CN113876929B - 肽MOTS-c在制备治疗帕金森药物中的应用 - Google Patents
肽MOTS-c在制备治疗帕金森药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种肽MOTS‑c在制备治疗帕金森药物中的应用，本发明所述肽MOTS‑c能够降低鱼藤酮诱导的帕金森大鼠动物模型中脑氧化应激损伤水平，保护大鼠中脑黑质纹状体多巴胺能神经元，并减少多巴胺能神经元的丢失，显著提高纹状体的多巴胺水平，有效改善帕金森大鼠神经行为学症状。因此，肽MOTS‑c在制备抗帕金森药物中具有重要的应用意义。
Description
技术领域
本发明涉及医药技术领域，特别涉及一种肽MOTS-c在制备治疗帕金森药物中的应用。
背景技术
帕金森病(Parkinson’s disease，PD)是全球第二大常见的神经退行性疾病，其发病率随着年龄增长而增高，严重影响中老年人的身体健康及生活质量。PD的主要病理特征是中脑黑质多巴胺神经元的缺失和路易小体(Lewy body)形成，随着纹状体多巴胺的减少，出现静止性震颤、肌肉强直、运动迟缓、姿态步态异常等常见的临床表现，症状呈进行性发展，严重时患者生活不能自理。目前其病因尚未完全清楚，主要认为是遗传，环境和衰老等多种因素共同相互作用的结果,尤其是环境因素在散发性PD发病机制中具有重要作用。大量的流行病和小动物实验研究发现农药鱼藤酮与PD发病相关，鱼藤酮染毒大鼠可成功构建拟PD的动物模型。在已经公认的PD发病机制中，氧化应激是导致黑质纹状体多巴胺能神经元损伤的重要因素，在PD发病进程中发挥重要的作用。
目前PD主要以药物治疗为主，常用的多巴胺前体左旋多巴可以通过血脑屏障进入脑内，代谢为多巴胺后补充内源性多巴胺缺乏，以缓解PD的症状。但是，大多数患者在长期用药后，其疗效逐渐减退，且出现各种运动并发症(包括开关现象，易动症等)，反而进一步增加了治疗难度。尽管目前临床上开始采用多种联合用药的方法以延长药物使用时间，但是仍然未能取得良好的临床治疗效果。因此寻找治疗PD的新药是神经科学方向重要的研究方向。随着对PD发病机制认识的深入，从多环节入手，着重保护多巴胺能神经元也成为了新的治疗PD药物的研发思路之一。
线粒体衍生肽(mitochondrial derived peptides，MDPs)是线粒体DNA(Mitochondrial DNA，mtDNA)开放阅读框编码的具有生物活性的短肽，参与机体代谢稳态的调节，在人体内发挥广泛的生理作用。已有的研究指出，MOTS-c作为目前已知的三种内源性MDPs之一，其可参与调节线粒体生物医学功能，影响线粒体介导的代谢过程，是研究线粒体相关疾病重要的靶点。此外，肽MOTS-c可在代谢应激条件下保护细胞，还参与糖脂代谢等过程，并发挥调节胰岛素抵抗，改善脂质沉积，调节骨代谢等多种作用。不仅如此，由于多肽类药物具有高效能、高选择性、作用靶点广泛及潜在毒性低等优点，使得肽MOTS-c具有巨大的研究价值和应用前景。
然而，肽MOTS-c发挥作用的机制还未完全阐明，目前也没有相关肽MOTS-c可用于治疗帕金森病的相关报道。
发明内容
本发明的目的是提供肽MOTS-c在制备治疗帕金森药物中的应用，所述肽MOTS-c治疗由鱼藤酮诱导的帕金森效果显著，其可以降低大鼠中脑的氧化损伤水平，保护多巴胺神经元，显著减少纹状体多巴胺神经元的丢失，并提高纹状体多巴胺的含量，进一步改善大鼠神经行为学症状，为帕金森病的治疗药物提供了新的选择。
本发明的技术方案是：
肽MOTS-c在制备治疗帕金森药物中的应用。
所述帕金森为由鱼藤酮诱导的。
所述肽MOTS-c的氨基酸序列如SEQ.ID.NO.1所示。
所述药物为多巴胺能神经元的保护剂。
所述药物增加纹状体多巴胺的含量。
所述药物采用腹腔注射方式给药。
所述药物的用量为3-5mg/kg/日。
本发明用鱼藤酮诱导帕金森大鼠模型，鱼藤酮使用梯度给药，第一阶段注射剂量为2mg/kg，共给药3天。第二阶段注射剂量为1mg/kg，共给药7天。第三阶段注射剂量为0.5mg/kg，共给药20天。合计给药30天，建立鱼藤酮诱导大鼠帕金森动物模型。治疗组给予模型组腹腔注射MOTS-c，持续30天，剂量为3-5mg/kg。
申请人的动物实验证明，肽MOTS-c能显著改善鱼藤酮诱导的帕金森大鼠的神经行为症状。肽MOTS-c能降低帕金森大鼠中脑内的氧化损伤水平，进一步保护大鼠多巴胺神经元，减少纹状体多巴胺神经元的丢失，并显著提高纹状体内的多巴胺的含量。在行为学方面，大鼠在新环境下的自主探索行为和运动协***况均得到了显著的改善。提示肽MOTS-c为治疗PD提供了一种新的思路。
附图说明
图1为大鼠中脑氧化损伤水平的检测结果，其中，
A为MDA(丙二醛)含量检测结果；图B为GSH(谷胱甘肽)含量的检测结果；图C为SOD(超氧化物歧化酶)活性的检测结果(注：*P<0.05，模型组与对照组比较；**P<0.01，模型组与对照组比较；#P<0.05，治疗组与模型组比较)
图2为大鼠纹状体HE染色结果，其中，箭头所指位置为细胞核。
图3为大鼠纹状体免疫组化的结果，其中，A为各组纹状体免疫组化染色(TH)结果；B为各组TH阳性面积的统计结果(注：*P<0.05，模型组与对照组比较；**P<0.01，模型组与对照组比较；#P<0.05，治疗组与模型组比较)。
图4大鼠纹状体蛋白免疫印迹的检测结果，其中，TH是多巴胺合成限速酶酪氨酸羟化酶，PSD95是突触后膜的标志物，SYP是突触前膜标志物。
图5为旷场实验结果，其中，A为大鼠运动总距离；B为大鼠运动轨迹的热图；C为大鼠后肢站立次数；D为静止不动时间(注：*P<0.05，模型组与对照组比较；**P<0.01，模型组与对照组比较；#P<0.05，治疗组与模型组比较)。
图6为转棒实验结果，图中，注：*P<0.05，模型组与对照组比较；**P<0.01，模型组与对照组比较；#P<0.05，治疗组与模型组比较。
具体实施方式
一、材料和试剂
1.动物
健康的SD雄性大鼠(5-7周龄)，体重220～270g之间，购于中国人民解放军陆军军医大学动物中心。
2.试剂
其他试剂均采用市售的分析纯产品。
3.主要仪器设备
二.实施例
实施例1动物处理
肽MOTS-c委托武汉百意欣生物科技有限公司合成，纯度为>95％，氨基酸序列如SEQ.ID.NO.1所示：
Met Arg Trp Gln Glu Met Gly Tyr Ile Phe Tyr Pro Arg Lys Leu Arg。
SD雄性大鼠44只，体重220～270g之间，饲养在室内温度为23±2℃的动物房，期间给大鼠提供自由饮水和进食。大鼠随机分为3组：对照组，模型组和治疗组。模型组(梯度剂量皮下注射)：第一阶段注射剂量为2mg/kg，共给药3天。第二阶段注射剂量为1mg/kg，共给药7天。第三阶段注射剂量为0.5mg/kg，共给药20天。合计给药30天，建立鱼藤酮诱导大鼠帕金森动物模型。治疗组：在模型组给药的同时，腹腔注射MOTS-c(3-5mg/kg)持续30天。处理结束后先检测大鼠行为学指标，包括旷场实验和转棒实验。行为学检测结束后麻醉处死大鼠，置于冰上迅速分离纹状体(一部分用多聚甲醛固定，一部分用液氮速冻)并收集其余脑组织。
实施例2实施各组大鼠神经行为学实验检测
1)旷场实验：旷场实验是评价实验动物在新环境中自主行为，探究行为以及紧张度的一种方法，以评价动物的活动度是否正常且均一。调整软件和***参数后，将大鼠至于旷场反应箱内，让其自由活动10分钟，***将记录大鼠的所有活动记录。实验结束后软件将记录分析其活动距离，活动轨迹和后肢站立次数等参数(每次实验前需要用酒精擦拭反应箱，以消除气味对动物的影响)。
2)转棒式疲劳仪实验：通过测量动物在滚轮上所停留的时间，用于评价动物平衡力，运动协调和中枢抑制等情况。实验开始前半小时将大鼠放到实验室适应环境。设置仪器转动参数，使得其以12rpm的速度匀速转动。实验动物依次放置于滚轮上，记录每只大鼠在转棒上保持平衡所能持续的时间，最长持续时间不超过3分钟。
实施例3大鼠纹状体多巴胺的含量(高分辨液质联用法)
将大鼠的纹状体称重并记录重量，精密加入1mL的甲醇(含0.1％甲酸)，漩涡震荡1min，匀浆3min。高速离心14000rpm，10min，取上清进样分析。色谱Waters T3(150×2.1mm，3μm)。流速0.3mL/min，水相0.1％甲酸水溶液，有机相0.1％甲酸乙腈，洗针液甲醇，柱温箱温度35℃。
实施例4免疫组织化学染色
纹状体组织用4％多聚甲醛固定后，常规脱水，石蜡包埋，切片，片厚5μm。组织切片经脱蜡水化，抗原修复，封闭内源性过氧化氢酶，抗体杂交(TH抗体)，DAB显色，复染，脱水，透明和封片后镜检。进一步用组织切片数字扫描仪扫描图像，应用赛维尔图像分析软件自动读取组织测量区域，首先进行阳性等级划分：阴性无着色，计为0分；弱阳淡黄色，计为1分，中阳性棕黄色，记为2分；强阳性棕褐色，计为3分，最终分析计算测量区域内阳性面积比，反应阳性面积的多少。
实施例5HE染色
纹状体组织用4％多聚甲醛固定后，常规脱水，石蜡包埋，切片，片厚5μm。切片经过脱蜡水化后按照HE试剂盒说明书进行苏木素-伊红染色，苏木素染核，盐酸乙醇分化，伊红染细胞质，透明化后封片。在光学显微镜下观察纹状体结构及损伤改变，并进行拍照。
实施例6免疫印迹法
纹状体组织提取总蛋白；BCA法进行蛋白浓度测定；然后进行SDS-聚丙烯酰胺凝胶电泳，转膜，封闭，一抗孵育过夜(4度)，对应种属二抗孵育(室温)，化学发光液进行显影。拍照后用Image J软件进行灰度值分析。
实施例7大鼠脑组织内GSH，MDA和SOD的含量测定
GSH，MDA和SOD检测试剂盒均购自上海碧云天生物科技有限公司，操作步骤均按照试剂盒说明书进行。
三.结果
1、肽MOTS-c可缓解鱼藤酮帕金森大鼠中脑氧化应激损伤
结果显示，治疗组大鼠大脑内MDA含量有所下降，同时GSH含量和SOD活性也有所提升，表明肽MOTS-c对鱼藤酮帕金森大鼠中脑内的氧化应激损伤水平有明显缓解作用(见图1)。
2、肽MOTS-c减轻鱼藤酮帕金森大鼠纹状体多巴胺神经元损伤
HE染色结果显示(见图2)，治疗组纹状体组织形态基本规则，细胞形态虽有回缩但接近于正常，核固缩现象明显好转。表明肽MOTS-c对鱼藤酮大鼠纹状体神经元有显著保护作用。
3、肽MOTS-c可减少帕金森大鼠纹状体多巴胺神经元丢失
如图所示(图3，图4)，模型组大鼠纹状体TH阳性着色减少明显，同时纹状体TH蛋白表达水平下降，提示帕金森大鼠纹状体多巴胺能神经元有显著丢失，这也是PD的重要标志。而治疗组大鼠纹状体的TH阳性细胞数量和TH蛋白表达水平明显提高。此外，治疗组纹状神经元突触标志物PSD95和SYP的表达水平相较模型组显著增高，上述结果提示MOTS-c对纹状体多巴胺神经元有明显的保护作用。
4、肽MOTS-c可增加帕金森大鼠纹状体多巴胺含量
表1结果显示，模型组纹状体DA含量显著降低，进一步说明模型复制成功；肽MOTS-c能显著提高纹状体多巴胺水平的作用。
表1：3组大鼠纹状体DA含量的测定结果
(注：*P<0.05，模型组与对照组比较；**P<0.01，模型组与对照组比较；#P<0.05，治疗组与模型组比较)
5、肽MOTS-c可改善鱼藤酮帕金森大鼠探究能力
旷场实验结果如图5所示，模型组大鼠后肢站立次数减少，运动距离缩短，休息时间增加，表明模型组动物在新环境中的自主探究行为能力显著下降，也提示帕金森模型复制成功。而治疗组的上述指标都得到了一定程度的好转，提示肽MOTS-c给药可以改善鱼藤酮致帕金森大鼠新环境下自主探究行为的能力。
6、肽MOTS-c可改善鱼藤酮帕金森大鼠运动协调能力
模型组大鼠运动协调障碍明显，且四肢活动欠佳，其在转棒上活动的时间显著减短，甚至个别大鼠活动极度不协调，很难在转棒上持续活动。而治疗组大鼠运动协调能力显著改善，在转棒上活动的时间明显延长。提示肽MOTS-c对缓解帕金森大鼠运动协调能力有积极的作用(见图6)。
综上，本发明建立鱼藤酮诱导帕金森大鼠模型，同时给予肽MOTS-c进行干预。实验结果显示肽MOTS-c能够缓解帕金森大鼠大脑的氧化应激损伤水平，保护多巴胺神经元，减少多巴胺神经元的丢失，且提高纹状体多巴胺的含量，改善帕金森大鼠的神经行为学症状。因此，肽MOTS-c对帕金森大鼠有明显的保护作用。肽MOTS-c能够应用于制备治疗帕金森病的药物。
序列表
<110> 中国人民解放军陆军军医大学
<120> 肽MOTS-c在制备治疗帕金森药物中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> 人工合成(Artificial synthesis)
<400> 1
Met Arg Trp Gln Glu Met Gly Tyr Ile Phe Tyr Pro Arg Lys Leu Arg
1 5 10 15
Claims (5)
1.肽MOTS-c在制备治疗帕金森药物中的应用，所述帕金森为由鱼藤酮诱导的，所述肽MOTS-c的氨基酸序列如SEQ.ID.NO.1所示。
2.根据权利要求1所述的应用，其特征在于：所述药物为多巴胺能神经元的保护剂。
3.根据权利要求1所述的应用，其特征在于：所述药物可增加纹状体多巴胺的含量。
4.根据权利要求1所述的应用，其特征在于：所述药物采用腹腔注射的方式给药。
5.根据权利要求1所述的应用，其特征在于：所述药物的用量为3-5mg/kg/日。
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