MPP~+诱导SH-SY5Y细胞慢性损伤帕金森病模型的建立及鹿茸多肽的保护作用
|
摘要
背景和目的帕金森病（Parkinson's disease，PD）是一种中老年常见的神经系统退行性疾病，其发病率的上升不仅严重影响患者的生活质量，而且给家庭和社会带来沉重的负担。PD的临床症状包括静态下震颤、反应迟钝、僵硬和姿势扭曲等。PD的主要病理特点是黑质致密部的多巴胺能神经元丢失，进而使纹状体内的多巴胺水平降低。
目前，PD的发病原因尚不完全清楚。各类体内和体外实4验显示，PD的发病机制涉及线粒体功能障碍、氧化应激、泛素化蛋白酶体等途径。最近的研究表明，PD发病机制还涉及未折叠蛋白聚积引起的内质网应激，即内质网应激（endoplasmic reticulum stress, ER stress）介导的细胞凋亡参与PD的发病过程。因此，深入了解PD发病机制，探寻有效的神经保护剂是目前治疗PD的重要策略。
PD发病机制的探讨和潜在治疗药物的筛选及评价，在很大程度上依赖于疾病相关体内外模型的建立及应用，特别是离体细胞模型的建立及应用。众所周知，PD是一种慢性疾病，具有缓慢、进展性的发病过程和特征，但是目前国内用于PD发病机制和药物筛选的离体细胞模型大多为急性损伤模型。我们的实践证明，采用常规急性损伤细胞模型已经不能客观反映PD缓慢、渐进性发展的规律和特征，必须建立PD稳定的慢性损伤的体外模型，以适应PD发病机制研究及探寻有效神经保护剂的需要。
基于以上的理解，本研究首先以1-甲基-4-苯基吡啶离子（1-methyl-4-phenylpyridinium, MPP~+）作为诱导剂，以SH-SY5Y细胞作为模型载体，建立PD急、慢性细胞损伤模型，确定建立急、慢性细胞模型的最佳条件。在比较急、慢性SH-SY5Y细胞损伤的规律和特点基础上，利用慢性SH-SY5Y细胞损伤模型探讨内质网应激介导的SH-SY5Y细胞凋亡机制。并在上述研究基础上观察鹿茸多肽（Velvet antler polypeptides, VAPs）对SH-SY5Y细胞凋亡的干预作用及初步机制探讨，为进一步开发抗PD的药物提供科学依据和研究思路。
方法
1.以新鲜梅花鹿茸为原材料，通过胶体磨匀浆、硫酸铵沉淀、冷冻干燥等方法制备VAPs；进一步利用SDS-聚丙烯酰胺凝胶电泳（polyacrylamide gelelectrophoresis，PAGE）、激光解吸电离飞行时间质谱和氨基酸组成分析等技术初步分析鹿茸总肽的理化性质。
2.采用MPP~+诱导人神经母细胞瘤SH-SY5Y细胞发生凋亡，设置不同浓度MPP~+分别作用24,48,72h，建立MPP~+急性损伤细胞模型；MTT法检测细胞活力，最终确定120.9μM MPP~+作用72h为建模浓度和时间。进而将培养的SH-SY5Y细胞分为空白对照组、MPP~+组和VAPs组。对照组给予细胞培养基培养，MPP~+组给予120.9μM的MPP~+处理，VAPs组给予不同浓度VAPs（62.5、125、250μg/mL）与120.9μM MPP~+同时处理。采用Hoechst33342染色检测细胞凋亡，罗丹明123染色检测线粒体膜电位（△Ψm），H2DCFDA染色检测细胞内活性氧簇（reactive oxygen species，ROS）的水平，Western blot和免疫细胞化学检测内质网分子伴侣重链结合蛋白/糖调节蛋白（Heavy-chain bindingprotein/glucose regulated protein78, Bip/GRP78）、CCAAT/增强子结合蛋白同源蛋白（C/EBP homologous protein, CCAAT/enhancer binding protein homologousprotein, CHOP）、磷酸化c-Jun氨基末端激酶/应激活化蛋白激酶（c-Jun N-terminalkinase/Stress-activated protein kinases, JNK/SAPK）、半胱氨酸天冬氨酸蛋白酶12（caspase-12）等4种蛋白的表达情况。
3.采用MPP~+诱导人神经母细胞瘤SH-SY5Y细胞发生凋亡，设置不同浓度MPP~+分别作用5,10,15天，建立MPP~+慢性损伤细胞模型；MTT法检测细胞活力。将培养的SH-SY5Y细胞分为空白对照组、MPP~+组和VAPs组。对照组给予细胞培养基培养，MPP~+组给予31.1μM的MPP~+处理，VAPs组给予不同浓度VAPs与31.1μM MPP~+同时处理。上述各组细胞均孵育5,10,15天后，采用MTT法检测细胞存活率。采用Hoechst33342染色检测细胞凋亡，罗丹明123染色检测△Ψm，H2DCFDA染色检测细胞内ROS的水平，Western blot检测GRP78、CHOP、磷酸化JNK（p-JNK）和caspase-12等蛋白表达情况。
结果
1.鹿茸总多肽（VAPs）
所得VAPs为白色粉末状，水溶性较好，蛋白质含量为54%。VAPs分子量主要分布在10kDa和20kDa区域范围内。进一步将分子量10kDa区域的多肽进行质谱分析，其分子量在3000～11000Da范围内。VAPs主要由天冬氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、苯丙氨酸、赖氨酸、精氨酸和脯氨酸组成，没有检测到半胱氨酸、酪氨酸和色氨酸。
2.急性损伤模型
⑴120.9μM的MPP~+可以诱导SH-SY5Y细胞存活率下降，凋亡率增加。125、250μg/mL的VAPs可减少细胞凋亡，以72h到达最大保护效应。MPP~+导致△Ψm降低，ROS（活性氧簇）含量增加。125、250μg/mL的VAPs拮抗MPP~+诱导产生的△Ψm和ROS的变化。
⑵MPP~+诱导caspase-12表达显著增加，不同浓度VAPs可使MPP~+诱导的Caspase-12表达有所降低。在MPP~+损伤的SH-SY5Y细胞未观察到GRP78、CHOP、p-JNK等内质网应激（ER）相关蛋白的显著表达。
综上，MPP~+致SH-SY5Y细胞损伤的主要特征为线粒体功能障碍和氧化应激介导的细胞凋亡，VAPs对该损伤有拮抗和纠正作用。在MPP~+诱导的急性损伤模型中，MPP~+可使SH-SY5Y细胞内的caspase-12水平显著提高，但没有看到其它内质网应激相关蛋白的过表达。内质网应激相关蛋白沉默表达提示，在急性损伤模型中caspase-12的激活及诱导细胞凋亡并非借助于内质网应激相关途径的启动，可能主要介导线粒体凋亡途径及氧化应激凋亡途径。
3.慢性损伤模型
（1）建立了MPP~+作用SH-SY5Y细胞5天（IC50为31.1μM）、10天（IC50为22.1μM）和15天（IC50为10.4μM）的慢性损伤模型，500μg/mL VAPs能有效提高SH-SY5Y细胞5天、10天和15天的存活率。
（2）在MPP~+慢性损伤SH-SY5Y细胞中，没有看到MPP~+急性损伤模型呈现的凋亡特征性改变和△Ψm的变化，VAPs也没有明显作用。但在MPP~+损伤5天的SH-SY5Y细胞内，ROS水平显著升高，VAPs能明显降低细胞内ROS含量。
（3）免疫细胞化学和Western blot检测均显示，MPP~+作用5天的SH-SY5Y细胞中，内质网应激相关蛋白caspase-12、GRP78、CHOP和p-JNK表达水平显著提高，VAPs与MPP~+共孵育可明显对抗caspase-12、GRP78、CHOP、p-JNK表达的上调。在MPP~+诱导10、15天的SH-SY5Y细胞中则看不到上述内质网应激相关蛋白的变化，VAPs也没有相应的作用。结果提示，MPP~+致SH-SY5Y细胞慢性损伤的主要特征为内质网应激相关蛋白的激活，是一种功能性损伤。
综上，在低浓度MPP~+（31.1μM）诱导5天的慢性损伤细胞模型中呈现出与高浓度MPP~+（120.9μM）诱导72小时急性损伤模型完全不同的损伤模式和特征性改变。研究表明，急性损伤模型是以线粒体功能障碍和氧化应激损伤介导的细胞结构性破坏，镜下看到大量凋亡细胞和细胞坏死残片。而慢性损伤模型则是以内质网应激和氧化应激介导的细胞功能性损伤，镜下没有典型的细胞凋亡和坏死。后者更接近于PD缓慢、进展性疾病的特点，是研究PD内质网应激相关机制的理想模型。鹿茸多肽对MPP~+诱发的SH-SY5Y细胞慢性损伤有保护作用，其机制可能是通过抑制内质网应激相关凋亡分子表达而实现的。
Background
Parkinson's disease (PD) is a common neurodegenerative disease in agedpopulation. The growing incidence of PD is a socio-economic problem affecting lifequality of PD patients. The syptoms of PD inclue tremor, bradykinesia, rigidity andflexed posture. Pathological features of PD implicated loss of dopaminergic neuronsin substanitial nigra, which cause the decreased dopamine level in the striata. Untilnow, the mechanism underlying PD onset is not fully understood. Most of the in vivoand ex vivo studies had illustrated that some of the mechanism involved in the PDpathogenesis, including mitochondial dysfunction, oxidative stress, ubiquitin-proteasomal pathway, etc. Recently, a misfolded protein related endoplasmicreticulum stress (ER stress) had been most intensively studied for its actions toneuronal cell death. Thus, understanding the process of ER stress is of great helpful toexplore novel neuroprotective agent for PD treatment. Clarifing the mechanisms ofPD onset and development of neuroprotective therapeutics are largely depend onestablishment of disease model, especially model in vitro. It is known that PD is achronic and progressive disease, but acute lesioned model in vitro was most commonused in domestic studies for PD. Our study indicated that the acute lesioned modelfailed to relect the chronic and progessive process of PD onset. Hence, a chroniclesioned model in vitro should be attentioned considerably in order to reveal the actualmechanism of PD and develop novel neuroprotective agents. Based on theseunderstandings,1-methyl-4-phenylpyridinium (MPP~+), a neurotoxin most commonlyused in establishment of PD model in vitro and human neuroblastoma SH-SY5Y cellline had been utilized to establish PD model. In this study, SH-SY5Y cell underoptimized acute or chronic MPP~+exposure was selected in comparison to features ofacute and chronic lesioned model. Based on these data, the chronic MPP~+lesionedmodel was selected to study ER stress mediated cell death, and the neuroprotectiveeffect of velvet antler polypeptides (VAPs), the peptides extracted from cervus nippon,was evaluated to develop the potential therapeutic agent for PD treatment.
Methods
1. VAPs was obtained from fresh velvet antler using colloid mill, ammonium sulfateprecipitation, lyophilizing procedure. For further study, SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), laser desorption ionization time-of-flight massspectrometry (MALDI-TOF-MS) and amino acid composition analysis wereperfomed to characterize the physical and chemical features of VAPs.
2. For acute MPP~+lesioned model in vitro, MPP~+with different concentrations wereexposed to human neuroblastoma SH-SY5Y cell line for24h,48h and72hrespectively. The half maximal inhibitory concentration (IC50) of MPP~+in SH-SY5Yand protective effect of VAPs against MPP~+cytotoxicity were measured using MTTassay. For acute MPP~+exposured model, a72h exposed model was selected. Forfurther study in this model, cells were divided into control, model and VAPs treatedgroup. Control cells were incubated with midium containing10%fetal bovine serum,MPP~+-treated cells were incubated with120.9μM MPP~+, VAPs group cells wereincubated with62.5、125、250μg/mL VAPs in the presence of120.9μM MPP~+. Cellviability was detected with MTT assay; Cell apoptosis rate and mitochondrialmembrane potential (ΔΨm) were measured using Hoechst33342and rhodamine123staining respectively; Intracellular reactive oxygen species (ROS) level was assayedwith H2DCFDA staining; Western blot and immunocytochemistry were performed todetect the level of ER stress related proteins, such as caspase-12, heavy-chain bindingprotein/glucose regulated protein78(Bip/GRP78), C/EBP homologous protein,CCAAT/enhancer binding protein homologous protein (CHOP) and c-Jun N-terminalkinase/Stress-activated protein kinases (JNK/SAPK).
3. For establishment of chronic MPP~+intoxication model, SH-SY5Y cells wereexposed to MPP~+for5days,10days and15days. Cell viability was measured withMTT assay; Cell apoptosis rate and ΔΨm were detected with Hoechst33342andrhodamine123staining; Intracellular ROS production was determined withH2DCFDA staining; Western blot and immunocytochemistry were processed forcaspase-12, GRP78, CHOP, phosphorylated JNK（p-JNK).
Results
1. Velvet antler polypeptides (VAPs)
A water soluble fluffy white powder containing54%protein had been obtained. Thelaser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)analysis indicated that molecular weigh of most of the peptides in VAPs ranged from10kDa~20kDa. Further purification and MALDI-TOF-MS analysis characterizedpeptides with molecular weigh of3~11kDa, which enrihed in asparagine, serine,glutamic acid, glycine, alanine, valine, leucine, phenylalanine, lysine, arginine,proline, but no cystine, tyrosine, tryptophan detected.
2. PD model received acute MPP~+exposure
⑴120.9μM MPP~+decreased the cell viability of SH-SY5Y cell and increased cellapoptotic rate.125and250μg/mL VAPs protected against MPP~+-induced cell deathin a significant way, which was much apparent for72h protection. MPP~+lead to△Ψ mdecrease and ROS production, but VAPs inhibited these changes in SH-SY5Ycell induced by MPP~+.
⑵MPP~+exposure result in a robust expression of caspase-12, and VAPs treatmentinhibited this process. GRP78, CHOP, p-JNK expression were not observed in cellsexposed to MPP~+. Collectively, these results show that a acute MPP~+exposurecharacterized with mitochondrial dysfunction and oxidative sress mediated cell death.VAPs counteract cell death induced by MPP~+. MPP~+incuced caspase-12expression ina significant way, but other RE stress associated proteins such as GRP78, CHOP andp-JNK were not apparent. It is conceivable that an acute MPP~+lesioned model in vitrocaused cell death depending on activation of mitochondrial pathway related caspasecascade and oxidative stress, but not determined by ER stress associated cell deathsignaling.
3. Chronic MPP~+intoxication model
⑴IC50values for5days,10days and15days MPP~+exposure were31.1μM,22.1μM and10.4μM respectively, which were used to establish MPP~+intoxication modelfor chronic exposure. MTT assay indicated500μg/mL VAPs protected against MPP~+induced decrease of cell viability.
⑵In MPP~+intoxication model for chronic exposure, either5days exposure or10days,15days exposure influenced cell apoptosis rate or△Ψm. But ROS productionwas visualized using fluorescent probe in cells exposed to31.1μM MPP~+for5days. 22.1μM MPP~+or10.4μM MPP~+for10days and15days exposure had no effect onROS production. VAPs treatment inhibited the ROS level in MPP~+lesioned model for5days.
⑶Western blot and immunocytochemistry indicated there was caspase-12, GRP78,CHOP and p-JNK expression in cells treated with MPP~+for5days. VAPs treatmentdownregulated these protein expression. But neither22.1μM MPP~+,10.4μM MPP~+exposure nor VAPs treatment for10days and15days induced ER stress associatedprotein expression, which indicated the chronic MPP~+exposure lead to ER stressactivation, but not initiation of mitochondrial pathway directly. These data show thatSH-SY5Y cell under31.1μM MPP~+exposure for5days and120.9μM exposure for72h appeared complate different cell signaling phenotypes. Our data identified acuteMPP~+injury induced mitochondrial dysfunction and oxidative stress mediated celldeath, which had been substantiated by cell apoptosis bodies in response to MPP~+exposure. A chronic MPP~+exposure induced ER stress and oxidative stress, but failedto result in cell death. We recognized that a chronic MPP~+intoxication model canmimic the progressive and long term neurodegenerative process of PD onset muchbetter than a acute and short term model. In conclusion, our model in vitro is ideal forstudying ER stress associated pathways. VAPs protect against chronic MPP~+intoxication and the mechanism underlying supression of ER stress associatedproteins activation.
目前，PD的发病原因尚不完全清楚。各类体内和体外实4验显示，PD的发病机制涉及线粒体功能障碍、氧化应激、泛素化蛋白酶体等途径。最近的研究表明，PD发病机制还涉及未折叠蛋白聚积引起的内质网应激，即内质网应激（endoplasmic reticulum stress, ER stress）介导的细胞凋亡参与PD的发病过程。因此，深入了解PD发病机制，探寻有效的神经保护剂是目前治疗PD的重要策略。
PD发病机制的探讨和潜在治疗药物的筛选及评价，在很大程度上依赖于疾病相关体内外模型的建立及应用，特别是离体细胞模型的建立及应用。众所周知，PD是一种慢性疾病，具有缓慢、进展性的发病过程和特征，但是目前国内用于PD发病机制和药物筛选的离体细胞模型大多为急性损伤模型。我们的实践证明，采用常规急性损伤细胞模型已经不能客观反映PD缓慢、渐进性发展的规律和特征，必须建立PD稳定的慢性损伤的体外模型，以适应PD发病机制研究及探寻有效神经保护剂的需要。
基于以上的理解，本研究首先以1-甲基-4-苯基吡啶离子（1-methyl-4-phenylpyridinium, MPP~+）作为诱导剂，以SH-SY5Y细胞作为模型载体，建立PD急、慢性细胞损伤模型，确定建立急、慢性细胞模型的最佳条件。在比较急、慢性SH-SY5Y细胞损伤的规律和特点基础上，利用慢性SH-SY5Y细胞损伤模型探讨内质网应激介导的SH-SY5Y细胞凋亡机制。并在上述研究基础上观察鹿茸多肽（Velvet antler polypeptides, VAPs）对SH-SY5Y细胞凋亡的干预作用及初步机制探讨，为进一步开发抗PD的药物提供科学依据和研究思路。
方法
1.以新鲜梅花鹿茸为原材料，通过胶体磨匀浆、硫酸铵沉淀、冷冻干燥等方法制备VAPs；进一步利用SDS-聚丙烯酰胺凝胶电泳（polyacrylamide gelelectrophoresis，PAGE）、激光解吸电离飞行时间质谱和氨基酸组成分析等技术初步分析鹿茸总肽的理化性质。
2.采用MPP~+诱导人神经母细胞瘤SH-SY5Y细胞发生凋亡，设置不同浓度MPP~+分别作用24,48,72h，建立MPP~+急性损伤细胞模型；MTT法检测细胞活力，最终确定120.9μM MPP~+作用72h为建模浓度和时间。进而将培养的SH-SY5Y细胞分为空白对照组、MPP~+组和VAPs组。对照组给予细胞培养基培养，MPP~+组给予120.9μM的MPP~+处理，VAPs组给予不同浓度VAPs（62.5、125、250μg/mL）与120.9μM MPP~+同时处理。采用Hoechst33342染色检测细胞凋亡，罗丹明123染色检测线粒体膜电位（△Ψm），H2DCFDA染色检测细胞内活性氧簇（reactive oxygen species，ROS）的水平，Western blot和免疫细胞化学检测内质网分子伴侣重链结合蛋白/糖调节蛋白（Heavy-chain bindingprotein/glucose regulated protein78, Bip/GRP78）、CCAAT/增强子结合蛋白同源蛋白（C/EBP homologous protein, CCAAT/enhancer binding protein homologousprotein, CHOP）、磷酸化c-Jun氨基末端激酶/应激活化蛋白激酶（c-Jun N-terminalkinase/Stress-activated protein kinases, JNK/SAPK）、半胱氨酸天冬氨酸蛋白酶12（caspase-12）等4种蛋白的表达情况。
3.采用MPP~+诱导人神经母细胞瘤SH-SY5Y细胞发生凋亡，设置不同浓度MPP~+分别作用5,10,15天，建立MPP~+慢性损伤细胞模型；MTT法检测细胞活力。将培养的SH-SY5Y细胞分为空白对照组、MPP~+组和VAPs组。对照组给予细胞培养基培养，MPP~+组给予31.1μM的MPP~+处理，VAPs组给予不同浓度VAPs与31.1μM MPP~+同时处理。上述各组细胞均孵育5,10,15天后，采用MTT法检测细胞存活率。采用Hoechst33342染色检测细胞凋亡，罗丹明123染色检测△Ψm，H2DCFDA染色检测细胞内ROS的水平，Western blot检测GRP78、CHOP、磷酸化JNK（p-JNK）和caspase-12等蛋白表达情况。
结果
1.鹿茸总多肽（VAPs）
所得VAPs为白色粉末状，水溶性较好，蛋白质含量为54%。VAPs分子量主要分布在10kDa和20kDa区域范围内。进一步将分子量10kDa区域的多肽进行质谱分析，其分子量在3000～11000Da范围内。VAPs主要由天冬氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、苯丙氨酸、赖氨酸、精氨酸和脯氨酸组成，没有检测到半胱氨酸、酪氨酸和色氨酸。
2.急性损伤模型
⑴120.9μM的MPP~+可以诱导SH-SY5Y细胞存活率下降，凋亡率增加。125、250μg/mL的VAPs可减少细胞凋亡，以72h到达最大保护效应。MPP~+导致△Ψm降低，ROS（活性氧簇）含量增加。125、250μg/mL的VAPs拮抗MPP~+诱导产生的△Ψm和ROS的变化。
⑵MPP~+诱导caspase-12表达显著增加，不同浓度VAPs可使MPP~+诱导的Caspase-12表达有所降低。在MPP~+损伤的SH-SY5Y细胞未观察到GRP78、CHOP、p-JNK等内质网应激（ER）相关蛋白的显著表达。
综上，MPP~+致SH-SY5Y细胞损伤的主要特征为线粒体功能障碍和氧化应激介导的细胞凋亡，VAPs对该损伤有拮抗和纠正作用。在MPP~+诱导的急性损伤模型中，MPP~+可使SH-SY5Y细胞内的caspase-12水平显著提高，但没有看到其它内质网应激相关蛋白的过表达。内质网应激相关蛋白沉默表达提示，在急性损伤模型中caspase-12的激活及诱导细胞凋亡并非借助于内质网应激相关途径的启动，可能主要介导线粒体凋亡途径及氧化应激凋亡途径。
3.慢性损伤模型
（1）建立了MPP~+作用SH-SY5Y细胞5天（IC50为31.1μM）、10天（IC50为22.1μM）和15天（IC50为10.4μM）的慢性损伤模型，500μg/mL VAPs能有效提高SH-SY5Y细胞5天、10天和15天的存活率。
（2）在MPP~+慢性损伤SH-SY5Y细胞中，没有看到MPP~+急性损伤模型呈现的凋亡特征性改变和△Ψm的变化，VAPs也没有明显作用。但在MPP~+损伤5天的SH-SY5Y细胞内，ROS水平显著升高，VAPs能明显降低细胞内ROS含量。
（3）免疫细胞化学和Western blot检测均显示，MPP~+作用5天的SH-SY5Y细胞中，内质网应激相关蛋白caspase-12、GRP78、CHOP和p-JNK表达水平显著提高，VAPs与MPP~+共孵育可明显对抗caspase-12、GRP78、CHOP、p-JNK表达的上调。在MPP~+诱导10、15天的SH-SY5Y细胞中则看不到上述内质网应激相关蛋白的变化，VAPs也没有相应的作用。结果提示，MPP~+致SH-SY5Y细胞慢性损伤的主要特征为内质网应激相关蛋白的激活，是一种功能性损伤。
综上，在低浓度MPP~+（31.1μM）诱导5天的慢性损伤细胞模型中呈现出与高浓度MPP~+（120.9μM）诱导72小时急性损伤模型完全不同的损伤模式和特征性改变。研究表明，急性损伤模型是以线粒体功能障碍和氧化应激损伤介导的细胞结构性破坏，镜下看到大量凋亡细胞和细胞坏死残片。而慢性损伤模型则是以内质网应激和氧化应激介导的细胞功能性损伤，镜下没有典型的细胞凋亡和坏死。后者更接近于PD缓慢、进展性疾病的特点，是研究PD内质网应激相关机制的理想模型。鹿茸多肽对MPP~+诱发的SH-SY5Y细胞慢性损伤有保护作用，其机制可能是通过抑制内质网应激相关凋亡分子表达而实现的。
Background
Parkinson's disease (PD) is a common neurodegenerative disease in agedpopulation. The growing incidence of PD is a socio-economic problem affecting lifequality of PD patients. The syptoms of PD inclue tremor, bradykinesia, rigidity andflexed posture. Pathological features of PD implicated loss of dopaminergic neuronsin substanitial nigra, which cause the decreased dopamine level in the striata. Untilnow, the mechanism underlying PD onset is not fully understood. Most of the in vivoand ex vivo studies had illustrated that some of the mechanism involved in the PDpathogenesis, including mitochondial dysfunction, oxidative stress, ubiquitin-proteasomal pathway, etc. Recently, a misfolded protein related endoplasmicreticulum stress (ER stress) had been most intensively studied for its actions toneuronal cell death. Thus, understanding the process of ER stress is of great helpful toexplore novel neuroprotective agent for PD treatment. Clarifing the mechanisms ofPD onset and development of neuroprotective therapeutics are largely depend onestablishment of disease model, especially model in vitro. It is known that PD is achronic and progressive disease, but acute lesioned model in vitro was most commonused in domestic studies for PD. Our study indicated that the acute lesioned modelfailed to relect the chronic and progessive process of PD onset. Hence, a chroniclesioned model in vitro should be attentioned considerably in order to reveal the actualmechanism of PD and develop novel neuroprotective agents. Based on theseunderstandings,1-methyl-4-phenylpyridinium (MPP~+), a neurotoxin most commonlyused in establishment of PD model in vitro and human neuroblastoma SH-SY5Y cellline had been utilized to establish PD model. In this study, SH-SY5Y cell underoptimized acute or chronic MPP~+exposure was selected in comparison to features ofacute and chronic lesioned model. Based on these data, the chronic MPP~+lesionedmodel was selected to study ER stress mediated cell death, and the neuroprotectiveeffect of velvet antler polypeptides (VAPs), the peptides extracted from cervus nippon,was evaluated to develop the potential therapeutic agent for PD treatment.
Methods
1. VAPs was obtained from fresh velvet antler using colloid mill, ammonium sulfateprecipitation, lyophilizing procedure. For further study, SDS-polyacrylamide gelelectrophoresis (SDS-PAGE), laser desorption ionization time-of-flight massspectrometry (MALDI-TOF-MS) and amino acid composition analysis wereperfomed to characterize the physical and chemical features of VAPs.
2. For acute MPP~+lesioned model in vitro, MPP~+with different concentrations wereexposed to human neuroblastoma SH-SY5Y cell line for24h,48h and72hrespectively. The half maximal inhibitory concentration (IC50) of MPP~+in SH-SY5Yand protective effect of VAPs against MPP~+cytotoxicity were measured using MTTassay. For acute MPP~+exposured model, a72h exposed model was selected. Forfurther study in this model, cells were divided into control, model and VAPs treatedgroup. Control cells were incubated with midium containing10%fetal bovine serum,MPP~+-treated cells were incubated with120.9μM MPP~+, VAPs group cells wereincubated with62.5、125、250μg/mL VAPs in the presence of120.9μM MPP~+. Cellviability was detected with MTT assay; Cell apoptosis rate and mitochondrialmembrane potential (ΔΨm) were measured using Hoechst33342and rhodamine123staining respectively; Intracellular reactive oxygen species (ROS) level was assayedwith H2DCFDA staining; Western blot and immunocytochemistry were performed todetect the level of ER stress related proteins, such as caspase-12, heavy-chain bindingprotein/glucose regulated protein78(Bip/GRP78), C/EBP homologous protein,CCAAT/enhancer binding protein homologous protein (CHOP) and c-Jun N-terminalkinase/Stress-activated protein kinases (JNK/SAPK).
3. For establishment of chronic MPP~+intoxication model, SH-SY5Y cells wereexposed to MPP~+for5days,10days and15days. Cell viability was measured withMTT assay; Cell apoptosis rate and ΔΨm were detected with Hoechst33342andrhodamine123staining; Intracellular ROS production was determined withH2DCFDA staining; Western blot and immunocytochemistry were processed forcaspase-12, GRP78, CHOP, phosphorylated JNK（p-JNK).
Results
1. Velvet antler polypeptides (VAPs)
A water soluble fluffy white powder containing54%protein had been obtained. Thelaser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)analysis indicated that molecular weigh of most of the peptides in VAPs ranged from10kDa~20kDa. Further purification and MALDI-TOF-MS analysis characterizedpeptides with molecular weigh of3~11kDa, which enrihed in asparagine, serine,glutamic acid, glycine, alanine, valine, leucine, phenylalanine, lysine, arginine,proline, but no cystine, tyrosine, tryptophan detected.
2. PD model received acute MPP~+exposure
⑴120.9μM MPP~+decreased the cell viability of SH-SY5Y cell and increased cellapoptotic rate.125and250μg/mL VAPs protected against MPP~+-induced cell deathin a significant way, which was much apparent for72h protection. MPP~+lead to△Ψ mdecrease and ROS production, but VAPs inhibited these changes in SH-SY5Ycell induced by MPP~+.
⑵MPP~+exposure result in a robust expression of caspase-12, and VAPs treatmentinhibited this process. GRP78, CHOP, p-JNK expression were not observed in cellsexposed to MPP~+. Collectively, these results show that a acute MPP~+exposurecharacterized with mitochondrial dysfunction and oxidative sress mediated cell death.VAPs counteract cell death induced by MPP~+. MPP~+incuced caspase-12expression ina significant way, but other RE stress associated proteins such as GRP78, CHOP andp-JNK were not apparent. It is conceivable that an acute MPP~+lesioned model in vitrocaused cell death depending on activation of mitochondrial pathway related caspasecascade and oxidative stress, but not determined by ER stress associated cell deathsignaling.
3. Chronic MPP~+intoxication model
⑴IC50values for5days,10days and15days MPP~+exposure were31.1μM,22.1μM and10.4μM respectively, which were used to establish MPP~+intoxication modelfor chronic exposure. MTT assay indicated500μg/mL VAPs protected against MPP~+induced decrease of cell viability.
⑵In MPP~+intoxication model for chronic exposure, either5days exposure or10days,15days exposure influenced cell apoptosis rate or△Ψm. But ROS productionwas visualized using fluorescent probe in cells exposed to31.1μM MPP~+for5days. 22.1μM MPP~+or10.4μM MPP~+for10days and15days exposure had no effect onROS production. VAPs treatment inhibited the ROS level in MPP~+lesioned model for5days.
⑶Western blot and immunocytochemistry indicated there was caspase-12, GRP78,CHOP and p-JNK expression in cells treated with MPP~+for5days. VAPs treatmentdownregulated these protein expression. But neither22.1μM MPP~+,10.4μM MPP~+exposure nor VAPs treatment for10days and15days induced ER stress associatedprotein expression, which indicated the chronic MPP~+exposure lead to ER stressactivation, but not initiation of mitochondrial pathway directly. These data show thatSH-SY5Y cell under31.1μM MPP~+exposure for5days and120.9μM exposure for72h appeared complate different cell signaling phenotypes. Our data identified acuteMPP~+injury induced mitochondrial dysfunction and oxidative stress mediated celldeath, which had been substantiated by cell apoptosis bodies in response to MPP~+exposure. A chronic MPP~+exposure induced ER stress and oxidative stress, but failedto result in cell death. We recognized that a chronic MPP~+intoxication model canmimic the progressive and long term neurodegenerative process of PD onset muchbetter than a acute and short term model. In conclusion, our model in vitro is ideal forstudying ER stress associated pathways. VAPs protect against chronic MPP~+intoxication and the mechanism underlying supression of ER stress associatedproteins activation.
引文
[1] Gao K, Deng X, Zheng W, et al. Genetic analysis of the FBXO42gene in ChineseHan patients with Parkinson's disease[J]. BMC Neurol,2013,13(1):125.
[2] Naohiro Egawa, Keisuke Yamamoto, Haruhisa Inoue, et al The EndoplasmicReticulum Stress Sensor,ATF6α,Protects against Neurotoxin-induced Dopaminer-gic Neuronal Death[J]. J Biol Chem,2011,286(10):7947–7957.
[3]王晟东，白洁.内质网应激与帕金森病[J].生命科学，2010，22（4）：326-330
[4] Brasnjevic I, Steinbusch HW, Schmitz C, et al. Delivery of peptide and proteindrugs over the blood-brain barrier[J]. Prog Neurobiol,2009,87(4):212-51.
[5]郝丽，李和平，严历.东北梅花鹿鹿茸尖端组织全长cDNA文库的构建[J].东北林业大学学报，2009，37（11）：120-122.
[6] Lu LJ, Chen L, Meng XT, et al. Biological effect of velvet antler polypeptides onneural stem cells from embryonic rat brain[J]. Chin Med J (Engl),2005,118(1):38-42.
[7] Li C, Stanton JA, Robertson TM, et al. Nerve growth factor mRNA expression inthe regenerating antler tip of red deer (Cervus elaphus)[J]. PLoS One,2007,2(1):e148.
[8] Lai AK, Hou WL, Verdon DJ, et al. The distribution of the growth factors FGF-2and VEGF, and their receptors, in growing red deer antler[J]. Tissue Cell,2007,39(1):35-46.
[9] Lord EA, Martin SK, Gray JP, et al. Cell cycle genes PEDF and CDKN1C ingrowing deer antlers[J]. Anat Rec (Hoboken),2007,290(8):994-1004.
[10]严铭铭，曲晓波，王旭，等.梅花鹿茸中活性多肽的纯化、测序及功能研究[J].高等学校化学学报，2007，28(10)：1893-1896.
[11] Guan SW, Duan LX, Li YY, et al. A novel polypeptide from Cervus nipponTemminck proliferation of epidermal cells and NIH3T3cell line[J]. ActaBiochimica Polonica,2006,53(2):395-397.
[12]张郑瑶，段冷昕，周秋丽，等.梅花鹿茸多肽的化学结构及生物活性[J].高等学校化学学报，2012，33（9）：2000-2004.
[13]郑帆，李仁宽，王辉林，等.鹿茸冻干粉的酶解及其酶解产物性质的研究[J].中国中药杂志，2010，35（19）：2628-2633.
[14] Srinivasulu Chigurupati, Zelan Wei, Cherine Belal, et al. The Homocysteine-inducibleEndoplasmic Reticulum Stress Protein Counteracts Calcium Store Depletion andInduction of CCAAT Enhancer-binding Protein Homologous Protein in aNeurotoxin Model of Parkinson Disease[J]. J Biol Chem,2009,284(27):18323-18333.
[15]王虎，蔡定芳.帕金森病与内质网应激[J].国际神经病学神经外科学杂志，2007，34（1）：86-90
[16]邓娟，白洁. TRPC1和Ca2+在帕金森病内质网应激中的作用[J].生命科学，2012，24（10）：1169-1173.
[17]高俊鹏，蔡定芳.内质网应激介导神经变性疾病错折叠蛋白的神经毒性[J].中国病理生理杂志，2008，24（1）：201-205.
[18] Pan C, Giraldo GS, Prentice H, et al. Taurine protection of PC12cells againstendoplasmic reticulum stress induced by oxidative stress[J]. Journal ofBiomedical Science,17(Suppl1): S17.
[19] Qu LiFeng, Liu Zhen, Zhang, H. F. M., et al. Endoplasmic reticulumstress-induced cell survival and apoptosis[J]. Journal of Chinese ClinicalMedicine,2009,4(8):452-459.
[20] Lebouvier T, Chaumette T, Paillusson S, et al. The second brain and Parkinson'sdisease[J]. Eur J Neurosci,2009,30(5):735-41.
[21]邱会卿，顾平，樊文峰.经颅脑实质超声诊断帕金森病的临床应用[J].临床误诊误治，2011，10：92-94.
[22]刘小平.利用果蝇模型研究帕金森病相关基因α-synuclein，PINK1和LRRK2的作用机制[D].中国优秀博硕士学位论文全文数据库（博士）：中南大学，2007.
[23]王晟东.环境毒性物质MPTP导致多巴胺能神经元损伤机理研究[D].中国优秀博硕士学位论文全文数据库（博士）：昆明理工大学，2011.
[24]陈茹.帕金森病研究进展[J].中国康复理论与实践，2007，13（7）：637-639
[25]刘人恺，肖波，杨晓苏，等.星形诺卡菌对多巴胺能细胞损伤的体外研究[J].卒中与神经疾病，2008，15（5）：259-263.
[26] Noyce AJ, Bestwick JP, Silveira-Moriyama L, et al. Meta-analysis of earlynonmotor features and risk factors for Parkinson disease[J]. Ann Neurol,2012,72(6):893-901.
[27]张玉梅.长期低剂量皮下注射鱼藤酮制备多巴胺神经元损伤模型及其机制研究[D].中国优秀博硕士学位论文全文数据库（博士）：大连医科大学，2009.
[28]李伟静，于群.辅酶Q10的生理作用及临床应用[J].生物技术通讯，2007，18（5）：882-884.
[29]姜宁.增龄和预运动训练在小鼠帕金森发病中的作用：线粒体与自噬途径的分子生理学机制研究[D].中国优秀博硕士学位论文全文数据库（博士）：中国人民解放军军事医学科学院，2012.
[30] Thomas RR, Keeney PM, Bennett JP. Impaired complex-I mitochondrialbiogenesis in Parkinson disease frontal cortex[J]. J Parkinsons Dis,2012,2(1):67-76.
[31] Sanders LH, Timothy Greenamyre J. Oxidative damage to macromolecules inhuman Parkinson disease and the rotenone model[J]. Free Radic Biol Med,2013,62:111-20.
[32]黄桂平.帕金森病的临床研究及展望[J].医学综述，2008，14（13）：2023-2026
[33]梁天佳，莫雪安.帕金森病发病机制研究进展[J].医学综述，2008，14（7）：1040-1042.
[34] Galligan JJ, Petersen DR. The human protein disulfide isomerase gene family[J].Hum Genomics,2012,6:6.
[35]项喜艳. DTT诱发内质网应激-自噬过程中活性氧的作用及其调节机制[D].中国优秀博硕士学位论文全文数据库（硕士）：吉林大学，2012.
[36]陈冬.帕金森病相关蛋白Parkin对自噬调节的研究[D].中国优秀博硕士学位论文全文数据库（博士）：中国科学技术大学，2010.
[37]叶青，郑民华.自噬的分子机制与病理生理意义[J].国际病理科学与临床杂志，2007，27（4）：358-362.
[38] Vogiatzi T, Xilouri M, Vekrellis K, et al. Wild Type-Synuclein Is Degraded byChaperone-mediated Autophagy and Macroautophagy in Neuronal Cells[J]. JBiol Chem,2008,283(35):23542-56.
[39] Zheng HF, Yang YP, Hu LF, et al. Autophagic impairment contributes to systemicinflammation-induced dopaminergic neuron loss in the midbrain[J]. PLoS One,2013,8(8): e70472.
[40]杨丽娟，董军，陆大祥，等.姜黄素对LPS激活小胶质细胞条件培养基诱导损伤的海马神经元保护作用及机制[J].中国病理生理杂志，2010，26（4）：742-747.
[41]陈生第.帕金森病.第1版[M].北京：人民卫生出版社，2006：110.
[42]张治国.黄芪甲苷对帕金森病神经元的保护作用及其机制研究[D].中国优秀博硕士学位论文全文数据库（硕士）：第四军医大学，2012.
[43]牛英才，潘志，李晓明，等.葛根异黄酮对MPP+诱导的PC12细胞凋亡的保护作用[J].中国药理学通报，2009，25（1）：112-115.
[44] Lee DH, Kim CS, Lee YJ. Astaxanthin protects against MPTP/MPP+-inducedmitochondrial dysfunction and ROS production in vivo and in vitro[J]. FoodChem Toxicol,2011,49(1):271-80.
[45] Zhang SP, Du XG, Pu XP.3-O-demethylswertipunicoside protects againstoxidative toxicity in PC12cells[J]. Biol Pharm Bull,2010,33(9):1529-33.
[46]黄金忠. PC12细胞中MPP+诱导的α-synuclein聚集和自噬对其降解机制研究
[D].中国优秀博硕士论文全文数据库，苏州大学，2010.
[47] Yim SB, Park SE, Lee CS. Protective effect of glycyrrhizin on1-methyl-4-phenylpyridinium-induced mitochondrial damage and cell death in differentiatedPC12cells[J]. J Pharmacol Exp Ther,2007,321(2):816-22.
[48]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.
[49] Zhang LJ, Xue YQ, Yang C, et al. Human Albumin Prevents6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and InVivo[J]. PLoS One,2012,7(7): e41226.
[50]王丹萍.6-羟基多巴胺诱导的SH-SY5Y细胞帕金森病模型差异蛋白质组学研究[D].中国优秀博硕士学位论文全文数据库（博士）：吉林大学，2010.
[51]孙家贺.帕金森病现代中医证治规律研究[D].中国优秀博硕士学位论文全文数据库（硕士）：山东中医药大学，2012.
[52]厚荣荣. EGCG对PQ诱导的PC12细胞凋亡的保护作用及其机制研究[D].中国优秀博硕士学位论文全文数据库（硕士）：第四军医大学，2007.
[53] Cabeza-Arvelaiz Y, Schiestl RH. Transcriptome analysis of a rotenone model ofparkinsonism reveals complex I-tied and-untied toxicity mechanisms common toneurodegenerative diseases[J]. PLoS One,2012,7(9): e44700.
[54]张劲松，栾春业，王强，等.代森锰锌对PC-12细胞凋亡的影响[J].中国药理学与毒理学杂志，2011，25（4）：349-353.
[55]贾琳琳，李术. α-突触核蛋白在神经损伤中的作用[J].畜牧与兽医，2011，43（2）：88-90.
[56] Choi WS, Abel G, Klintworth H, et al. JNK3mediates paraquat-and rotenone-induced dopaminergic neuron death[J]. J Neuropathol Exp Neurol,2010,69(5):511-20.
[57]李海龙，粟艳，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.
[58] Sun ZW, Zhang L, Zhu SJ, et al. Excitotoxicity effects of glutamate on humanneuroblastoma SH-SY5Y cells via oxidative damage[J]. Neurosci Bull,2010,26(1):8-16.
[59]何秀全.罗格列酮通过抑制小胶质细胞活化而保护多巴胺能神经元的研究[D].中国优秀博硕士学位论文全文数据库（博士）：山东大学，2012.
[60] Kim SJ, Park JH, Kim KH, et al. The Protective Effect of Apamin onLPS/Fat-Induced Atherosclerotic Mice[J]. Evid Based Complement Alternat Med,2012,2012:305454.
[61] Son DJ, Lee JW, Lee YH, et al. Therapeutic application of anti-arthritis,pain-releasing, and anti-cancer effects of bee venom and its constituentcompounds[J]. Pharmacol Ther,2007,115(2):246-70.
[62]王忠田.蜂毒有助于治疗帕金森病[J].蜜蜂杂志，2013，1：31.
[63] Doo A-R, Kim S, Kim S, et al. Neuroprotective effects of bee venompharmaceutical acupuncture in acute1-Parkinson's disease[J]. NeurologicalResearch Suppl,2010,1:88–91.
[64] Kim J, Yang EJ, Lee M, et al. Bee venom reduces neuroinflammation in theMPTP-induced model of Parkinson's disease[J]. Int J Neurosci,2011,121:209–217.
[65] Doo AR, Kim SN, Kim ST, et al. Bee venom protects SH-SY5Y humanneuroblastoma cells from1-methyl-4-phenylpyridinium-induced apoptotic celldeath[J]. Brain Res,2012,1429:106-15.
[66] Alvarez-Fischer D, Noelker C, Vulinovi F, et al. Bee Venom and Its ComponentApamin as Neuroprotective Agents in a Parkinson Disease Mouse Model[J].PLoS ONE,2013,6(11): e61700.
[67]肖昭扬，殷盛明，于德钦，等.蝎毒对C57BL/6帕金森病小鼠脑内前脑啡肽原表达的影响[J].中国临床康复，2006，10（19）：33-36.
[68]殷盛明，于德钦，安宁，等.蝎毒耐热蛋白对MPTP诱导的帕金森病小鼠脑内小胶质细胞的影响[J].中国应用生理学杂志，2009，25（1）：79-80，90.
[69]殷盛明，于德钦，彭岩，等.神经元营养活性肽对帕金森病大鼠小胶质细胞保护作用及机制研究[J].中草药，2010，41（11）：1838-1842.
[70] Trejo E, Borges A, Na ez B, et al. Tityus zulianus venom induces massivecatecholamine release from PC12cells and in a mouse envenomation model[J].Toxicon,2012,59(1):117-23.
[71]陈健.东亚钳蝎毒素Martentoxin的分离纯化与功能研究[D].中国优秀博硕士学位论文全文数据库（硕士）：南京医科大学，2011.
[72]罗素兰，长孙东亭，吴勇，等.海南产芋螺毒素在疼痛与成瘾治疗中的应用前景[J].中国药理学与毒理学杂志，2012，（3）：439-440.
[73] Balsara R, Li N, Weber-Adrian D, et al. Opposing action of conantokin-G onsynaptically and extrasynaptically-activated NMDA receptors[J]. Neurophar-macology,2012,62(7):2227-38.
[74] Gowd KH, Watkins M, Twede VD, et al. Characterization of conantokin Rl-A:molecular phylogeny as structure/function study[J]. J Pept Sci,2010,16(8):375-82.
[75] Sheng Z, Liang Z, Geiger JH, et al. The selectivity of conantokin-G for ionchannel inhibition of NR2B subunit-containing NMDA receptors is regulated byamino acid residues in the S2region of NR2B[J]. Neuropharmacology,2009,57(2):127-36.
[76]任展宏，朱永平.芋螺毒素conantokin的药理作用及构效关系研究进展[J].中国药学杂志，2007，42（23）：1761-1764.
[77] Surin AM, Kriukova EV, Strukov AS, et al. Effect of alpha-conotoxin MII and itsN-terminal derivatives on Ca2+and Na+signals induced by nicotine inneuroblastoma cell line SH-SY5Y[J]. Bioorg Khim,2012,38(2):214-22.
[78] Zheng L, Su G, Ren J, et al. Isolation and characterization of an oxygen radicalabsorbance activity peptide from defatted peanut meal hydrolysate and itsantioxidant properties[J]. J Agric Food Chem,2012,6(21):5431-7.
[79]郝丽，李和平，严历.梅花鹿鹿茸尖端组织ESTs分析[J].遗传，2011，33（4）：371-377.
[80]张郑瑶，尚禹东，牟凤辉，等.鹿茸多肽及其生长因子制备工艺的研究进展[J].特产研究，2011，3：69-72.
[81] Li C, Stanton JA, Robertson TM, et al. Nerve growth factor mRNA expression inthe regenerating antler tip of red deer (Cervus elaphus)[J]. PLoS One,2007,2(1):e148.
[82] Lai AK, Hou WL, Verdon DJ, et al. The distribution of the growth factors FGF-2and VEGF, and their receptors, in growing red deer antler[J]. Tissue Cell,2007,39(1):35-46.
[83]严铭铭，曲晓波，王旭，等.梅花鹿茸中活性多肽的纯化、测序及功能研究[J].高等学校化学学报,2007,28(10):1893-1896.
[84] Guan SW, Duan LX, Li YY, et al. A novel polypeptide from Cervus nipponTemminck proliferation of epidermal cells and NIH3T3cell line[J]. ActaBiochimica Polonica,2006,53(2):395-397.
[85]张郑瑶，段冷昕，周秋丽，等.梅花鹿茸多肽的化学结构及生物活性[J].高等学校化学学报，2012，33（9）：2000-2004.
[86]柯李晶，聂毅磊，叶秀云，等.梅花鹿鹿茸中促PC12细胞增殖蛋白的分离和活性研究[J].中草药，2009，40（5）：715-718.
[87] Bourgault S, Vaudry D, Botia B, et al. Novel stable PACAP analogs with potentactivity towards the PAC1receptor[J]. Peptides.2008,29(6):919-32.
[88] Ayako Miura, Naoko Odahara, Aiko Tominaga, et al. Regulatory mechanism ofPAC1gene expression via Sp1by nerve growth factor in PC12cells[J]. FEBSLetters,2012,568,1731-1735.
[89] Reglodi D, Kiss P, Lubics A, et al. Review on the protective effects of PACAP inmodels of neurodegenerative diseases in vitro and in vivo[J]. Curr Pharm Des,2011,17(10):962-72.
[90] Manecka DL, Mahmood SF, Grumolato L, et al. Pituitary adenylatecyclase-activating polypeptide (PACAP) promotes both survival andneuritogenesis in PC12cells through activation of nuclear factor κB (NF-κB)pathway: involvement of extracellular signal-regulated kinase (ERK), calcium,and c-REL[J]. J Biol Chem,2013,288(21):14936-48.
[91] Emery AC, Eiden LE. Signaling through the neuropeptide GPCR PAC1inducesneuritogenesis via a single linear cAMP-and ERK-dependent pathway using anovel cAMP sensor[J]. FASEB J,2012,26(8):3199-211.
[92] Zhang W, Smith A, Liu JP, et al. GSK3beta modulates PACAP-inducedneuritogenesis in PC12cells by acting downstream of Rap1in a caveolae-dependent manner[J]. Cell Signal,2009,21(2):237-45.
[93] Dejda A, Chan P, Seaborn T, et al. Involvement of stathmin1in the neurotrophiceffects of PACAP in PC12cells[J]. J Neurochem,2010,114(5):1498-510.
[94] Miura A, Odahara N, Tominaga A, et al. Regulatory mechanism of PAC1geneexpression via Sp1by nerve growth factor in PC12cells[J]. FEBS Lett,2012,586(12):1731-5.
[95] Raoult E, Roussel BD, Bénard M, et al. Pituitary adenylate cyclase-activatingpolypeptide (PACAP) stimulates the expression and the release of tissueplasminogen activator (tPA) in neuronal cells: involvement of tPA in theneuroprotective effect of PACAP[J]. J Neurochem,2011,119(5):920-31.
[96] Mahata M, Zhang K, Gayen JR, et al. Catecholamine biosynthesis and secretion:physiological and pharmacological effects of secretin[J]. Cell Tissue Res,2011,345(1):87-102.
[97]毛姗姗，张咏梅. PACAP对神经系统疾病保护作用的研究进展[J].生理科学进展，2011，42（4）：276-280.
[98] Lingyan Zhang, Wen Liu, Karen K. Szumlinskid, et al. p10, the N-terminaldomain of p35, protects against CDK5/p25-induced neurotoxicity[J]. PNAS,2012,109(49):20041–20046.
[99] Oh YM, Jang EH, Ko JH, et al. Inhibition of6-hydroxydopamine-inducedendoplasmic reticulum stress by l-carnosine in SH-SY5Y cells. Neurosci Lett,2009,459(1):7-10.
[100] Boldyrev AA, Stvolinsky SL, Fedorova TN, et al. Carnosine as a naturalantioxidant and geroprotector: from molecular mechanisms to clinical trials[J].Rejuvenation Res,2010,13(2-3):156-8.
[101]沈耀，胡薇薇，陈忠.肌肽和中枢神经系统疾病[J].浙江大学学报（医学版），2007，3（2）：199-203.
[102] oban J, Bingül I, Yesil-Mizrak K, et al. Effects of carnosine plus vitamin E andbetaine treatments on oxidative stress in some tissues of aged rats[J]. Curr AgingSci,2013,6(2):199-205.
[103] Banerjee S, Poddar MK. Platelet monoamine oxidase-A activity and aging: effectof carnosine[J]. J Physiol Sci,2013,63(4):279-85.
[104] Kulebyakin K, Karpova L, Lakonsteva E, et al. Carnosine protects neuronsagainst oxidative stress and modulates the time profile of MAPK cascadesignaling[J]. Amino Acids,2012,43(1):91-6.
[105]朱传兵，余国英.脑苷肌肽治疗帕金森病的临床观察[J].中国老年保健医学，2005，3（3）：43-44.
[106] Grasso GI, Bellia F, Arena G, et al. Noncovalent interaction-driven stereose-lectivity of copper(II) complexes with cyclodextrin derivatives of L-andD-carnosine[J]. Inorg Chem,2011,50(11):4917-24.
[107] Bellia F, Vecchio G, Rizzarelli E. Carnosine derivatives: new multifunctionaldrug-like molecules[J]. Amino Acids,2012,43(1):153-63.
[108] Monti JM, Torterolo P, Lagos P. Melanin-concentrating hormone control ofsleep-wake behavior[J]. Sleep Med Rev,2013,17(4):293-8.
[109]孙丽丽，张艳，王君，等.伏隔核黑色素聚集激素在大鼠甲基苯丙胺行为敏化中的作用[J].中国药理学与毒理学杂志，2012，26（3）：430.
[110] Cotta-Grand N, Rovère C, Guyon, A et al. Melanin-concentrating hormoneinduces neurite outgrowth in human neuroblastoma SH-SY5Y cells through p53and MAPKinase signaling pathways[J]. Peptides,2009,30(11):2014-24.
[111]张建敏，高培，魏静.帕金森病患者睡眠障碍现况调查及护理对策[J].中国社区医师：医学专业，2010，8：175-176.
[112]吴佳英，李琳，刘振国.帕金森病患者睡眠障碍发病机制的研究[J].上海交通大学学报：医学版，2009，29（8）：1001-1004.
[113] Oz S, Ivashko-Pachima Y, Gozes I. The ADNP derived peptide, NAP modulatesthe tubulin pool: implication for neurotrophic and neuroprotective activities[J].PLoS One,2012,7(12): e51458.
[114] Fleming SM, Mulligan CK, Richter F, et al. A pilot trial of themicrotubule-interacting peptide (NAP) in mice overexpressing alpha-synucleinshows improvement in motor function and reduction of alpha-synucleininclusions[J]. Mol Cell Neurosci,2011,46(3):597-606.
[115] Sharma NK, Sethy NK, Meena RN, et al. Activity-dependent neuroprotectiveprotein (ADNP)-derived peptide (NAP) ameliorates hypobaric hypoxia inducedoxidative stress in rat brain[J]. Peptides,2011,32(6):1217-24.
[116]杨宇，吴江，杨欣，等.分泌表达神经保护短肽重组慢病毒对SK-N-SH细胞的保护作用[J].中国老年学杂志，2007，27（3）：213-216.
[117] Liu Z, Gao X, Kang T, et al. B6peptide-modified PEG-PLA nanoparticles forenhanced brain delivery of neuroprotective peptide[J]. Bioconjug Chem,2013,24(6):997-1007.
[118] Liu Z, Jiang M, Kang T, et al. Lactoferrin-modified PEG-co-PCL nanoparticlesfor enhanced brain delivery of NAP peptide following intranasal administration[J].Biomaterials,2013,34(15):3870-81.
[119]郭芬，黄晓锋，李实骞，等.鞘脂激活蛋白原Prosaspoin与Rhox5间的功能性相互作用[J].遗传学报：英文版，2007，34（5）：392-399.
[120] Gao HL, Li C, Nabeka H, et al. Attenuation of MPTP/MPP(+) toxicity in vivoand in vitro by an18-mer peptide derived from prosaposin[J]. Neuroscience,2013,236:373-93.
[121] Khan RS, Yu C, Kastin AJ, et al. Brain Activation by Peptide Pro-Leu-Gly-NH2(MIF-1)[J]. Int J Pept,2010. doi:10.1155/2010/537639.
[122]唐丽灵，李大军，王远亮.外力作为信号诱导基因的选择性剪接与力生长因子表达[J].生物化学与生物物理进展，2007，34（5）：454-459.
[123] Quesada A, Micevych P, Handforth A. C-terminal mechano growth factor protectsdopamine neurons: a novel peptide that induces heme oxygenase-1[J]. ExpNeurol,2009,220(2):255-66.
[124] Pirnik Z, Maixnerova J, Matyskova R, et al. Effect of anorexinergic peptides,cholecystokinin (CCK) and cocaine and amphetamine regulated transcript (CART)peptide, on the activity of neurons in hypothalamic structures of C57Bl/6miceinvolved in the food intake regulation[J]. Peptides,2009,31:139–144.
[125] Jones DC, Kuhar MJ. CART receptor binding in primary cell cultures of the ratnucleus accumbens[J]. Synapse,2008,62:122–127.
[126] Lin Y, Hall RA, Kuhar MJ. CART peptide stimulation of G protein-mediatedsignaling in differentiated PC12cells: identification of PACAP6-38as a CARTreceptor antagonist[J]. Neuropeptides,2011,45(5):351-8.
[127] Chan NY, Robador PA, Levi R. Natriuretic peptide-induced catecholamine releasefrom cardiac sympathetic neurons: inhibition by histamine H3and H4receptoractivation[J]. J Pharmacol Exp Ther,2012,343(3):568-77.
[128] Wu Y, Shang Y, Sun SG, et al. Protective effect of erythropoietin against1-methyl-4-phenylpyridinium-induced neurodegenaration in PC12cells[J].Neurosci Bull,2007,23(3):156-64.
[129] Phani S, Jablonski M, Pelta-Heller J, et al. Gremlin is a novel VTA derivedneuroprotective factor for dopamine neurons[J]. Brain Res,2013,1500:88-98.
[130] Liu Y, Pan W, Yang S, et al. Interleukin-22protects rat PC12pheochromocytomacells from serum deprivation-induced cell death[J]. Mol Cell Biochem,2012,371(1-2):137-46.
[131]潘文艳. IL-22在大鼠嗜铬瘤细胞PC12中生物学作用的研究[D].中国优秀博硕士学位论文全文数据库（硕士）：扬州大学，2012.
[132] Duarte AI, Candeias E, Correia SC, et al. Crosstalk between diabetes and brain:glucagon-like peptide-1mimetics as a promising therapy against neurodegener-ation[J]. Biochim Biophys Acta,2013,1832(4):527-41.
[133]张蕾，周舟，张广斌，等.微波暴露对大鼠GLP-1及其受体表达的影响及与学习记忆功能损伤的关系[J].辐射研究与辐射工艺学报，2007，25（3）：180-184.
[134] Biswas SC, Buteau J, Greene LA. Glucagon-like peptide-1(GLP-1) diminishesneuronal degeneration and death caused by NGF deprivation by suppressing Biminduction[J]. Neurochem Res,2008,33(9):1845-51.
[135] Li Y, Tweedie D, Mattson MP, et al. Enhancing the GLP-1receptor signalingpathway leads toproliferation and neuroprotection in human neuroblastomacells[J]. J Neurochem,2010,113(6):1621-31.
[136]全会标，高勇义. Exendin-4临床研究进展[J].中国热带医学，2011，11（3）：388-390.
[137] Luciani P, Deledda C, Benvenuti S, et al. Exendin-4induces cell adhesion anddifferentiation and counteracts the invasive potential of human neuroblastomacells[J]. PLoS One,2013,8(8): e71716.
[138] Holscher C. Central effects of GLP-1: new opportunities for treatments ofneurodegenerative diseases[J]. J Endocrinol,2013, doi:10.1530/JOE-13-0221.
[139] Harkavyi A, Abuirmeileh A, Lever R, et al. Glucagon-like peptide1receptorstimulation reverses key deficits in distinct rodent models of Parkinson'sdisease[J]. J Neuroinflammation,2008,5:19.
[140] Bertilsson G, Patrone C, Zachrisson O, et al. Peptide hormone exendin-4stimulates subventricular zone neurogenesis in the adult rodent brain and inducesrecovery in an animal model of Parkinson's disease[J]. J Neurosci Res,2008,86(2):326-38.
[141] Li Y, Perry T, Kindy MS, et al. GLP-1receptor stimulation preserves primarycortical and dopaminergic neurons in cellular and rodent models of stroke andParkinsonism[J]. Proc Natl Acad Sci U S A,2009,106(4):1285-90.
[142]段冷昕，李瑞芳，金瑾，等.黄连解毒汤对MPTP所致帕金森病小鼠的防治作用[J].中国老年学杂志，2010，30（14）：2004-2006.
[143] Heales SJ, Menzes A, Davey GP. Depletion of glutathione does not affect electrontransport chain complex activity in brain mitochondria: Implications forParkinson disease and postmortem studies[J]. Free Radic Biol Med,2011,50(7):899-902.
[144] Yürekli VA, Gürler S, Naz rolu M, et al. Zonisamide attenuates MPP+-inducedoxidative toxicity through modulation of Ca2+signaling and caspase-3activity inneuronal PC12cells[J]. Cell Mol Neurobiol,2013,33(2):205-12.
[145] Delic M, Rebnegger C, Wanka F, et al. Oxidative protein folding and unfoldedprotein response elicit differing redox regulation in endoplasmic reticulum andcytosol of yeast[J]. Free Radic Biol Med,2012,52(9):2000-12.
[146] Lee M, Kwon BM, Suk K, et al. Effects of obovatol on GSH depletedglia-mediated neurotoxicity and oxidative damage[J]. J Neuroimmune Pharmacol,2012,7(1):173-86.
[147] Zhou W, Bercury K, Cummiskey J, et al. Phenylbutyrate up-regulates the DJ-1protein and protects neurons in cell culture and in animal models of Parkinsondisease[J]. J Biol Chem,2011,286(17):14941-51.
[148]罗蔚锋，包仕尧，刘春风，等.还原型谷胱苷肽治疗帕金森病大鼠模型的实验研究[J].临床神经病学杂志，2006，19（6）：441-443.
[149]陆建明，周厚广，鲍远程.谷胱甘肽合中药对帕金森病动物模型抗氧化指标及细胞形态学的影响[J].中西医结合心脑血管病杂志，2004，2（1）：27-30.
[150]曹园园. P-糖蛋白抑制剂粉防己碱联合还原性谷胱甘肽对帕金森病大鼠脑内多巴胺能系统的影响研究[D].中国优秀博硕士学位论文全文数据库（硕士）：苏州大学，2008.
[151]张波，汪瀚，鲍远程，等.抗震止痉胶囊合谷胱甘肽治疗帕金森病临床观察[J].中国中医急症，2010，7：1104-1105，1113.
[152]汪瀚，鲍远程，张波，等.还原型谷胱甘肽治疗帕金森病38例临床观察[J].中国实用医药，2006，3：13-15.
[153] Mischley LK, Vespignani MF, Finnell JS. Safety survey of intranasal glutathione[J]. J Altern Complement Med,2013,19(5):459-63.
[154] Gu L, Zhao M, Li W, et al. Chemical and cellular antioxidant activity of twonovel peptides designed based on glutathione structure[J]. Food Chem Toxicol,2012,50(11):4085-91.
[155] Martynova KV, Andreeva LA, Klimova PA, et al. Structural-functional study ofglycine-and-proline-containing peptides (glyprolines) as potential neuroprotectors[J]. Bioorg Khim,2009,35(2):165-71.
[156] Zhao HK, Chen BY, Chang R, et al. Vasonatrin peptide, a novel protector ofdopaminergic neurons against the injuries induced byn-methyl-4-phenylpyridiniums[J]. Peptides,2013,47:117-22.
[157] Choi H, Hwang JS, Kim H, et al. Antifungal effect of CopA3monomer peptidevia membrane-active mechanism and stability to proteolysis of enantiomericd-CopA3[J]. Biochem Biophys Res Commun,2013,440(1):94-8.
[158] Nam HJ, Oh AR, Nam ST, et al. The insect peptide CopA3inhibitslipopolysaccharide-induced macrophage activation[J]. J Pept Sci,2012,18(10):650-6.
[159] Kim SJ, Kim IW, Kwon YN, et al. Synthetic Coprisin analog peptide, D-CopA3has antimicrobial activity and pro-apoptotic effects in human leukemia cells[J]. JMicrobiol Biotechnol,2012,22(2):264-9.
[160] Nam ST, Kim DH, Lee MB, et al. Insect peptide CopA3-induced proteindegradation of p27Kip1stimulates proliferation and protects neuronal cells fromapoptosis[J]. Biochem Biophys Res Commun,2013,437(1):35-40.
[161] Borlongan CV, Wang Y, Su TP. Delta opioid peptide (D-Ala2, D-Leu5)enkephalin: linking hibernation and neuroprotection[J]. Front Biosci,2004,9:3392-8.
[162] Sen D, Huchital M, Chen YL. Crosstalk between Delta Opioid Receptor andNerve Growth Factor Signaling Modulates Neuroprotection and Differentiation inRodent Cell Models[J]. Int J Mol Sci,2013,14(10):21114-39.
[163]段冷昕.梅花鹿茸多肽的化学结构及其抗肝纤维化作用[D].中国优秀博硕士学位论文全文数据库（博士）：吉林大学，2007.
[164] Wang M, Zhang Z, Cheang LC, et al. Eriocaulon buergerianum extract protectsPC12cells and neurons in zebrafish against6-hydroxydopamine-induceddamage[J]. Chin Med.2011,6:16.
[165]孟金兰，兰爱平，郭瑞鲜，等.硫化氢对化学性缺氧诱导PC12细胞凋亡的影响[J].中山大学学报(医学科学版)，2010，31(1)：79-84.
[166] Jian-Wei Gao, Takuya Yamane, Hiroshi Maita, et al. DJ-1–Mediated ProtectiveEffect of Protocatechuic Aldehyde Against Oxidative Stress in SH-SY5Y Cells[J].J Pharmacol Sci,2011,115:36-44.
[167] Huang JZ, Chen YZ, Su M, et al. dl-3-n-Butylphthalide prevents oxidativedamage and reduces mitochondrial dysfunction in an MPP+-induced cellularmodel of Parkinson's disease[J]. Neurosci Lett,2010,475(2):89-94.
[168] Isaac G. Onyango. Mitochondrial Dysfunction and Oxidative Stress in Parkinson'sDisease[J]. Neurochem Res,2008,33(3):589-597.
[169] Perfeito R, Cunha-Oliveira T, Rego AC. Revisiting oxidative stress andmitochondrial dysfunction in the pathogenesis of Parkinson disease--resemblanceto the effect of amphetamine drugs of abuse[J]. Free Radic Biol Med,2012,53(9):1791-806.
[170] Hauser DN, Hastings TG. Mitochondrial dysfunction and oxidative stress inParkinson's disease and monogenic parkinsonism[J]. Neurobiol Dis,2013,51:35-42.
[171] Lim KL, Tan JM. Role of the ubiquitin proteasome system in Parkinson'sdisease[J]. BMC Biochem,2007,8, Suppl1: S13.
[172] Dudek EJ, Lampi KJ, Lampi JA, et al. Ubiquitin proteasome pathway-mediateddegradation of proteins: effects due to site-specific substrate deamidation[J].Invest Ophthalmol Vis Sci,2010,51(8):4164-73.
[173] Lee DH, Kim CS, Lee YJ. Astaxanthin protects against MPTP/MPP+-inducedmitochondrial dysfunction and ROS production in vivo and in vitro[J]. FoodChem Toxicol,2011,49(1):271-80.
[174] Johnson KA, Conn PJ, Niswender CM. Glutamate receptors as therapeutic targetsfor Parkinson's disease[J]. CNS Neurol Disord Drug Targets,2009,8(6):475-91.
[175] Won-Seok Choi, Shane E. Kruse, Richard D. Palmiter, et al. Mitochondrialcomplex I inhibition is not required for dopaminergic neuron death induced byrotenone, MPP+, or paraquat[J]. PNAS,2008,105(39):15136–15141.
[176] Rodriguez-Rocha H, Garcia-Garcia A, Pickett C, et al. Compartmentalizedoxidative stress in dopaminergic cell death induced by pesticides and complex Iinhibitors: Distinct roles of superoxide anion and superoxide dismutases[J]. FreeRadic Biol Med,2013,61C:370-383.
[177] Zhang S, Ding JH, Zhou F, et al. Iptakalim ameliorates MPP+-induced astrocytemitochondrial dysfunction by increasing mitochondrial complex activity besidesopening mitoK(ATP) channels[J]. J Neurosci Res,2009,87(5):1230-9.
[178] Ying R, Liang HL, Whelan HT, et al. Pretreatment with near-infrared light vialight-emitting diode provides added benefit against rotenone-and MPP+-inducedneurotoxicity[J]. Brain Res,2008,1243:167-73.
[179] Qing Zhu, Jing Wang, Yunjian Zhang, et al. Mechanisms of MPP+-induced PC12cell apoptosis via reactive oxygen species[J]. J Huazhong U Sci-Med,2012,32(6):861-866.
[180] Wang M, Wey S, Zhang Y, et al. Role of the Unfolded Protein ResponseRegulator GRP78/BiP in Development, Cancer, and Neurological Disorders[J].Antioxid Redox Signal,2009,11(9):2307-16.
[181] Holtz WA, Turetzky JM, Jong YJ, et al. Oxidative stress-triggered unfoldedprotein response is upstream of intrinsic cell death evoked by parkinsonianmimetics[J]. J Neurochem,2006,99(1):54-69.
[182] Szegezdi E, Logue SE, Gorman AM, et al. Mediators of endoplasmic reticulumstress-induced apoptosis[J]. EMBO Rep,2006,7(9):880-5.
[183] Nishitoh H, Matsuzawa A, Tobiume K, et al. ASK1is essential for endoplasmicreticulum stress-induced neuronal cell death triggered by expanded polyglutaminerepeats[J]. Genes&Dev,2002,16:1345-1355.
[184] Antoine Fouillet, Clémence Levet, Angélique Virgone, et al. ER stress inhibitsneuronal death by promoting autophagy[J]. Autophagy,2012,8(6):915-26.
[2] Naohiro Egawa, Keisuke Yamamoto, Haruhisa Inoue, et al The EndoplasmicReticulum Stress Sensor,ATF6α,Protects against Neurotoxin-induced Dopaminer-gic Neuronal Death[J]. J Biol Chem,2011,286(10):7947–7957.
[3]王晟东，白洁.内质网应激与帕金森病[J].生命科学，2010，22（4）：326-330
[4] Brasnjevic I, Steinbusch HW, Schmitz C, et al. Delivery of peptide and proteindrugs over the blood-brain barrier[J]. Prog Neurobiol,2009,87(4):212-51.
[5]郝丽，李和平，严历.东北梅花鹿鹿茸尖端组织全长cDNA文库的构建[J].东北林业大学学报，2009，37（11）：120-122.
[6] Lu LJ, Chen L, Meng XT, et al. Biological effect of velvet antler polypeptides onneural stem cells from embryonic rat brain[J]. Chin Med J (Engl),2005,118(1):38-42.
[7] Li C, Stanton JA, Robertson TM, et al. Nerve growth factor mRNA expression inthe regenerating antler tip of red deer (Cervus elaphus)[J]. PLoS One,2007,2(1):e148.
[8] Lai AK, Hou WL, Verdon DJ, et al. The distribution of the growth factors FGF-2and VEGF, and their receptors, in growing red deer antler[J]. Tissue Cell,2007,39(1):35-46.
[9] Lord EA, Martin SK, Gray JP, et al. Cell cycle genes PEDF and CDKN1C ingrowing deer antlers[J]. Anat Rec (Hoboken),2007,290(8):994-1004.
[10]严铭铭，曲晓波，王旭，等.梅花鹿茸中活性多肽的纯化、测序及功能研究[J].高等学校化学学报，2007，28(10)：1893-1896.
[11] Guan SW, Duan LX, Li YY, et al. A novel polypeptide from Cervus nipponTemminck proliferation of epidermal cells and NIH3T3cell line[J]. ActaBiochimica Polonica,2006,53(2):395-397.
[12]张郑瑶，段冷昕，周秋丽，等.梅花鹿茸多肽的化学结构及生物活性[J].高等学校化学学报，2012，33（9）：2000-2004.
[13]郑帆，李仁宽，王辉林，等.鹿茸冻干粉的酶解及其酶解产物性质的研究[J].中国中药杂志，2010，35（19）：2628-2633.
[14] Srinivasulu Chigurupati, Zelan Wei, Cherine Belal, et al. The Homocysteine-inducibleEndoplasmic Reticulum Stress Protein Counteracts Calcium Store Depletion andInduction of CCAAT Enhancer-binding Protein Homologous Protein in aNeurotoxin Model of Parkinson Disease[J]. J Biol Chem,2009,284(27):18323-18333.
[15]王虎，蔡定芳.帕金森病与内质网应激[J].国际神经病学神经外科学杂志，2007，34（1）：86-90
[16]邓娟，白洁. TRPC1和Ca2+在帕金森病内质网应激中的作用[J].生命科学，2012，24（10）：1169-1173.
[17]高俊鹏，蔡定芳.内质网应激介导神经变性疾病错折叠蛋白的神经毒性[J].中国病理生理杂志，2008，24（1）：201-205.
[18] Pan C, Giraldo GS, Prentice H, et al. Taurine protection of PC12cells againstendoplasmic reticulum stress induced by oxidative stress[J]. Journal ofBiomedical Science,17(Suppl1): S17.
[19] Qu LiFeng, Liu Zhen, Zhang, H. F. M., et al. Endoplasmic reticulumstress-induced cell survival and apoptosis[J]. Journal of Chinese ClinicalMedicine,2009,4(8):452-459.
[20] Lebouvier T, Chaumette T, Paillusson S, et al. The second brain and Parkinson'sdisease[J]. Eur J Neurosci,2009,30(5):735-41.
[21]邱会卿，顾平，樊文峰.经颅脑实质超声诊断帕金森病的临床应用[J].临床误诊误治，2011，10：92-94.
[22]刘小平.利用果蝇模型研究帕金森病相关基因α-synuclein，PINK1和LRRK2的作用机制[D].中国优秀博硕士学位论文全文数据库（博士）：中南大学，2007.
[23]王晟东.环境毒性物质MPTP导致多巴胺能神经元损伤机理研究[D].中国优秀博硕士学位论文全文数据库（博士）：昆明理工大学，2011.
[24]陈茹.帕金森病研究进展[J].中国康复理论与实践，2007，13（7）：637-639
[25]刘人恺，肖波，杨晓苏，等.星形诺卡菌对多巴胺能细胞损伤的体外研究[J].卒中与神经疾病，2008，15（5）：259-263.
[26] Noyce AJ, Bestwick JP, Silveira-Moriyama L, et al. Meta-analysis of earlynonmotor features and risk factors for Parkinson disease[J]. Ann Neurol,2012,72(6):893-901.
[27]张玉梅.长期低剂量皮下注射鱼藤酮制备多巴胺神经元损伤模型及其机制研究[D].中国优秀博硕士学位论文全文数据库（博士）：大连医科大学，2009.
[28]李伟静，于群.辅酶Q10的生理作用及临床应用[J].生物技术通讯，2007，18（5）：882-884.
[29]姜宁.增龄和预运动训练在小鼠帕金森发病中的作用：线粒体与自噬途径的分子生理学机制研究[D].中国优秀博硕士学位论文全文数据库（博士）：中国人民解放军军事医学科学院，2012.
[30] Thomas RR, Keeney PM, Bennett JP. Impaired complex-I mitochondrialbiogenesis in Parkinson disease frontal cortex[J]. J Parkinsons Dis,2012,2(1):67-76.
[31] Sanders LH, Timothy Greenamyre J. Oxidative damage to macromolecules inhuman Parkinson disease and the rotenone model[J]. Free Radic Biol Med,2013,62:111-20.
[32]黄桂平.帕金森病的临床研究及展望[J].医学综述，2008，14（13）：2023-2026
[33]梁天佳，莫雪安.帕金森病发病机制研究进展[J].医学综述，2008，14（7）：1040-1042.
[34] Galligan JJ, Petersen DR. The human protein disulfide isomerase gene family[J].Hum Genomics,2012,6:6.
[35]项喜艳. DTT诱发内质网应激-自噬过程中活性氧的作用及其调节机制[D].中国优秀博硕士学位论文全文数据库（硕士）：吉林大学，2012.
[36]陈冬.帕金森病相关蛋白Parkin对自噬调节的研究[D].中国优秀博硕士学位论文全文数据库（博士）：中国科学技术大学，2010.
[37]叶青，郑民华.自噬的分子机制与病理生理意义[J].国际病理科学与临床杂志，2007，27（4）：358-362.
[38] Vogiatzi T, Xilouri M, Vekrellis K, et al. Wild Type-Synuclein Is Degraded byChaperone-mediated Autophagy and Macroautophagy in Neuronal Cells[J]. JBiol Chem,2008,283(35):23542-56.
[39] Zheng HF, Yang YP, Hu LF, et al. Autophagic impairment contributes to systemicinflammation-induced dopaminergic neuron loss in the midbrain[J]. PLoS One,2013,8(8): e70472.
[40]杨丽娟，董军，陆大祥，等.姜黄素对LPS激活小胶质细胞条件培养基诱导损伤的海马神经元保护作用及机制[J].中国病理生理杂志，2010，26（4）：742-747.
[41]陈生第.帕金森病.第1版[M].北京：人民卫生出版社，2006：110.
[42]张治国.黄芪甲苷对帕金森病神经元的保护作用及其机制研究[D].中国优秀博硕士学位论文全文数据库（硕士）：第四军医大学，2012.
[43]牛英才，潘志，李晓明，等.葛根异黄酮对MPP+诱导的PC12细胞凋亡的保护作用[J].中国药理学通报，2009，25（1）：112-115.
[44] Lee DH, Kim CS, Lee YJ. Astaxanthin protects against MPTP/MPP+-inducedmitochondrial dysfunction and ROS production in vivo and in vitro[J]. FoodChem Toxicol,2011,49(1):271-80.
[45] Zhang SP, Du XG, Pu XP.3-O-demethylswertipunicoside protects againstoxidative toxicity in PC12cells[J]. Biol Pharm Bull,2010,33(9):1529-33.
[46]黄金忠. PC12细胞中MPP+诱导的α-synuclein聚集和自噬对其降解机制研究
[D].中国优秀博硕士论文全文数据库，苏州大学，2010.
[47] Yim SB, Park SE, Lee CS. Protective effect of glycyrrhizin on1-methyl-4-phenylpyridinium-induced mitochondrial damage and cell death in differentiatedPC12cells[J]. J Pharmacol Exp Ther,2007,321(2):816-22.
[48]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.
[49] Zhang LJ, Xue YQ, Yang C, et al. Human Albumin Prevents6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and InVivo[J]. PLoS One,2012,7(7): e41226.
[50]王丹萍.6-羟基多巴胺诱导的SH-SY5Y细胞帕金森病模型差异蛋白质组学研究[D].中国优秀博硕士学位论文全文数据库（博士）：吉林大学，2010.
[51]孙家贺.帕金森病现代中医证治规律研究[D].中国优秀博硕士学位论文全文数据库（硕士）：山东中医药大学，2012.
[52]厚荣荣. EGCG对PQ诱导的PC12细胞凋亡的保护作用及其机制研究[D].中国优秀博硕士学位论文全文数据库（硕士）：第四军医大学，2007.
[53] Cabeza-Arvelaiz Y, Schiestl RH. Transcriptome analysis of a rotenone model ofparkinsonism reveals complex I-tied and-untied toxicity mechanisms common toneurodegenerative diseases[J]. PLoS One,2012,7(9): e44700.
[54]张劲松，栾春业，王强，等.代森锰锌对PC-12细胞凋亡的影响[J].中国药理学与毒理学杂志，2011，25（4）：349-353.
[55]贾琳琳，李术. α-突触核蛋白在神经损伤中的作用[J].畜牧与兽医，2011，43（2）：88-90.
[56] Choi WS, Abel G, Klintworth H, et al. JNK3mediates paraquat-and rotenone-induced dopaminergic neuron death[J]. J Neuropathol Exp Neurol,2010,69(5):511-20.
[57]李海龙，粟艳，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.
[58] Sun ZW, Zhang L, Zhu SJ, et al. Excitotoxicity effects of glutamate on humanneuroblastoma SH-SY5Y cells via oxidative damage[J]. Neurosci Bull,2010,26(1):8-16.
[59]何秀全.罗格列酮通过抑制小胶质细胞活化而保护多巴胺能神经元的研究[D].中国优秀博硕士学位论文全文数据库（博士）：山东大学，2012.
[60] Kim SJ, Park JH, Kim KH, et al. The Protective Effect of Apamin onLPS/Fat-Induced Atherosclerotic Mice[J]. Evid Based Complement Alternat Med,2012,2012:305454.
[61] Son DJ, Lee JW, Lee YH, et al. Therapeutic application of anti-arthritis,pain-releasing, and anti-cancer effects of bee venom and its constituentcompounds[J]. Pharmacol Ther,2007,115(2):246-70.
[62]王忠田.蜂毒有助于治疗帕金森病[J].蜜蜂杂志，2013，1：31.
[63] Doo A-R, Kim S, Kim S, et al. Neuroprotective effects of bee venompharmaceutical acupuncture in acute1-Parkinson's disease[J]. NeurologicalResearch Suppl,2010,1:88–91.
[64] Kim J, Yang EJ, Lee M, et al. Bee venom reduces neuroinflammation in theMPTP-induced model of Parkinson's disease[J]. Int J Neurosci,2011,121:209–217.
[65] Doo AR, Kim SN, Kim ST, et al. Bee venom protects SH-SY5Y humanneuroblastoma cells from1-methyl-4-phenylpyridinium-induced apoptotic celldeath[J]. Brain Res,2012,1429:106-15.
[66] Alvarez-Fischer D, Noelker C, Vulinovi F, et al. Bee Venom and Its ComponentApamin as Neuroprotective Agents in a Parkinson Disease Mouse Model[J].PLoS ONE,2013,6(11): e61700.
[67]肖昭扬，殷盛明，于德钦，等.蝎毒对C57BL/6帕金森病小鼠脑内前脑啡肽原表达的影响[J].中国临床康复，2006，10（19）：33-36.
[68]殷盛明，于德钦，安宁，等.蝎毒耐热蛋白对MPTP诱导的帕金森病小鼠脑内小胶质细胞的影响[J].中国应用生理学杂志，2009，25（1）：79-80，90.
[69]殷盛明，于德钦，彭岩，等.神经元营养活性肽对帕金森病大鼠小胶质细胞保护作用及机制研究[J].中草药，2010，41（11）：1838-1842.
[70] Trejo E, Borges A, Na ez B, et al. Tityus zulianus venom induces massivecatecholamine release from PC12cells and in a mouse envenomation model[J].Toxicon,2012,59(1):117-23.
[71]陈健.东亚钳蝎毒素Martentoxin的分离纯化与功能研究[D].中国优秀博硕士学位论文全文数据库（硕士）：南京医科大学，2011.
[72]罗素兰，长孙东亭，吴勇，等.海南产芋螺毒素在疼痛与成瘾治疗中的应用前景[J].中国药理学与毒理学杂志，2012，（3）：439-440.
[73] Balsara R, Li N, Weber-Adrian D, et al. Opposing action of conantokin-G onsynaptically and extrasynaptically-activated NMDA receptors[J]. Neurophar-macology,2012,62(7):2227-38.
[74] Gowd KH, Watkins M, Twede VD, et al. Characterization of conantokin Rl-A:molecular phylogeny as structure/function study[J]. J Pept Sci,2010,16(8):375-82.
[75] Sheng Z, Liang Z, Geiger JH, et al. The selectivity of conantokin-G for ionchannel inhibition of NR2B subunit-containing NMDA receptors is regulated byamino acid residues in the S2region of NR2B[J]. Neuropharmacology,2009,57(2):127-36.
[76]任展宏，朱永平.芋螺毒素conantokin的药理作用及构效关系研究进展[J].中国药学杂志，2007，42（23）：1761-1764.
[77] Surin AM, Kriukova EV, Strukov AS, et al. Effect of alpha-conotoxin MII and itsN-terminal derivatives on Ca2+and Na+signals induced by nicotine inneuroblastoma cell line SH-SY5Y[J]. Bioorg Khim,2012,38(2):214-22.
[78] Zheng L, Su G, Ren J, et al. Isolation and characterization of an oxygen radicalabsorbance activity peptide from defatted peanut meal hydrolysate and itsantioxidant properties[J]. J Agric Food Chem,2012,6(21):5431-7.
[79]郝丽，李和平，严历.梅花鹿鹿茸尖端组织ESTs分析[J].遗传，2011，33（4）：371-377.
[80]张郑瑶，尚禹东，牟凤辉，等.鹿茸多肽及其生长因子制备工艺的研究进展[J].特产研究，2011，3：69-72.
[81] Li C, Stanton JA, Robertson TM, et al. Nerve growth factor mRNA expression inthe regenerating antler tip of red deer (Cervus elaphus)[J]. PLoS One,2007,2(1):e148.
[82] Lai AK, Hou WL, Verdon DJ, et al. The distribution of the growth factors FGF-2and VEGF, and their receptors, in growing red deer antler[J]. Tissue Cell,2007,39(1):35-46.
[83]严铭铭，曲晓波，王旭，等.梅花鹿茸中活性多肽的纯化、测序及功能研究[J].高等学校化学学报,2007,28(10):1893-1896.
[84] Guan SW, Duan LX, Li YY, et al. A novel polypeptide from Cervus nipponTemminck proliferation of epidermal cells and NIH3T3cell line[J]. ActaBiochimica Polonica,2006,53(2):395-397.
[85]张郑瑶，段冷昕，周秋丽，等.梅花鹿茸多肽的化学结构及生物活性[J].高等学校化学学报，2012，33（9）：2000-2004.
[86]柯李晶，聂毅磊，叶秀云，等.梅花鹿鹿茸中促PC12细胞增殖蛋白的分离和活性研究[J].中草药，2009，40（5）：715-718.
[87] Bourgault S, Vaudry D, Botia B, et al. Novel stable PACAP analogs with potentactivity towards the PAC1receptor[J]. Peptides.2008,29(6):919-32.
[88] Ayako Miura, Naoko Odahara, Aiko Tominaga, et al. Regulatory mechanism ofPAC1gene expression via Sp1by nerve growth factor in PC12cells[J]. FEBSLetters,2012,568,1731-1735.
[89] Reglodi D, Kiss P, Lubics A, et al. Review on the protective effects of PACAP inmodels of neurodegenerative diseases in vitro and in vivo[J]. Curr Pharm Des,2011,17(10):962-72.
[90] Manecka DL, Mahmood SF, Grumolato L, et al. Pituitary adenylatecyclase-activating polypeptide (PACAP) promotes both survival andneuritogenesis in PC12cells through activation of nuclear factor κB (NF-κB)pathway: involvement of extracellular signal-regulated kinase (ERK), calcium,and c-REL[J]. J Biol Chem,2013,288(21):14936-48.
[91] Emery AC, Eiden LE. Signaling through the neuropeptide GPCR PAC1inducesneuritogenesis via a single linear cAMP-and ERK-dependent pathway using anovel cAMP sensor[J]. FASEB J,2012,26(8):3199-211.
[92] Zhang W, Smith A, Liu JP, et al. GSK3beta modulates PACAP-inducedneuritogenesis in PC12cells by acting downstream of Rap1in a caveolae-dependent manner[J]. Cell Signal,2009,21(2):237-45.
[93] Dejda A, Chan P, Seaborn T, et al. Involvement of stathmin1in the neurotrophiceffects of PACAP in PC12cells[J]. J Neurochem,2010,114(5):1498-510.
[94] Miura A, Odahara N, Tominaga A, et al. Regulatory mechanism of PAC1geneexpression via Sp1by nerve growth factor in PC12cells[J]. FEBS Lett,2012,586(12):1731-5.
[95] Raoult E, Roussel BD, Bénard M, et al. Pituitary adenylate cyclase-activatingpolypeptide (PACAP) stimulates the expression and the release of tissueplasminogen activator (tPA) in neuronal cells: involvement of tPA in theneuroprotective effect of PACAP[J]. J Neurochem,2011,119(5):920-31.
[96] Mahata M, Zhang K, Gayen JR, et al. Catecholamine biosynthesis and secretion:physiological and pharmacological effects of secretin[J]. Cell Tissue Res,2011,345(1):87-102.
[97]毛姗姗，张咏梅. PACAP对神经系统疾病保护作用的研究进展[J].生理科学进展，2011，42（4）：276-280.
[98] Lingyan Zhang, Wen Liu, Karen K. Szumlinskid, et al. p10, the N-terminaldomain of p35, protects against CDK5/p25-induced neurotoxicity[J]. PNAS,2012,109(49):20041–20046.
[99] Oh YM, Jang EH, Ko JH, et al. Inhibition of6-hydroxydopamine-inducedendoplasmic reticulum stress by l-carnosine in SH-SY5Y cells. Neurosci Lett,2009,459(1):7-10.
[100] Boldyrev AA, Stvolinsky SL, Fedorova TN, et al. Carnosine as a naturalantioxidant and geroprotector: from molecular mechanisms to clinical trials[J].Rejuvenation Res,2010,13(2-3):156-8.
[101]沈耀，胡薇薇，陈忠.肌肽和中枢神经系统疾病[J].浙江大学学报（医学版），2007，3（2）：199-203.
[102] oban J, Bingül I, Yesil-Mizrak K, et al. Effects of carnosine plus vitamin E andbetaine treatments on oxidative stress in some tissues of aged rats[J]. Curr AgingSci,2013,6(2):199-205.
[103] Banerjee S, Poddar MK. Platelet monoamine oxidase-A activity and aging: effectof carnosine[J]. J Physiol Sci,2013,63(4):279-85.
[104] Kulebyakin K, Karpova L, Lakonsteva E, et al. Carnosine protects neuronsagainst oxidative stress and modulates the time profile of MAPK cascadesignaling[J]. Amino Acids,2012,43(1):91-6.
[105]朱传兵，余国英.脑苷肌肽治疗帕金森病的临床观察[J].中国老年保健医学，2005，3（3）：43-44.
[106] Grasso GI, Bellia F, Arena G, et al. Noncovalent interaction-driven stereose-lectivity of copper(II) complexes with cyclodextrin derivatives of L-andD-carnosine[J]. Inorg Chem,2011,50(11):4917-24.
[107] Bellia F, Vecchio G, Rizzarelli E. Carnosine derivatives: new multifunctionaldrug-like molecules[J]. Amino Acids,2012,43(1):153-63.
[108] Monti JM, Torterolo P, Lagos P. Melanin-concentrating hormone control ofsleep-wake behavior[J]. Sleep Med Rev,2013,17(4):293-8.
[109]孙丽丽，张艳，王君，等.伏隔核黑色素聚集激素在大鼠甲基苯丙胺行为敏化中的作用[J].中国药理学与毒理学杂志，2012，26（3）：430.
[110] Cotta-Grand N, Rovère C, Guyon, A et al. Melanin-concentrating hormoneinduces neurite outgrowth in human neuroblastoma SH-SY5Y cells through p53and MAPKinase signaling pathways[J]. Peptides,2009,30(11):2014-24.
[111]张建敏，高培，魏静.帕金森病患者睡眠障碍现况调查及护理对策[J].中国社区医师：医学专业，2010，8：175-176.
[112]吴佳英，李琳，刘振国.帕金森病患者睡眠障碍发病机制的研究[J].上海交通大学学报：医学版，2009，29（8）：1001-1004.
[113] Oz S, Ivashko-Pachima Y, Gozes I. The ADNP derived peptide, NAP modulatesthe tubulin pool: implication for neurotrophic and neuroprotective activities[J].PLoS One,2012,7(12): e51458.
[114] Fleming SM, Mulligan CK, Richter F, et al. A pilot trial of themicrotubule-interacting peptide (NAP) in mice overexpressing alpha-synucleinshows improvement in motor function and reduction of alpha-synucleininclusions[J]. Mol Cell Neurosci,2011,46(3):597-606.
[115] Sharma NK, Sethy NK, Meena RN, et al. Activity-dependent neuroprotectiveprotein (ADNP)-derived peptide (NAP) ameliorates hypobaric hypoxia inducedoxidative stress in rat brain[J]. Peptides,2011,32(6):1217-24.
[116]杨宇，吴江，杨欣，等.分泌表达神经保护短肽重组慢病毒对SK-N-SH细胞的保护作用[J].中国老年学杂志，2007，27（3）：213-216.
[117] Liu Z, Gao X, Kang T, et al. B6peptide-modified PEG-PLA nanoparticles forenhanced brain delivery of neuroprotective peptide[J]. Bioconjug Chem,2013,24(6):997-1007.
[118] Liu Z, Jiang M, Kang T, et al. Lactoferrin-modified PEG-co-PCL nanoparticlesfor enhanced brain delivery of NAP peptide following intranasal administration[J].Biomaterials,2013,34(15):3870-81.
[119]郭芬，黄晓锋，李实骞，等.鞘脂激活蛋白原Prosaspoin与Rhox5间的功能性相互作用[J].遗传学报：英文版，2007，34（5）：392-399.
[120] Gao HL, Li C, Nabeka H, et al. Attenuation of MPTP/MPP(+) toxicity in vivoand in vitro by an18-mer peptide derived from prosaposin[J]. Neuroscience,2013,236:373-93.
[121] Khan RS, Yu C, Kastin AJ, et al. Brain Activation by Peptide Pro-Leu-Gly-NH2(MIF-1)[J]. Int J Pept,2010. doi:10.1155/2010/537639.
[122]唐丽灵，李大军，王远亮.外力作为信号诱导基因的选择性剪接与力生长因子表达[J].生物化学与生物物理进展，2007，34（5）：454-459.
[123] Quesada A, Micevych P, Handforth A. C-terminal mechano growth factor protectsdopamine neurons: a novel peptide that induces heme oxygenase-1[J]. ExpNeurol,2009,220(2):255-66.
[124] Pirnik Z, Maixnerova J, Matyskova R, et al. Effect of anorexinergic peptides,cholecystokinin (CCK) and cocaine and amphetamine regulated transcript (CART)peptide, on the activity of neurons in hypothalamic structures of C57Bl/6miceinvolved in the food intake regulation[J]. Peptides,2009,31:139–144.
[125] Jones DC, Kuhar MJ. CART receptor binding in primary cell cultures of the ratnucleus accumbens[J]. Synapse,2008,62:122–127.
[126] Lin Y, Hall RA, Kuhar MJ. CART peptide stimulation of G protein-mediatedsignaling in differentiated PC12cells: identification of PACAP6-38as a CARTreceptor antagonist[J]. Neuropeptides,2011,45(5):351-8.
[127] Chan NY, Robador PA, Levi R. Natriuretic peptide-induced catecholamine releasefrom cardiac sympathetic neurons: inhibition by histamine H3and H4receptoractivation[J]. J Pharmacol Exp Ther,2012,343(3):568-77.
[128] Wu Y, Shang Y, Sun SG, et al. Protective effect of erythropoietin against1-methyl-4-phenylpyridinium-induced neurodegenaration in PC12cells[J].Neurosci Bull,2007,23(3):156-64.
[129] Phani S, Jablonski M, Pelta-Heller J, et al. Gremlin is a novel VTA derivedneuroprotective factor for dopamine neurons[J]. Brain Res,2013,1500:88-98.
[130] Liu Y, Pan W, Yang S, et al. Interleukin-22protects rat PC12pheochromocytomacells from serum deprivation-induced cell death[J]. Mol Cell Biochem,2012,371(1-2):137-46.
[131]潘文艳. IL-22在大鼠嗜铬瘤细胞PC12中生物学作用的研究[D].中国优秀博硕士学位论文全文数据库（硕士）：扬州大学，2012.
[132] Duarte AI, Candeias E, Correia SC, et al. Crosstalk between diabetes and brain:glucagon-like peptide-1mimetics as a promising therapy against neurodegener-ation[J]. Biochim Biophys Acta,2013,1832(4):527-41.
[133]张蕾，周舟，张广斌，等.微波暴露对大鼠GLP-1及其受体表达的影响及与学习记忆功能损伤的关系[J].辐射研究与辐射工艺学报，2007，25（3）：180-184.
[134] Biswas SC, Buteau J, Greene LA. Glucagon-like peptide-1(GLP-1) diminishesneuronal degeneration and death caused by NGF deprivation by suppressing Biminduction[J]. Neurochem Res,2008,33(9):1845-51.
[135] Li Y, Tweedie D, Mattson MP, et al. Enhancing the GLP-1receptor signalingpathway leads toproliferation and neuroprotection in human neuroblastomacells[J]. J Neurochem,2010,113(6):1621-31.
[136]全会标，高勇义. Exendin-4临床研究进展[J].中国热带医学，2011，11（3）：388-390.
[137] Luciani P, Deledda C, Benvenuti S, et al. Exendin-4induces cell adhesion anddifferentiation and counteracts the invasive potential of human neuroblastomacells[J]. PLoS One,2013,8(8): e71716.
[138] Holscher C. Central effects of GLP-1: new opportunities for treatments ofneurodegenerative diseases[J]. J Endocrinol,2013, doi:10.1530/JOE-13-0221.
[139] Harkavyi A, Abuirmeileh A, Lever R, et al. Glucagon-like peptide1receptorstimulation reverses key deficits in distinct rodent models of Parkinson'sdisease[J]. J Neuroinflammation,2008,5:19.
[140] Bertilsson G, Patrone C, Zachrisson O, et al. Peptide hormone exendin-4stimulates subventricular zone neurogenesis in the adult rodent brain and inducesrecovery in an animal model of Parkinson's disease[J]. J Neurosci Res,2008,86(2):326-38.
[141] Li Y, Perry T, Kindy MS, et al. GLP-1receptor stimulation preserves primarycortical and dopaminergic neurons in cellular and rodent models of stroke andParkinsonism[J]. Proc Natl Acad Sci U S A,2009,106(4):1285-90.
[142]段冷昕，李瑞芳，金瑾，等.黄连解毒汤对MPTP所致帕金森病小鼠的防治作用[J].中国老年学杂志，2010，30（14）：2004-2006.
[143] Heales SJ, Menzes A, Davey GP. Depletion of glutathione does not affect electrontransport chain complex activity in brain mitochondria: Implications forParkinson disease and postmortem studies[J]. Free Radic Biol Med,2011,50(7):899-902.
[144] Yürekli VA, Gürler S, Naz rolu M, et al. Zonisamide attenuates MPP+-inducedoxidative toxicity through modulation of Ca2+signaling and caspase-3activity inneuronal PC12cells[J]. Cell Mol Neurobiol,2013,33(2):205-12.
[145] Delic M, Rebnegger C, Wanka F, et al. Oxidative protein folding and unfoldedprotein response elicit differing redox regulation in endoplasmic reticulum andcytosol of yeast[J]. Free Radic Biol Med,2012,52(9):2000-12.
[146] Lee M, Kwon BM, Suk K, et al. Effects of obovatol on GSH depletedglia-mediated neurotoxicity and oxidative damage[J]. J Neuroimmune Pharmacol,2012,7(1):173-86.
[147] Zhou W, Bercury K, Cummiskey J, et al. Phenylbutyrate up-regulates the DJ-1protein and protects neurons in cell culture and in animal models of Parkinsondisease[J]. J Biol Chem,2011,286(17):14941-51.
[148]罗蔚锋，包仕尧，刘春风，等.还原型谷胱苷肽治疗帕金森病大鼠模型的实验研究[J].临床神经病学杂志，2006，19（6）：441-443.
[149]陆建明，周厚广，鲍远程.谷胱甘肽合中药对帕金森病动物模型抗氧化指标及细胞形态学的影响[J].中西医结合心脑血管病杂志，2004，2（1）：27-30.
[150]曹园园. P-糖蛋白抑制剂粉防己碱联合还原性谷胱甘肽对帕金森病大鼠脑内多巴胺能系统的影响研究[D].中国优秀博硕士学位论文全文数据库（硕士）：苏州大学，2008.
[151]张波，汪瀚，鲍远程，等.抗震止痉胶囊合谷胱甘肽治疗帕金森病临床观察[J].中国中医急症，2010，7：1104-1105，1113.
[152]汪瀚，鲍远程，张波，等.还原型谷胱甘肽治疗帕金森病38例临床观察[J].中国实用医药，2006，3：13-15.
[153] Mischley LK, Vespignani MF, Finnell JS. Safety survey of intranasal glutathione[J]. J Altern Complement Med,2013,19(5):459-63.
[154] Gu L, Zhao M, Li W, et al. Chemical and cellular antioxidant activity of twonovel peptides designed based on glutathione structure[J]. Food Chem Toxicol,2012,50(11):4085-91.
[155] Martynova KV, Andreeva LA, Klimova PA, et al. Structural-functional study ofglycine-and-proline-containing peptides (glyprolines) as potential neuroprotectors[J]. Bioorg Khim,2009,35(2):165-71.
[156] Zhao HK, Chen BY, Chang R, et al. Vasonatrin peptide, a novel protector ofdopaminergic neurons against the injuries induced byn-methyl-4-phenylpyridiniums[J]. Peptides,2013,47:117-22.
[157] Choi H, Hwang JS, Kim H, et al. Antifungal effect of CopA3monomer peptidevia membrane-active mechanism and stability to proteolysis of enantiomericd-CopA3[J]. Biochem Biophys Res Commun,2013,440(1):94-8.
[158] Nam HJ, Oh AR, Nam ST, et al. The insect peptide CopA3inhibitslipopolysaccharide-induced macrophage activation[J]. J Pept Sci,2012,18(10):650-6.
[159] Kim SJ, Kim IW, Kwon YN, et al. Synthetic Coprisin analog peptide, D-CopA3has antimicrobial activity and pro-apoptotic effects in human leukemia cells[J]. JMicrobiol Biotechnol,2012,22(2):264-9.
[160] Nam ST, Kim DH, Lee MB, et al. Insect peptide CopA3-induced proteindegradation of p27Kip1stimulates proliferation and protects neuronal cells fromapoptosis[J]. Biochem Biophys Res Commun,2013,437(1):35-40.
[161] Borlongan CV, Wang Y, Su TP. Delta opioid peptide (D-Ala2, D-Leu5)enkephalin: linking hibernation and neuroprotection[J]. Front Biosci,2004,9:3392-8.
[162] Sen D, Huchital M, Chen YL. Crosstalk between Delta Opioid Receptor andNerve Growth Factor Signaling Modulates Neuroprotection and Differentiation inRodent Cell Models[J]. Int J Mol Sci,2013,14(10):21114-39.
[163]段冷昕.梅花鹿茸多肽的化学结构及其抗肝纤维化作用[D].中国优秀博硕士学位论文全文数据库（博士）：吉林大学，2007.
[164] Wang M, Zhang Z, Cheang LC, et al. Eriocaulon buergerianum extract protectsPC12cells and neurons in zebrafish against6-hydroxydopamine-induceddamage[J]. Chin Med.2011,6:16.
[165]孟金兰，兰爱平，郭瑞鲜，等.硫化氢对化学性缺氧诱导PC12细胞凋亡的影响[J].中山大学学报(医学科学版)，2010，31(1)：79-84.
[166] Jian-Wei Gao, Takuya Yamane, Hiroshi Maita, et al. DJ-1–Mediated ProtectiveEffect of Protocatechuic Aldehyde Against Oxidative Stress in SH-SY5Y Cells[J].J Pharmacol Sci,2011,115:36-44.
[167] Huang JZ, Chen YZ, Su M, et al. dl-3-n-Butylphthalide prevents oxidativedamage and reduces mitochondrial dysfunction in an MPP+-induced cellularmodel of Parkinson's disease[J]. Neurosci Lett,2010,475(2):89-94.
[168] Isaac G. Onyango. Mitochondrial Dysfunction and Oxidative Stress in Parkinson'sDisease[J]. Neurochem Res,2008,33(3):589-597.
[169] Perfeito R, Cunha-Oliveira T, Rego AC. Revisiting oxidative stress andmitochondrial dysfunction in the pathogenesis of Parkinson disease--resemblanceto the effect of amphetamine drugs of abuse[J]. Free Radic Biol Med,2012,53(9):1791-806.
[170] Hauser DN, Hastings TG. Mitochondrial dysfunction and oxidative stress inParkinson's disease and monogenic parkinsonism[J]. Neurobiol Dis,2013,51:35-42.
[171] Lim KL, Tan JM. Role of the ubiquitin proteasome system in Parkinson'sdisease[J]. BMC Biochem,2007,8, Suppl1: S13.
[172] Dudek EJ, Lampi KJ, Lampi JA, et al. Ubiquitin proteasome pathway-mediateddegradation of proteins: effects due to site-specific substrate deamidation[J].Invest Ophthalmol Vis Sci,2010,51(8):4164-73.
[173] Lee DH, Kim CS, Lee YJ. Astaxanthin protects against MPTP/MPP+-inducedmitochondrial dysfunction and ROS production in vivo and in vitro[J]. FoodChem Toxicol,2011,49(1):271-80.
[174] Johnson KA, Conn PJ, Niswender CM. Glutamate receptors as therapeutic targetsfor Parkinson's disease[J]. CNS Neurol Disord Drug Targets,2009,8(6):475-91.
[175] Won-Seok Choi, Shane E. Kruse, Richard D. Palmiter, et al. Mitochondrialcomplex I inhibition is not required for dopaminergic neuron death induced byrotenone, MPP+, or paraquat[J]. PNAS,2008,105(39):15136–15141.
[176] Rodriguez-Rocha H, Garcia-Garcia A, Pickett C, et al. Compartmentalizedoxidative stress in dopaminergic cell death induced by pesticides and complex Iinhibitors: Distinct roles of superoxide anion and superoxide dismutases[J]. FreeRadic Biol Med,2013,61C:370-383.
[177] Zhang S, Ding JH, Zhou F, et al. Iptakalim ameliorates MPP+-induced astrocytemitochondrial dysfunction by increasing mitochondrial complex activity besidesopening mitoK(ATP) channels[J]. J Neurosci Res,2009,87(5):1230-9.
[178] Ying R, Liang HL, Whelan HT, et al. Pretreatment with near-infrared light vialight-emitting diode provides added benefit against rotenone-and MPP+-inducedneurotoxicity[J]. Brain Res,2008,1243:167-73.
[179] Qing Zhu, Jing Wang, Yunjian Zhang, et al. Mechanisms of MPP+-induced PC12cell apoptosis via reactive oxygen species[J]. J Huazhong U Sci-Med,2012,32(6):861-866.
[180] Wang M, Wey S, Zhang Y, et al. Role of the Unfolded Protein ResponseRegulator GRP78/BiP in Development, Cancer, and Neurological Disorders[J].Antioxid Redox Signal,2009,11(9):2307-16.
[181] Holtz WA, Turetzky JM, Jong YJ, et al. Oxidative stress-triggered unfoldedprotein response is upstream of intrinsic cell death evoked by parkinsonianmimetics[J]. J Neurochem,2006,99(1):54-69.
[182] Szegezdi E, Logue SE, Gorman AM, et al. Mediators of endoplasmic reticulumstress-induced apoptosis[J]. EMBO Rep,2006,7(9):880-5.
[183] Nishitoh H, Matsuzawa A, Tobiume K, et al. ASK1is essential for endoplasmicreticulum stress-induced neuronal cell death triggered by expanded polyglutaminerepeats[J]. Genes&Dev,2002,16:1345-1355.
[184] Antoine Fouillet, Clémence Levet, Angélique Virgone, et al. ER stress inhibitsneuronal death by promoting autophagy[J]. Autophagy,2012,8(6):915-26.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |