黄芩苷对多巴胺神经元的保护作用及机制
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摘要
帕金森病(Parkinson's disease, PD)又名震颤麻痹,是继阿尔茨海默病(Alzheirmer's disease, AD)之后第二大神经系统慢性退行性疾病,主要症状为震颤、肌肉强直、运动减少和姿势平衡障碍。随着中国老龄化的进展,PD的发病率逐年升高。目前,PD的治疗药物和策略只能对症治疗,不能阻止多巴胺能(Dopamine, DA)神经元的进行性变性和缺失。因此,寻找有效的抗帕金森病药物弥补当前治疗的不足已成为PD研究的当务之急。
动物模型是研究帕金森病的发病机理、筛选药物和治疗策略的重要工具。MPTP诱导的小鼠模型无论在病理改变还是在行为学方面与PD患者有类似的变化特点,是目前公认较为理想的PD动物模型。基于动物模型上建立敏感稳定的行为学检测方法是构建PD药物筛选平台的重要组成部分。
黄芩苷(Baicalin)是唇形科植物黄芩中提取的黄酮类单体化合物,具有抑菌抗炎、降压、利尿及抗变态反应的作用。近年来大量体内外研究证实黄芩苷也具有对抗缺血性脑损伤、减轻感染性脑水肿及抗脑内炎症的作用。
本研究通过腹腔注射MPTP制作亚急性帕金森病小鼠模型,并应用计算机控制的CatWalk步态分析系统与Western Blotting、免疫组化和Open field test相结合的方法,首次分析MPTP诱导的PD亚急性模型小鼠的步态及姿势变化,并将各步态和姿势指标与黑质区TH蛋白水平做相关性分析,筛选出步幅、对角双足支撑模式、三足支撑模式、速度变异率、两后足间距、双后足的支撑率、双前足的初次接触时间等敏感稳定的步态指标,以期为PD的治疗研究建立一套有效可靠的评估体系。基于以上的评估平台我们进行了大量的药物筛选,发现黄芩苷可有效地改善PD模型小鼠的运动功能障碍和DA神经元的变性损伤。
基于以上的发现,我们提出以下问题：1.黄芩苷是通过哪条信号通路发挥DA神经元保护作用?2.黄芩苷发挥DA神经元保护作用的关键性蛋白是什么?本研究应用双向凝胶电泳和质谱技术检测黄芩苷处理前后差异表达的蛋白谱,并采用基于复杂网络理论的生物信息学方法,进一步阐明黄芩苷的生物学活性及其对DA神经元的保护作用的关键性蛋白,以期为PD的治疗提供新的治疗方向和靶标。本研究共分为4部分,摘要如下：
第一部分MPTP诱导的帕金森病小鼠模型及评估平台的建立
目的：建立PD亚急性小鼠模型,利用CatWalk步态分析系统定量分析PD小鼠的步态和姿势的变化,并与黑质区TH蛋白水平做相关性分析,筛选出敏感稳定的步态和姿势指标。
方法：
1. MPTP粉末配制成4mg/ml注射液,每天注射一次,连续注射5天,正常对照组注射同体积生理盐水。
2.最后一次MPTP注射后第21天,取小鼠全脑,行TH免疫组织化学染色,检测各组小鼠黑质区TH阳性神经元的形态和数量,及纹状体区TH阳性神经末梢的含量。
3.最后一次MPTP注射后第4,7,12,16,21天,各组小鼠进行Open filed test,检测小鼠的自发活动性。
4.最后一次MPTP注射前2天,及注射后第2,4,7,12,16,21天,冰上迅速取小鼠中脑黑质区和纹状体区新鲜脑组织,以Western Blotting检测黑质和纹状体区TH蛋白水平。
5.以计算机控制的CatWalk步态分析系统分析模型小鼠的步态和姿势的变化,并与黑质区TH蛋白做相关性分析,筛选出敏感稳定的步态和姿势指标。
结果：
1.最后一次MPTP注射后第21天,TH免疫组织化学染色显示,PD小鼠黑质致密区DA神经元约减至正常组的35%,证明PD小鼠模型制作成功。
2. Open field test结果显示,造模后第4天,PD小鼠的自发活动性明显降低；而第7天,PD小鼠的活动性高于正常组小鼠：造模后第12和16天,PD小鼠的活动性与正常对照组无差别；造模后第21天,PD小鼠的活动性明显低于正常组小鼠。
3. Western Blotting结果显示,最后一次MPTP注射前2天,PD小鼠黑质和纹状体区TH蛋白水平开始下降,至造模结束后第4天达到最低点,造模后第7,12,16天,TH的表达表现为上升趋势,至造模后第21天,PD小鼠黑质和纹状体区TH的蛋白水平达最低。黑质和纹状体区TH蛋白表达的趋势一致。
4. CatWalk test结果显示,PD小鼠穿过玻璃板的时间、速度变异率、步频、摇摆速度、步幅、静止时间、接触率、步伐周期、初次双足接触时间、末次双足接触时间、两后足间距、对角双足支撑模式、三足支撑模式、四足支撑模式发生明显的变化。步态与姿势指标与TH做相关性分析发现,步幅、对角双足支撑模式、三足支撑模式、速度变异率、两后足间距、双后足的支撑率和双前足的初次双足接触时间等指标敏感稳定,以上指标可以客观有效地评估PD模型小鼠的运动功能。
结论：应用MPTP腹腔注射诱导的亚急性PD小鼠模型,能模拟人类PD的病理和行为学特征。计算机控制的CatWalk自动化步态分析系统可快速客观地评价模型小鼠的运动功能,操作简单,为PD的治疗研究建立了稳定敏感的筛选评估平台。
第二部分黄芩苷对MPTP帕金森病小鼠的保护作用及机制
目的：应用第一部分建立的PD小鼠模型及评估体系评价黄芩苷的抗帕金森病效果。
方法：
1.随机将小鼠分为正常对照组、模型组、模型+黄芩苷组及黄芩苷处理组。模型组和模型+黄芩苷组小鼠,腹腔注射MPTP制作PD模型；MPTP注射后第7天,治疗组和黄芩苷处理组小鼠腹腔注射黄芩苷,连续注射20天；正常对照组小鼠全程注射生理盐水。
2.各组小鼠于MPTP注射完毕后第7天,进行Open field test.
3.各组小鼠于MPTP注射完毕后第8天,进行CatWalk test。
4.各组小鼠行心脏灌注固定后,取脑,O.C.T包埋后,制作冰冻切片,行TH免疫组织化学染色,观察各组小鼠黑质区DA神经元的形态,采用立体细胞计数法计算各组小鼠黑质区DA神经元的数量,并观察各组小鼠纹状体区TH阳性纤维末端的含量。
5.各组小鼠冰上快速取中脑黑质和纹状体区脑组织,裂解后,以Western Blotting的方法检测各组小鼠黑质区TH、Bax、cleaved-Caspase-3和iNOS的水平。
结果：
1.黄芩苷治疗后,能明显升高PD小鼠的自发性活动,10分钟内行走的距离及运动时间比PD组小鼠明显升高,而休息时间显著降低。
2. CatWalk检测结果显示,MPTP注射后第8天,步幅、对角双足支撑模式、双后足的支撑率及摇摆速度明显降低,三足支撑模式、速度变异率、两后足爪间距、双前足的初次接触时间明显增大。给予黄芩苷治疗后,上述运动功能障碍情况均得到明显的改善。
3.免疫组织化学染色结果显示,MPTP注射可导致小鼠中脑黑质致密区TH阳性细胞明显减少,纹状体区TH阳性纤维末端明显减少。黄芩苷干预后,可明显缓解DA神经元的损伤情况。
4. Western Blotting结果显示,MPTP注射后,小鼠黑质致密区TH蛋白水平较正常组小鼠明显降低,而Bax、cleaved-Caspase-3和NOS表达显著升高。黄芩苷可以明显逆转上述蛋白变化情况。
结论：黄芩苷可明显缓解MPTP诱导的DA神经元的损伤,改善PD小鼠的步态紊乱等运动功能障碍。黄芩苷可能涉及抗凋亡和抗炎症的生物活性发挥神经保护作用。
第三部分黄芩苷对多巴胺能神经元保护作用的蛋白组学研究
目的：应用双向凝胶电泳和质谱技术,对比分析黄芩苷处理前后PD小鼠黑质区差异表达的蛋白质谱,并以RT-PCR的方法进一步检测其基因的表达变化,进一步探讨黄芩苷发挥DA神经元保护作用的机制及生物活性。
方法：
1.PD及黄芩苷处理的PD组小鼠,于黄芩苷注射完毕后,断头,冰上取新鲜脑组织。
2.脑组织裂解后,以双向凝胶电泳法分离蛋白,银染后人工找出差异蛋白,将差异蛋白从凝胶中挖出,行质谱分析,以MASCOT软件检索肽质量指纹图谱鉴定蛋白质。
3.脑组织裂解后,以RT-PCR的方法,检测PD及黄芩苷处理的PD组小鼠黑质区中差异表达蛋白的mRNA的表达差异。
结果：
1.黄芩苷治疗后,PD小鼠黑质区蛋白表达发生变化。经软件和质谱分析鉴定出36个差异表达蛋白,初步鉴定差异表达的蛋白涉及能量代谢、细胞增殖、细胞骨架、凋亡、炎症及信号转导等多种生理活动。
2. RT-PCR检测结果显示,差异蛋白的基因表达趋势与蛋白表达的趋势基本一致。
结论：黄芩苷可能通过正性调节PD小鼠黑质区的蛋白异常折叠和聚集、能量代谢及氧化应激等过程,增强神经元内环境的稳态,发挥神经元保护的作用。
第四部分黄芩苷多巴胺能神经保护作用中蛋白质组生物信息学分析
目的：应用String、KEGG、Panthwaylinker等软件,分析差异表达蛋白之间的相互作用及参与的生物通路。综合蛋白组学和生物信息学的分析,预测出关键的蛋白和信号通路并以Western Blotting的方法检测关键性蛋白和信号通路中的蛋白的表达。
方法：
1.应用String在线软件,检测差异表达蛋白之间的相互作用。
2.应用KEGG和Pathwaylinker等软件,预测黄芩苷发生作用的信号通路。
3.综合蛋白组学和生物信息学分析,预测参与黄芩苷的神经保护作用的关键性蛋白和信号通路。
4.应用Western Blotting的方法,检测关键性蛋白和信号通路中蛋白的表达。
结果：
1. String在线软件分析发现,一共有22个蛋白之间发生相互作用,所有蛋白可以分成5个亚组,主要涉及骨架相关蛋白、CRMP/DPYL家族蛋白和Septin家族蛋白。
2.通过Pathwayliner和KEGG软件分析发现,差异表达的基因主要涉及帕金森病通路、阿尔茨海默病通路、actin骨架蛋白调节通路、轴突导向通路、大肠杆菌感染通路和溶酶体通路等14个生物通路。
3.综合蛋白组学和生物信息学,我们预测GFAP、GAPDH和Stip1等关键性蛋白及MAPK/ERK信号通路参与了黄芩苷的神经保护作用。
4. Western Blotting结果显示,PD小鼠黑质区LC3-Ⅱ和HSP70表达下降,黄芩苷处理后,LC-Ⅱ和HSP70表达升高。PD小鼠黑质区ERK1/2表达显著升高,黄芩苷处理后,ERK1/2表达明显降低。
结论：在本研究中,我们利用生物信息学的方法分析了差异表达蛋白之间的相互作用关系和参与的生物学通路。我们对差异蛋白所处的分子功能和生物学过程进行了分析,预测GAPDH GFPA和Stip1等关键性蛋白和MAPK/ERK通路可能参与黄芩苷的DA神经元保护作用。
小结：
本课题研究发现,基于CatWalk步态分析系统的步态检测是一种稳定可靠的运动功能障碍的检测方法。我们应用已建立的PD小鼠模型和相关的评估体系,发现黄芩苷能有效地缓解由MPTP引起的C57/BL6小鼠黑质纹状体通路中的多巴胺能神经元的变性丢失,并明显改善了MPTP诱导的C57/BL6小鼠步态紊乱和自发性活动减少等运动功能障碍。进一步,蛋白组学和生物信息学方法结合分析,我们预测到关键性蛋白GFAP、GAPDH和Stip1,及MAPK/ERK信号通路在黄芩苷的DA神经元保护作用中起到重要的调节作用。
Parkinson's disease (PD) also named Paralysis agitans, is the second chronic neurodegenerative disease after Alzheimer's Disease (AD). The main clinical features of PD include resting tremor, bradykinesia, postural instability and rigidity. As the population of the elders is growing, the incidence of PD increases in China.Though PD is aneurodegenerative disease with effective symptomatic treatment, modern medicine has not found an effective neuroprotective treatment or strategy to prevent the loss of dopaminergic neurons in PD. Therefore, development of new drugs with better curative effect and fewer side effects for PD is urgently needed.
Animal models are invaluable tools in the identification of pathogenic mechanisms and testing new therapeutic strategies for PD.The1-methyl4-pheny1-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model is the most commonly used animal model in PD research, neuropathology and motor deficits induced by MPTP is strikingly similar to PD patients. Establishment of a stable and sensitive evaluation platform for the animal model is very important for the treatment screening in PD.
Scutellaria baicalensis Georgi, an important medicinalherb, has been widely used in China to treat the inflammatory diseases and ischemic stroke for thousands of years.Baicalin, a flavonoid compound isolated from S. baicalensisGeorgi, possesses anti-oxidant, anti-inflammatory and anti-apoptotic properties.
In present study, we made the PD mice model by intraperitoneal administration of MPTP to the C57/BL6mice. Then, we investigated the gait and postural changes in PD mice using the computer-assisted CatWalk system, and screened the sensitive parameters by correlating the gait and postural parameters with the TH protein levels in the substantial nigra. The gait and postural readouts, includingstride length,diagonal dual support, three-point supports,variation of walking speed, base of support between hind limbs, duty cycle in the hind limbs and initial dual stance of the forelimbswere proved to be the satble and sensitive parameters for evaluating the movement disorders in PD mice. We have screened a lot of drugs for PD, and found that Bacalin can effectively alleviate the functional disorder and the degeneration of dopaminergic neurons. Some questions arise:1. Through which pathway Bacalin prevent dopaminergic neurons degeneration?2. Which protein is the key one during Baicalin's neuroprotective role?
To answer these questions, we profile the quantitative proteomic differences of substantial nigra in PD mice with or without Bacaline treatment by two-dimensional gel electrophoresis (2-DE) and Mass Spectrometry (MS) analysis. Integration of the quantitative proteomics with global protein interaction data using the String software leads to the identification of several novel proteins, which may be important in find new targets for PD therapy.
This study was divided into4parts:
PART I ESTABLISHMENTOF THE MPTP-INDUCED PD MICE MODEL AND ITS EVALUATION SYSTEM
Objective:To establish the MPTP-induced PD mice model and screen the sensitive and stable gait parameters by correlating the CatWalk test with TH protein levels in substantial nigra of PD mice.
Methods:
1. The C57/BL6mice received one injection of MPTP-HClintraperitoneally at the dose of40mg/kg for5consecutive days, the control mice were treated with the same volume of normal saline.
2. On the21day after the last MPTP injection, brain of the C57/BL6mice in both control and PD groups were dissected for TH immunohistochemistry analysis
3. On the4,7,12,16,21day after the last MPTP injection, C57/BL6mice in both control and PD groups performed the open field test.
4. On the2day before MPTP injection, and2,4,7,12,16,21day after the last MPTP injection, the substantial nigra and striatum of mice in both control and PD groups were dissected for detecting the TH protein level by Western Blotting analysis.
5. On the4,7,12,16,21day after the last MPTP injection, C57/BL6mice in both control and PD groups performed the CatWalk test. We will screen the sensitive and stable gait parameters by correlating the gait parameters and TH level in substantial nigra.
Results:
1. The dopaminergic neurons subjecting to this regimen of MPTP injection induced a significant loss to30%of that of control group after the last injection of MPTP. The challenge with MPTP also resulted in a great depletion of TH-ir in striatum.
2. In the open field test, the walking distance of PD mice decreased significantlyon the4th day after the last MPTP injection, increased slightly on the7th day. There is no different between PD mice and control mice on the12and16day. On the21day, the distance of PD mice decreased stably and significantly.
3. The gait parameters including stride length,diagonal dual support, three-point supports,variation of walking speed, base of support between hind limbs, duty cycle in the hind limbs and initial dual stance of the forelimbscorrelated well with the TH protein levels in substantial nigra of PD mice. They are served as the stable and sensitive parameters for analysis gait in MPTP-induced PD mice.
4. A significant fall in TH was evident in both substantianigra andstriatum of mice starting on day2of MPTP administration, followed by a slight rebound at twoweeks. TH then remained at the lowest levels on three weeks post-MPTP injection. It was noted that the down regulationof TH by MPTP in the striatum was more profound than that in the substantia nigra.
5. The correlation analysis between CatWalk test and TH level shows significant positive correlations of the levels of TH in the substantia nigra of MPTP-treatedmice with readouts derived from CatWalk tests of diagonaldual support, stride length in all limbs, and swing speed inthe forelimbs three weeks post MPTP administration. Substantially negative correlations of TH levels with stancein the hind limbs, step cycle, duty cycle, initial dual stance, terminal dual stance, three-point support, four-point support,walking speed variation, cadence,and base of supportbetween hind limbs were noted. On the other hand, thelevels of TH correlated poorly with the trajectories in theopen field tests.
Conclusion:The MPTP-induced subacute PD mice model can effectively mimic the pathological and behavioral feature in PD patients. The computer-assisted CatWalk system can evaluate the motor deficits in PD mice in a friendly, automated,fast and objective way, and is an important part of the evaluation system in PD model.
PART II THE PROTECTIVE EFFECT OF BACAILIN ON THE MPTP PD MICE
Objective:To screen the therapeutic drugs on the MPTP-induced PD mice using the evaluation system established in the first part.
Method:
1. Animals were divided into four groups:Control, PD, PD+Baicalin,Baicalin. Mice in PD and PD+Baicalin group were injected with MPTP, mice in PD+Baicalin and Bacalin group were treated with Bacalin, and mice in the Control group were injected with normal saline.
2. The open field test was performed on the7th day after MPTP/Saline injection.
3. The CatWalk test was performed on the8th day after MPTP/Saline injection.
4. After the baicalin/saline treatment, mice in different groups were perfused and fixed transcardially. The brain were dissected and embedded in Tissue-Tek O.C.T compound, after immunostaining with TH antibody, total number of TH-positive neurons inSNpc and the TH positive terminal in striatum in all groups was estimated.
5. After the baicalin/saline treatment, the substantial nigra and striatum of mice in all groups were dissected for detecting the TH and other proteins level by Western Blotting analysis.
Results:
1. After Baicalin treatment, the traveling distance in10minutes and time of mice in PD+Baicalin group increased while rest time decreased greatly than mice in PD group.
2. After MPTP treatment, the stride length,diagonal dual support, duty cycle in the hind limbs and swing speed decreased significantly, and the three-point supports,variation of walking speed, base of support between hind limbs, and initial dual stance of the forelimbs increased significantly. Bacalin treatment can effectively improve the gait and postural disorders in PD mice.
3. After the subacute MPTP treatment, TH-irpositive neurons decreased greatly in the pars compacta. Baicalin treatment can effectively prevent the neuron loss in PD mice.
4. After the subacute MPTP treatment, the protein levels of TH and cleaved-Caspase-3decreased, while Bax and iNOS increased in the substantial nigra pars compactagreatly. Baicalin treatment can effectively reverse the change in substantial nigra in PD mice.
Conclusion:
Baicalin can effectively inhibit the damage in DA neurons and alleviate the movement disorders in PD mice. The protective mechanism of Baicalin may related with its anti-apoptosis and anti-inflammation effects.
PARTⅢ QUANTITATIVE PROTEOMIC ANALYSIS OF THE ANTIPARKINSONIAN ROLE OF BAICALIN
Objective:To identify and analyze the key proteins associated with the antiparkinsonian effects of baicalin. Comparativeanalyse of2-DE protein patterns between the PD and PD+Baiclin groups were done using computerized image analysis. Differentially expressed proteins (at least two-fold) were identified by MASCOT software. RT-PCR was carried out to confirm the gene expression patterns of those screened proteins.
Methods:
1. After the baicalin treatment, mice brain of PD and PD+Baicalin group were removed, and the substantial nigra was dissected and lysis.
2. The total proteins from substantial nigra were separated by2-DE. Comparativeanalyses of2-DE protein patterns between the PD and PD+Baiclin groups were done using computerized image analysis. Differentially expressed proteins (at least two-fold) were identified by MASCOT software.
3. RT-PCR was carried out to confirm the gene expression patterns of those screened proteins.
Results:
1. A total of36differentially expressed proteinswere identified by the MASCOT software.
2. The RT-PCR results domistrated that the gene expression patterns of the screened proteins were almost in accordance with the protein expressing pattern.
Conclusion:
Baicalin may play neuroprotective role by regulating the aggregation of protein and energy metabolizing, inhibiting the oxidative stress and modulating some sigal pathways.
PART IV BIOINFORMATIC ANALYSIS UNDERLYING THE ANTIPARKINSONIAN EFFECTS OF BAICALIN
Objective:To investigate the protein-protein interaction and biological pathways associated with antiparkinsonian effects of baicalin using String, KEGG and Pathwaylinkersoftware. To confirm the role of proteins and pathways by Western Blotting to provide new thoughts for therapeutic study of PD.
Methods:
1. Predict the key proteins and pathways involved in the antiparkinsonian effects of baicalin using KEGG and cytoscape software.
2. Confirm the role of the key proteins and pathways using Western Blotting analysis.
Results:
1. All the differentially expressed proteins screened using the2-D method involve in about14biological pathways, including the Parkinson's disease pathway, the Alzermer's disease pathway, the actin regulating pathway, the axon guidance pathway and the Coli infection pathway and soon on.
2. There are22interacting proteins in the differentially expressed proteins. All the interacting proteins can be devided into5sub-groups including the actin-related protein group, CRMP/DPYL family proteins and Septin family proteins.
3. Integrating the information of the proteomic and bioinformatic results, we predict that the proteins including GFAP, GAPDH and Stipl and the MAPK/ERK pathway may play important role in the protective effects of Baicalin.
4. Western Blotting results showed that the expressing of GFAP and ERK1/2increased after MPTP injection, while HSP70and LC-Ⅱdecreased. After treated with Baicalin, expression of GFAP and ERK1/2decreased while HSP70and LC-Ⅱincreased significantly.
Conclusion:
The protective effects of Baicalin may mainly relating with the protein processing and energy metabolism. The antiparkinsonian effects of baicalin may involve multipleproteins including GFAP, GAPDH and Stipl. The MAPK/ERK pathway may also play important role in the antiparkinsonian effects of baicalin.
Summary:We found that the computer-assisted CatWalk system can provide sensitive and stable parameters for analysising the motor disorders in PD mice. Baicalin can effectively inhibit the damage in DA neurons and alleviate the movement disorders in PD mice. Baicalin may play neuroprotective role through the anti-inflammatory, anti-apoptosis, anti-aggregation and regulating the energy metabolizing and some signal pathways. The antiparkinsonian effects of baicalin involve multiple proteins including GFAP, GAPDH and Stipl. The MAPK/ERK pathway may play important role in the antiparkinsonian effects of baicalin.
动物模型是研究帕金森病的发病机理、筛选药物和治疗策略的重要工具。MPTP诱导的小鼠模型无论在病理改变还是在行为学方面与PD患者有类似的变化特点,是目前公认较为理想的PD动物模型。基于动物模型上建立敏感稳定的行为学检测方法是构建PD药物筛选平台的重要组成部分。
黄芩苷(Baicalin)是唇形科植物黄芩中提取的黄酮类单体化合物,具有抑菌抗炎、降压、利尿及抗变态反应的作用。近年来大量体内外研究证实黄芩苷也具有对抗缺血性脑损伤、减轻感染性脑水肿及抗脑内炎症的作用。
本研究通过腹腔注射MPTP制作亚急性帕金森病小鼠模型,并应用计算机控制的CatWalk步态分析系统与Western Blotting、免疫组化和Open field test相结合的方法,首次分析MPTP诱导的PD亚急性模型小鼠的步态及姿势变化,并将各步态和姿势指标与黑质区TH蛋白水平做相关性分析,筛选出步幅、对角双足支撑模式、三足支撑模式、速度变异率、两后足间距、双后足的支撑率、双前足的初次接触时间等敏感稳定的步态指标,以期为PD的治疗研究建立一套有效可靠的评估体系。基于以上的评估平台我们进行了大量的药物筛选,发现黄芩苷可有效地改善PD模型小鼠的运动功能障碍和DA神经元的变性损伤。
基于以上的发现,我们提出以下问题：1.黄芩苷是通过哪条信号通路发挥DA神经元保护作用?2.黄芩苷发挥DA神经元保护作用的关键性蛋白是什么?本研究应用双向凝胶电泳和质谱技术检测黄芩苷处理前后差异表达的蛋白谱,并采用基于复杂网络理论的生物信息学方法,进一步阐明黄芩苷的生物学活性及其对DA神经元的保护作用的关键性蛋白,以期为PD的治疗提供新的治疗方向和靶标。本研究共分为4部分,摘要如下：
第一部分MPTP诱导的帕金森病小鼠模型及评估平台的建立
目的：建立PD亚急性小鼠模型,利用CatWalk步态分析系统定量分析PD小鼠的步态和姿势的变化,并与黑质区TH蛋白水平做相关性分析,筛选出敏感稳定的步态和姿势指标。
方法：
1. MPTP粉末配制成4mg/ml注射液,每天注射一次,连续注射5天,正常对照组注射同体积生理盐水。
2.最后一次MPTP注射后第21天,取小鼠全脑,行TH免疫组织化学染色,检测各组小鼠黑质区TH阳性神经元的形态和数量,及纹状体区TH阳性神经末梢的含量。
3.最后一次MPTP注射后第4,7,12,16,21天,各组小鼠进行Open filed test,检测小鼠的自发活动性。
4.最后一次MPTP注射前2天,及注射后第2,4,7,12,16,21天,冰上迅速取小鼠中脑黑质区和纹状体区新鲜脑组织,以Western Blotting检测黑质和纹状体区TH蛋白水平。
5.以计算机控制的CatWalk步态分析系统分析模型小鼠的步态和姿势的变化,并与黑质区TH蛋白做相关性分析,筛选出敏感稳定的步态和姿势指标。
结果：
1.最后一次MPTP注射后第21天,TH免疫组织化学染色显示,PD小鼠黑质致密区DA神经元约减至正常组的35%,证明PD小鼠模型制作成功。
2. Open field test结果显示,造模后第4天,PD小鼠的自发活动性明显降低；而第7天,PD小鼠的活动性高于正常组小鼠：造模后第12和16天,PD小鼠的活动性与正常对照组无差别；造模后第21天,PD小鼠的活动性明显低于正常组小鼠。
3. Western Blotting结果显示,最后一次MPTP注射前2天,PD小鼠黑质和纹状体区TH蛋白水平开始下降,至造模结束后第4天达到最低点,造模后第7,12,16天,TH的表达表现为上升趋势,至造模后第21天,PD小鼠黑质和纹状体区TH的蛋白水平达最低。黑质和纹状体区TH蛋白表达的趋势一致。
4. CatWalk test结果显示,PD小鼠穿过玻璃板的时间、速度变异率、步频、摇摆速度、步幅、静止时间、接触率、步伐周期、初次双足接触时间、末次双足接触时间、两后足间距、对角双足支撑模式、三足支撑模式、四足支撑模式发生明显的变化。步态与姿势指标与TH做相关性分析发现,步幅、对角双足支撑模式、三足支撑模式、速度变异率、两后足间距、双后足的支撑率和双前足的初次双足接触时间等指标敏感稳定,以上指标可以客观有效地评估PD模型小鼠的运动功能。
结论：应用MPTP腹腔注射诱导的亚急性PD小鼠模型,能模拟人类PD的病理和行为学特征。计算机控制的CatWalk自动化步态分析系统可快速客观地评价模型小鼠的运动功能,操作简单,为PD的治疗研究建立了稳定敏感的筛选评估平台。
第二部分黄芩苷对MPTP帕金森病小鼠的保护作用及机制
目的：应用第一部分建立的PD小鼠模型及评估体系评价黄芩苷的抗帕金森病效果。
方法：
1.随机将小鼠分为正常对照组、模型组、模型+黄芩苷组及黄芩苷处理组。模型组和模型+黄芩苷组小鼠,腹腔注射MPTP制作PD模型；MPTP注射后第7天,治疗组和黄芩苷处理组小鼠腹腔注射黄芩苷,连续注射20天；正常对照组小鼠全程注射生理盐水。
2.各组小鼠于MPTP注射完毕后第7天,进行Open field test.
3.各组小鼠于MPTP注射完毕后第8天,进行CatWalk test。
4.各组小鼠行心脏灌注固定后,取脑,O.C.T包埋后,制作冰冻切片,行TH免疫组织化学染色,观察各组小鼠黑质区DA神经元的形态,采用立体细胞计数法计算各组小鼠黑质区DA神经元的数量,并观察各组小鼠纹状体区TH阳性纤维末端的含量。
5.各组小鼠冰上快速取中脑黑质和纹状体区脑组织,裂解后,以Western Blotting的方法检测各组小鼠黑质区TH、Bax、cleaved-Caspase-3和iNOS的水平。
结果：
1.黄芩苷治疗后,能明显升高PD小鼠的自发性活动,10分钟内行走的距离及运动时间比PD组小鼠明显升高,而休息时间显著降低。
2. CatWalk检测结果显示,MPTP注射后第8天,步幅、对角双足支撑模式、双后足的支撑率及摇摆速度明显降低,三足支撑模式、速度变异率、两后足爪间距、双前足的初次接触时间明显增大。给予黄芩苷治疗后,上述运动功能障碍情况均得到明显的改善。
3.免疫组织化学染色结果显示,MPTP注射可导致小鼠中脑黑质致密区TH阳性细胞明显减少,纹状体区TH阳性纤维末端明显减少。黄芩苷干预后,可明显缓解DA神经元的损伤情况。
4. Western Blotting结果显示,MPTP注射后,小鼠黑质致密区TH蛋白水平较正常组小鼠明显降低,而Bax、cleaved-Caspase-3和NOS表达显著升高。黄芩苷可以明显逆转上述蛋白变化情况。
结论：黄芩苷可明显缓解MPTP诱导的DA神经元的损伤,改善PD小鼠的步态紊乱等运动功能障碍。黄芩苷可能涉及抗凋亡和抗炎症的生物活性发挥神经保护作用。
第三部分黄芩苷对多巴胺能神经元保护作用的蛋白组学研究
目的：应用双向凝胶电泳和质谱技术,对比分析黄芩苷处理前后PD小鼠黑质区差异表达的蛋白质谱,并以RT-PCR的方法进一步检测其基因的表达变化,进一步探讨黄芩苷发挥DA神经元保护作用的机制及生物活性。
方法：
1.PD及黄芩苷处理的PD组小鼠,于黄芩苷注射完毕后,断头,冰上取新鲜脑组织。
2.脑组织裂解后,以双向凝胶电泳法分离蛋白,银染后人工找出差异蛋白,将差异蛋白从凝胶中挖出,行质谱分析,以MASCOT软件检索肽质量指纹图谱鉴定蛋白质。
3.脑组织裂解后,以RT-PCR的方法,检测PD及黄芩苷处理的PD组小鼠黑质区中差异表达蛋白的mRNA的表达差异。
结果：
1.黄芩苷治疗后,PD小鼠黑质区蛋白表达发生变化。经软件和质谱分析鉴定出36个差异表达蛋白,初步鉴定差异表达的蛋白涉及能量代谢、细胞增殖、细胞骨架、凋亡、炎症及信号转导等多种生理活动。
2. RT-PCR检测结果显示,差异蛋白的基因表达趋势与蛋白表达的趋势基本一致。
结论：黄芩苷可能通过正性调节PD小鼠黑质区的蛋白异常折叠和聚集、能量代谢及氧化应激等过程,增强神经元内环境的稳态,发挥神经元保护的作用。
第四部分黄芩苷多巴胺能神经保护作用中蛋白质组生物信息学分析
目的：应用String、KEGG、Panthwaylinker等软件,分析差异表达蛋白之间的相互作用及参与的生物通路。综合蛋白组学和生物信息学的分析,预测出关键的蛋白和信号通路并以Western Blotting的方法检测关键性蛋白和信号通路中的蛋白的表达。
方法：
1.应用String在线软件,检测差异表达蛋白之间的相互作用。
2.应用KEGG和Pathwaylinker等软件,预测黄芩苷发生作用的信号通路。
3.综合蛋白组学和生物信息学分析,预测参与黄芩苷的神经保护作用的关键性蛋白和信号通路。
4.应用Western Blotting的方法,检测关键性蛋白和信号通路中蛋白的表达。
结果：
1. String在线软件分析发现,一共有22个蛋白之间发生相互作用,所有蛋白可以分成5个亚组,主要涉及骨架相关蛋白、CRMP/DPYL家族蛋白和Septin家族蛋白。
2.通过Pathwayliner和KEGG软件分析发现,差异表达的基因主要涉及帕金森病通路、阿尔茨海默病通路、actin骨架蛋白调节通路、轴突导向通路、大肠杆菌感染通路和溶酶体通路等14个生物通路。
3.综合蛋白组学和生物信息学,我们预测GFAP、GAPDH和Stip1等关键性蛋白及MAPK/ERK信号通路参与了黄芩苷的神经保护作用。
4. Western Blotting结果显示,PD小鼠黑质区LC3-Ⅱ和HSP70表达下降,黄芩苷处理后,LC-Ⅱ和HSP70表达升高。PD小鼠黑质区ERK1/2表达显著升高,黄芩苷处理后,ERK1/2表达明显降低。
结论：在本研究中,我们利用生物信息学的方法分析了差异表达蛋白之间的相互作用关系和参与的生物学通路。我们对差异蛋白所处的分子功能和生物学过程进行了分析,预测GAPDH GFPA和Stip1等关键性蛋白和MAPK/ERK通路可能参与黄芩苷的DA神经元保护作用。
小结：
本课题研究发现,基于CatWalk步态分析系统的步态检测是一种稳定可靠的运动功能障碍的检测方法。我们应用已建立的PD小鼠模型和相关的评估体系,发现黄芩苷能有效地缓解由MPTP引起的C57/BL6小鼠黑质纹状体通路中的多巴胺能神经元的变性丢失,并明显改善了MPTP诱导的C57/BL6小鼠步态紊乱和自发性活动减少等运动功能障碍。进一步,蛋白组学和生物信息学方法结合分析,我们预测到关键性蛋白GFAP、GAPDH和Stip1,及MAPK/ERK信号通路在黄芩苷的DA神经元保护作用中起到重要的调节作用。
Parkinson's disease (PD) also named Paralysis agitans, is the second chronic neurodegenerative disease after Alzheimer's Disease (AD). The main clinical features of PD include resting tremor, bradykinesia, postural instability and rigidity. As the population of the elders is growing, the incidence of PD increases in China.Though PD is aneurodegenerative disease with effective symptomatic treatment, modern medicine has not found an effective neuroprotective treatment or strategy to prevent the loss of dopaminergic neurons in PD. Therefore, development of new drugs with better curative effect and fewer side effects for PD is urgently needed.
Animal models are invaluable tools in the identification of pathogenic mechanisms and testing new therapeutic strategies for PD.The1-methyl4-pheny1-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice model is the most commonly used animal model in PD research, neuropathology and motor deficits induced by MPTP is strikingly similar to PD patients. Establishment of a stable and sensitive evaluation platform for the animal model is very important for the treatment screening in PD.
Scutellaria baicalensis Georgi, an important medicinalherb, has been widely used in China to treat the inflammatory diseases and ischemic stroke for thousands of years.Baicalin, a flavonoid compound isolated from S. baicalensisGeorgi, possesses anti-oxidant, anti-inflammatory and anti-apoptotic properties.
In present study, we made the PD mice model by intraperitoneal administration of MPTP to the C57/BL6mice. Then, we investigated the gait and postural changes in PD mice using the computer-assisted CatWalk system, and screened the sensitive parameters by correlating the gait and postural parameters with the TH protein levels in the substantial nigra. The gait and postural readouts, includingstride length,diagonal dual support, three-point supports,variation of walking speed, base of support between hind limbs, duty cycle in the hind limbs and initial dual stance of the forelimbswere proved to be the satble and sensitive parameters for evaluating the movement disorders in PD mice. We have screened a lot of drugs for PD, and found that Bacalin can effectively alleviate the functional disorder and the degeneration of dopaminergic neurons. Some questions arise:1. Through which pathway Bacalin prevent dopaminergic neurons degeneration?2. Which protein is the key one during Baicalin's neuroprotective role?
To answer these questions, we profile the quantitative proteomic differences of substantial nigra in PD mice with or without Bacaline treatment by two-dimensional gel electrophoresis (2-DE) and Mass Spectrometry (MS) analysis. Integration of the quantitative proteomics with global protein interaction data using the String software leads to the identification of several novel proteins, which may be important in find new targets for PD therapy.
This study was divided into4parts:
PART I ESTABLISHMENTOF THE MPTP-INDUCED PD MICE MODEL AND ITS EVALUATION SYSTEM
Objective:To establish the MPTP-induced PD mice model and screen the sensitive and stable gait parameters by correlating the CatWalk test with TH protein levels in substantial nigra of PD mice.
Methods:
1. The C57/BL6mice received one injection of MPTP-HClintraperitoneally at the dose of40mg/kg for5consecutive days, the control mice were treated with the same volume of normal saline.
2. On the21day after the last MPTP injection, brain of the C57/BL6mice in both control and PD groups were dissected for TH immunohistochemistry analysis
3. On the4,7,12,16,21day after the last MPTP injection, C57/BL6mice in both control and PD groups performed the open field test.
4. On the2day before MPTP injection, and2,4,7,12,16,21day after the last MPTP injection, the substantial nigra and striatum of mice in both control and PD groups were dissected for detecting the TH protein level by Western Blotting analysis.
5. On the4,7,12,16,21day after the last MPTP injection, C57/BL6mice in both control and PD groups performed the CatWalk test. We will screen the sensitive and stable gait parameters by correlating the gait parameters and TH level in substantial nigra.
Results:
1. The dopaminergic neurons subjecting to this regimen of MPTP injection induced a significant loss to30%of that of control group after the last injection of MPTP. The challenge with MPTP also resulted in a great depletion of TH-ir in striatum.
2. In the open field test, the walking distance of PD mice decreased significantlyon the4th day after the last MPTP injection, increased slightly on the7th day. There is no different between PD mice and control mice on the12and16day. On the21day, the distance of PD mice decreased stably and significantly.
3. The gait parameters including stride length,diagonal dual support, three-point supports,variation of walking speed, base of support between hind limbs, duty cycle in the hind limbs and initial dual stance of the forelimbscorrelated well with the TH protein levels in substantial nigra of PD mice. They are served as the stable and sensitive parameters for analysis gait in MPTP-induced PD mice.
4. A significant fall in TH was evident in both substantianigra andstriatum of mice starting on day2of MPTP administration, followed by a slight rebound at twoweeks. TH then remained at the lowest levels on three weeks post-MPTP injection. It was noted that the down regulationof TH by MPTP in the striatum was more profound than that in the substantia nigra.
5. The correlation analysis between CatWalk test and TH level shows significant positive correlations of the levels of TH in the substantia nigra of MPTP-treatedmice with readouts derived from CatWalk tests of diagonaldual support, stride length in all limbs, and swing speed inthe forelimbs three weeks post MPTP administration. Substantially negative correlations of TH levels with stancein the hind limbs, step cycle, duty cycle, initial dual stance, terminal dual stance, three-point support, four-point support,walking speed variation, cadence,and base of supportbetween hind limbs were noted. On the other hand, thelevels of TH correlated poorly with the trajectories in theopen field tests.
Conclusion:The MPTP-induced subacute PD mice model can effectively mimic the pathological and behavioral feature in PD patients. The computer-assisted CatWalk system can evaluate the motor deficits in PD mice in a friendly, automated,fast and objective way, and is an important part of the evaluation system in PD model.
PART II THE PROTECTIVE EFFECT OF BACAILIN ON THE MPTP PD MICE
Objective:To screen the therapeutic drugs on the MPTP-induced PD mice using the evaluation system established in the first part.
Method:
1. Animals were divided into four groups:Control, PD, PD+Baicalin,Baicalin. Mice in PD and PD+Baicalin group were injected with MPTP, mice in PD+Baicalin and Bacalin group were treated with Bacalin, and mice in the Control group were injected with normal saline.
2. The open field test was performed on the7th day after MPTP/Saline injection.
3. The CatWalk test was performed on the8th day after MPTP/Saline injection.
4. After the baicalin/saline treatment, mice in different groups were perfused and fixed transcardially. The brain were dissected and embedded in Tissue-Tek O.C.T compound, after immunostaining with TH antibody, total number of TH-positive neurons inSNpc and the TH positive terminal in striatum in all groups was estimated.
5. After the baicalin/saline treatment, the substantial nigra and striatum of mice in all groups were dissected for detecting the TH and other proteins level by Western Blotting analysis.
Results:
1. After Baicalin treatment, the traveling distance in10minutes and time of mice in PD+Baicalin group increased while rest time decreased greatly than mice in PD group.
2. After MPTP treatment, the stride length,diagonal dual support, duty cycle in the hind limbs and swing speed decreased significantly, and the three-point supports,variation of walking speed, base of support between hind limbs, and initial dual stance of the forelimbs increased significantly. Bacalin treatment can effectively improve the gait and postural disorders in PD mice.
3. After the subacute MPTP treatment, TH-irpositive neurons decreased greatly in the pars compacta. Baicalin treatment can effectively prevent the neuron loss in PD mice.
4. After the subacute MPTP treatment, the protein levels of TH and cleaved-Caspase-3decreased, while Bax and iNOS increased in the substantial nigra pars compactagreatly. Baicalin treatment can effectively reverse the change in substantial nigra in PD mice.
Conclusion:
Baicalin can effectively inhibit the damage in DA neurons and alleviate the movement disorders in PD mice. The protective mechanism of Baicalin may related with its anti-apoptosis and anti-inflammation effects.
PARTⅢ QUANTITATIVE PROTEOMIC ANALYSIS OF THE ANTIPARKINSONIAN ROLE OF BAICALIN
Objective:To identify and analyze the key proteins associated with the antiparkinsonian effects of baicalin. Comparativeanalyse of2-DE protein patterns between the PD and PD+Baiclin groups were done using computerized image analysis. Differentially expressed proteins (at least two-fold) were identified by MASCOT software. RT-PCR was carried out to confirm the gene expression patterns of those screened proteins.
Methods:
1. After the baicalin treatment, mice brain of PD and PD+Baicalin group were removed, and the substantial nigra was dissected and lysis.
2. The total proteins from substantial nigra were separated by2-DE. Comparativeanalyses of2-DE protein patterns between the PD and PD+Baiclin groups were done using computerized image analysis. Differentially expressed proteins (at least two-fold) were identified by MASCOT software.
3. RT-PCR was carried out to confirm the gene expression patterns of those screened proteins.
Results:
1. A total of36differentially expressed proteinswere identified by the MASCOT software.
2. The RT-PCR results domistrated that the gene expression patterns of the screened proteins were almost in accordance with the protein expressing pattern.
Conclusion:
Baicalin may play neuroprotective role by regulating the aggregation of protein and energy metabolizing, inhibiting the oxidative stress and modulating some sigal pathways.
PART IV BIOINFORMATIC ANALYSIS UNDERLYING THE ANTIPARKINSONIAN EFFECTS OF BAICALIN
Objective:To investigate the protein-protein interaction and biological pathways associated with antiparkinsonian effects of baicalin using String, KEGG and Pathwaylinkersoftware. To confirm the role of proteins and pathways by Western Blotting to provide new thoughts for therapeutic study of PD.
Methods:
1. Predict the key proteins and pathways involved in the antiparkinsonian effects of baicalin using KEGG and cytoscape software.
2. Confirm the role of the key proteins and pathways using Western Blotting analysis.
Results:
1. All the differentially expressed proteins screened using the2-D method involve in about14biological pathways, including the Parkinson's disease pathway, the Alzermer's disease pathway, the actin regulating pathway, the axon guidance pathway and the Coli infection pathway and soon on.
2. There are22interacting proteins in the differentially expressed proteins. All the interacting proteins can be devided into5sub-groups including the actin-related protein group, CRMP/DPYL family proteins and Septin family proteins.
3. Integrating the information of the proteomic and bioinformatic results, we predict that the proteins including GFAP, GAPDH and Stipl and the MAPK/ERK pathway may play important role in the protective effects of Baicalin.
4. Western Blotting results showed that the expressing of GFAP and ERK1/2increased after MPTP injection, while HSP70and LC-Ⅱdecreased. After treated with Baicalin, expression of GFAP and ERK1/2decreased while HSP70and LC-Ⅱincreased significantly.
Conclusion:
The protective effects of Baicalin may mainly relating with the protein processing and energy metabolism. The antiparkinsonian effects of baicalin may involve multipleproteins including GFAP, GAPDH and Stipl. The MAPK/ERK pathway may also play important role in the antiparkinsonian effects of baicalin.
Summary:We found that the computer-assisted CatWalk system can provide sensitive and stable parameters for analysising the motor disorders in PD mice. Baicalin can effectively inhibit the damage in DA neurons and alleviate the movement disorders in PD mice. Baicalin may play neuroprotective role through the anti-inflammatory, anti-apoptosis, anti-aggregation and regulating the energy metabolizing and some signal pathways. The antiparkinsonian effects of baicalin involve multiple proteins including GFAP, GAPDH and Stipl. The MAPK/ERK pathway may play important role in the antiparkinsonian effects of baicalin.
引文
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