立体定向注射制作PD大鼠模型。将24只大鼠随机分为3组(每组8只):模型组、GSH组、假手术组,分别给予相应处理,共45d,给药前后均进行行为学测试,给药后测定各组大鼠黑质区谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、活性氧(ROS)及线粒体呼吸链酶复合体水平,并行免疫组化检测。结果(1)GSH对大鼠旋转行为无明显影响(P>0.05)。(2)GSH能减轻黑质区氧化应激损伤。(3)GSH能增强黑质呼吸链酶复合体I活性。(4)免疫组化发现GSH组TH-IR神经元数量较模型组明显增多。结论GSH能减轻PD模型大鼠黑质区氧化应激损伤,并对线粒体呼吸链及细胞形态具有一定保护作用。     

还原型谷胱甘肽对帕金森病保护性治疗的实验研究

目的观察还原型谷胱甘肽对帕金森病(PD)大鼠模型的保护性治疗作用,并探讨其作用机理.方法应用6-羟基多巴制作PD大鼠模型.将大鼠模型随机分为4组(每组8只):模型组,谷胱甘肽(GSH)组,左旋多巴(Ldopa)组,谷胱甘肽 左旋多巴组,另设对照组(8只),分别给予相应处理,共45天,给药前后均进行行为学测试,给药结束后行免疫组化和电镜观察,并测定黑质区谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、活性氧(ROS)及线粒体呼吸链酶复合体Ⅰ水平.结果 (1)GSH对其旋转行为无明显影响(P＞0.05);(2)GSH 能减轻黑质区氧化应激损伤;(3)GSH能增强黑质呼吸链酶复合体Ⅰ活性;(4)免疫组化发现GSH组TH-IR神经元较模型组明显增多(P＜0.001);(5)电镜下发现GSH可部分延缓凋亡进程.结论 (1)GSH能减轻黑质区氧化应激损伤,并对线粒体呼吸链具有一定保护作用;(2)GSH与L-dopa合用既可有效改善症状,又对残存黑质多巴胺能神经元具有保护性治疗作用.     

还原型谷胱甘肽对黑质多巴胺能细胞保护作用的实验研究

目的　观察还原型谷胱甘肽 (GSH)对帕金森病 (PD)大鼠模型的保护性治疗作用 ,并探讨其作用机制。方法　应用 6 羟基多巴制作PD大鼠模型 ,随机分为 4组 :模型组 ,GSH组 ,左旋多巴 (L dopa)组 ,GSH并L dopa组 ,并设正常组 ,给药结束后行电镜观察 ,并测定各组其余大鼠黑质区谷胱甘肽过氧化物酶 (GSH Px)、丙二醛 (MDA)、活性氧 (ROS)及线粒体呼吸链酶复合体Ⅰ水平 ,测定尾状核头部多巴胺 (DA)、高香草酸 (HVA)及单胺氧化酶 B(MAO B)水平。结果　①GSH组旋转行为无明显变化 ,但GSH并L dopa组可使大鼠旋转行为明显改善 ;②GSH能减轻黑质区氧化应激损伤 ;③GSH能增强黑质呼吸链酶复合体Ⅰ活性 ;④GSH组对MAO B活性及DA、HVA含量和DA/HVA比值均无明显影响 ;⑤电镜下发现模型组存在大量中、晚期凋亡细胞 ,而GSH组以早、中期凋亡为主。结论　①GSH能减轻PD模型大鼠黑质区氧化应激损伤 ,并对线粒体呼吸链具有一定保护作用 ;②GSH与L dopa合用 ,既可有效改善症状 ,又对残存DA能神经元具有保护性治疗作用。     

还原型谷胱甘肽拮抗6-OHDA对骨髓基质细胞的毒性作用

目的:探讨6-羟基多巴胺(6-OHDA)对骨髓基质细胞(BMSCs)的毒性作用及还原型谷胱甘肽(GSH)对其的拮抗作用。方法:取体重60～90g的SD大鼠股骨、胫骨及肱骨BMSCs,体外培养至传3代。采用MTT法检测不同剂量6-OHDA对BMSCs的毒性作用,相同条件下同时检测GSH的细胞保护作用。结果:MTT显示GSH可使传3代BMSCs活性升高(P<0.05);6-OHDA可使传3代BMSCs的活性下降(P<0.05);GSH干预能提高6-OHDA作用后的传3代BMSCs的活性(P<0.05)。结论:一定剂量的6-OHDA对BMSCs具有毒性作用,GSH能够明显拮抗6-OHDA对BMSCs的毒性作用。     

还原型谷胱甘肽合用粉防己碱对帕金森病大鼠抗氧化效应的研究

目的:观察还原型谷胱甘肽合用粉防己碱(Tet)对帕金森病(PD)大鼠抗氧化效应的影响。方法:应用定向注射6-羟基多巴胺法制作(PD)大鼠模型,将成功模型分4组给予不同药物50d,观察大鼠旋转行为,测定黑质谷胱甘肽(GSH)和纹状体丙二醛、活性氧水平。结果:GSH合用Tet能:①显著改善模型大鼠旋转行为;②减轻氧化应激损伤,纹状体丙二醛、活性氧水平显著降低;③显著增加黑质区GSH含量。结论:Tet能增强GSH对PD大鼠的抗氧化能力。     

还原型谷胱甘肽治疗帕金森病的临床研究

目的:评价还原型谷胱甘肽治疗帕金森病的疗效。方法:13例早期帕金森病组和6例晚期帕金森病组,每例患者给予还原型谷胱甘肽1200～1800 mg,静脉滴注,1次/日,共21d。治疗前和治疗后第1、2、3、4个月末进行UPDRS评分以观察其疗效。结果:早期帕金森病患者组精神活动、行为和精神评分在治疗后第1、2、3个月较治疗前有显著性降低。日常生活活动评分和运动检查评分第1、2、3、4个月较治疗前有显著性降低。晚期帕金森病组UPDRS评分在治疗后无明显改善。结论:还原型谷胱甘肽能够暂时改善早期帕金森病患者病情。     

还原型谷胱甘肽治疗帕金森病的临床研究

目的探讨还原型谷胱甘肽(GSH)治疗帕金森病(PD)患者的临床疗效。方法 63例帕金森病患者随机分为2组,治疗组32例,对照组31例,均予抗PD药物治疗,其中治疗组加用0.9%氯化钠注射液100 mL+还原型谷胱甘肽1.2～2.4 g静滴,1次/d,共21 d。治疗前后分别采用PD统一评分量表(UPDRS)、Hoehn-Yahr分级量表进行临床评价,行血清谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)测定。结果与对照组比较,GSH组UPDRS评分、Hoehn-Yahr分级有显著改善(P0.05或0.01),GSH-Px、SOD值较治疗前明显升高(P0.01),而对照组改变不明显。结论 GSH治疗帕金森病疗效显著。     

粉防己碱联合谷胱甘肽对帕金森病大鼠的治疗作用

目的探讨粉防己碱(Tet)联合还原型谷胱甘肽(GSH)对帕金森病(PD)大鼠的治疗作用。方法应用定向注射6-羟基多巴胺制作PD大鼠模型;将成功模型随机分为PD组、GSH组、左旋多巴(L-dopa)组、GSH Tet L-dopa组、GSH Tet组,并给予相应药物腹腔注射治疗;给药后对各组大鼠进行阿朴吗啡旋转试验、脑组织黑质谷胱甘肽、纹状体单胺氧化酶B(MAO-B)含量测定;免疫组化法观察脑组织多巴胺能神经元数目;RT-PCR技术检测酪氨酸羟化酶(TH)mRNA含量。结果(1)L-dopa组、GSH Tet L-dopa组、GSH Tet组阿朴吗啡试验旋转圈数较治疗前明显减少(P<0.05～0.01);(2)GSH Tet组脑组织GSH含量明显高于其他治疗组,MAO-B含量明显低于其他治疗组(均P<0.05);(3)GSH Tet组脑组织TH阳性神经元数目显著多于其他各治疗组(均P<0.05);(4)GSH Tet L-dopa组脑组织THmRNA含量高于其他治疗组及PD组(P<0.05～0.01)。结论Tet联合GSH能增强对PD大鼠的治疗作用。     

左旋多巴和多巴胺对中脑原代细胞的毒性作用

目的：观察左旋多巴和DA对中脑原代培养细胞的毒性作用。方法：采用大鼠胚胎中脑原代细胞培养法，运用TH免疫荧光染色和[^3H]DA摄取率检测DA能神经元的存活数和功能；GFAP免疫荧光染色检测星形胶质细胞的存活数；以及MTT检测非DA能神经元的存活数。结果：左旋多巴或DA处理后的TH阳性和GFAP阳性细胞数以及细胞存活率均显著低于加药前基数，且呈剂量依赖性；同时残存细胞体积变小，突起减少，变短或断裂。TH阳性细胞和GFAP阳性细胞比非DA能神经元更易受损。结论：左旋多巴和DA对中脑原代细胞培养中的DA能神经元和非DA能神经元均有毒性作用。     

Glutathione depletion in rat brain does not cause nigrostriatal pathway degeneration

Summary Nigral cell death in Parkinson's disease (PD) may involve oxidative stress and mitochondrial dysfunction initiated by a decrease in reduced glutathione (GSH) levels in substantia nigra. L-buthionine-(S,R)-sulphoximine (BSO; 4.8 and 9.6 mg/kg/day), an irreversible inhibitor of -glutamyl cysteine synthetase, was chronically infused into the left lateral ventricle of rats over a period of 28 days and markedly reduced GSH concentrations in substantia nigra (approx. 59% and 65% in 4.8 and 9.6 mg/kg/d BSO respectively) and the striatum (approx. 63% and 80% in 4.8 and 9.6 mg/kg/d BSO respectively). However, the number of tyrosine hydroxylase (TH)-positive cells in substantia nigra was not altered by BSO-treatment compared to control animals. Similarly, there was no difference in specific [³H]-mazindol binding in the striatum and nucleus accumbens of BSO-treated rats compared to control rats. In conclusion, depletion of GSH following chronic administration of BSO in the rat brain does not cause damage to the nigrostriatal pathway and suggests that loss of GSH alone is not responsible for nigrostriatal damage in PD. Rather, GSH depletion may enhance the susceptibility of substantia nigra to destruction by endogenous or exogenous toxins.     

Alterations in the distribution of glutathione in the substantia nigra in Parkinson's disease

Summary Depletion of reduced glutathione occurs in the substantia nigra in Parkinson's disease and in incidental Lewy body disease (presymptomatic Parkinson's disease) which may implicate oxidative stress in the neurode-generative process. In this study mercury orange fluorescent staining and immunostaining with an antibody to reduced glutathione have been used to determine the distribution of reduced glutathione in the substantia nigra in Parkinson's disease compared with normal individuals.Mercury orange staining showed moderate background levels of fluorescence in the neuropil in both control and Parkinson's disease substantia nigra and localised reduced glutathione to the somata of melanized nigral neurons and glial elements of the neuropil. Neuronal nuclei revealed a relative lack of fluorescence after mercury orange staining. There was a significant depletion of reduced glutathione in surviving neurons in Parkinson's disease compared to nerve cell populations in control tissue. Mercury orange fluorescence indicated a high concentration of reduced glutathione in a subpopulation of non-neuronal cells, most likely astrocytes or microglia.Immunohistochemical examination of nigral tissue from the same Parkinson's disease and control patients with an antibody to glutathione showed staining in neuronal perikarya and axonodendritic processes of melanized nigral neurons which was generally most intense in control neurons. Moderately intense staining of the background neuropil, most prominent in control nigras, and staining of capillary walls was also detected. Intense staining was seen in cells with the morphological features of glial cells in both control and PD nigra.These data show a significant presence of reduced glutathione in the cell bodies and axons of nigral neurons. They are in agreement with biochemical studies showing depletion of reduced glutathione in substantia nigra in Parkinson's disease, and indicate a significant loss of neuronal reduced glutathione in surviving nigral neurons in Parkinson's disease.Abbreviations GFAP glial fibrillary acidic protein - GSH reduced glutathione - GSSG oxidized glutathione - ILBD incidental Lewy body disease - PBS phosphate-buffered saline - PD Parkinson's disease     

Oxidative stress as a cause of Parkinson's disease

The cause of dopamine cell death in Parkinson's disease remains unknown. Present interest centres on the possible involvement of a toxin mediated mechanism such as that produced, by MPTP. In post-mortem studies there is evidence in the substantia nigra for an on-going toxic process involving increased lipid peroxidation, altered iron metabolism and impairment of mitochondrial function at the level of complex I. Although the precise realtionship between these biochemical changes is not known, present evidence points to oxidative stress as an important factor contributing to neuronal loss. Altered mitochondrial function and increased iron levels may not initiate Parkinson's disease but rather act to accelerate cell death. Future strategies for the treatment of Parkinson's disease should be aimed at preventing oxidative stress and stopping or slowing the progression of the underlying pathology.     

Glutathione peroxidase 1 and glutathione are required to protect mouse astrocytes from iron-mediated hydrogen peroxide toxicity

The enzyme glutathione peroxidase 1 (GPx1) is involved in the cellular detoxification of peroxides. To test for the consequences of GPx deficiency in astrocytes, astrocyte-rich primary cultures from wild-type and GPx1-deficient [GPx1(-/-)] mice were exposed to H(2)O(2). In GPx1(-/-) astrocytes, the clearance rate of H(2)O(2) was slower than in wild-type cells. In contrast to GPx1-deficient astrocytes, wild-type cells exhibited, within 2 min of H(2)O(2) application, a rapid and transient accumulation of cellular glutathione disulfide that amounted to 60% of total glutathione. The peroxide treatment did not affect the viability of wild-type astrocytes, whereas 45% of the GPx1(-/-) cells died within 8 hr. However, the viability of both types of astrocytes was strongly compromised by lowering cellular glutathione content before peroxide application. In contrast, inactivation of catalase caused substantial cell death only in GPx1(-/-) cells but not in wild-type astrocytes. The cell death observed was prevented by the iron chelators deferoxamine, 1,10-phenathroline, or 2,2'-dipyridyl, whereas preincubation with ferric ammonium citrate increased the toxicity of peroxide treatments. These results demonstrate that GPx1 contributes to the rapid clearance of H(2)O(2) by mouse astrocytes and that both GPx1 and a high concentration of glutathione are required to protect these cells from iron-dependent peroxide damage.     

基因治疗帕金森病的实验研究

本研究把Ⅰ型人酪氨酸羟化酶(human tyrosine hydroxylase type Ⅰ;HTH_1)cDNA连接到逆转录病毒载体LNSX上,构建成真核表达载体 LNSHTH_1,然后通过in vivo途径转染帕金森病模型鼠的纹状体细胞。外源的HTH_1基因在宿主脑内得到了表达,并使其病理行为改善约70％。这种新的基因治疗帕金森病的方式,有其独特的优越性。     

COMT-inhibition increases serum levels of dihydroxyphenylacetic acid (DOPAC) in patients with advanced Parkinson's disease

Summary. Inhibition of the catechol-O-methyltransferase (COMT) is an effective treatment for end-of-dose fluctuations in advanced Parkinson's disease. The aim of the present investigation was to analyse the consequences of subsequent alterations in levodopa metabolism under common treat-ment conditions when the levodopa dose is adjusted due to the occurrence of dyskinesias after initiation of the COMT-inhibitor. Ten patients with advanced Parkinson's disease (Hoehn & Yahr stage IV) were medicated with tolcapone. Prior to and five to ten days after the initiation of tolca-pone 300 mg/d, serum level profiles of levodopa and its metabolites (3-O-methyldopa (3-OMD), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)) were performed. The mean daily levodopa dose was reduced from 894 ± 248 mg to 646 ± 252 mg (p = 0.003). There was a significant increase in the area under the curve (AUC) of DOPAC during COMT-inhibition compared to the baseline profile (p = 0.009). There were significant decreases of the AUC of HAV (p = 0.001) and the ratios of the AUC HVA / AUC DOPAC (p = 0.0001) and AUC 3-OMD / AUC levodopa (p = 0.0001). Conclusion: The elevation of DOPAC and the decrease of HVA and HVA / DOPAC reflect a shift of the levodopa metabolism towards the MAO-B dependent oxidative pathway. This might contribute to production of hydroxyl radicals and induction of oxidative stress. Received July 9, 2001; accepted September 28, 2001