CN114042078A - 罗汉果苷v在制备对帕金森病神经元损伤具有保护作用的药物中的应用 - Google Patents
罗汉果苷v在制备对帕金森病神经元损伤具有保护作用的药物中的应用 Download PDFInfo
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Abstract
本发明公开了罗汉果苷V(Mogroside V，MV)在制备对帕金森病(Parkinson’s disease，PD)神经元损伤具有保护作用的药物中的应用，研究MV对鱼藤酮(Rotenone，Rot)诱导的神经毒性起保护作用，通过建立了PD的体内、外模型，然后，检测模型小鼠行为学及神经损伤、细胞活力、ROS水平、线粒体膜电势(Mitochondrial membrane potential，MMP)、线粒体呼吸链功能、早期凋亡和ATP水平。最后，通过上调Sirtuin 3(SIRT3)，探讨了MV在PD神经保护中的作用机制。
Description
【技术领域】
本发明属于治疗帕金森病药物技术领域，涉及罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用。
【背景技术】
帕金森病(Parkinson’s disease，PD)是人类最常见的神经退行性运动障碍之一，仅次于阿尔茨海默病(Alzheimer’s disease，AD)。PD的特征性病理特征是中脑黑质(Substantia nigra，SN)多巴胺能神经元的进行性丢失，导致纹状体多巴胺(Dopamine，DA)明显减少。DA的丢失导致黑质纹状体系统的损伤和PD的发生。PD的病因尚不清楚。在分子水平上，线粒体功能障碍已被证明是导致PD患者多巴胺能神经元进行性丢失的关键因素。尸检研究发现线粒体复合物I(complex I)活性和泛素酮辅酶Q10(CQ10)在PD脑SN 中。线粒体是细胞内活性氧(Reactive oxygen species，ROS)的主要来源，其是氧化磷酸化过程中未配对电子与分子O2相互作用产生的。人们普遍认为，作为呼吸链的一个组成部分，ROS破坏complex I，诱导进一步抑制，导致更多ROS形成。线粒体损伤与氧化应激形成恶性循环，参与PD的发病过程，并可能引发神经退行性疾病的后续下游事件，导致多巴胺能神经元进行性变性。神经毒素鱼藤酮(Rotenone，Rot)和1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP) 都是典型的complex I抑制剂，是已知的PD诱导物。它们可以在PD动物模型中引起氧化应激和多巴胺能神经元变性，进一步证明了线粒体功能障碍在PD发病机制中的重要性。
Sirtuin(SIRT)将一个乙酰基从蛋白底物转移到腺苷二磷酸核糖(ADPR)的2’-OH基上，形成3'-o-乙酰基-ADPR，进而使靶蛋白去乙酰化，以调节其功能。SIRTs在体内和体外均被证明参与了PD的发病机制。哺乳动物有7种SIRTs(SIRT1-SIRT7)，每种SIRT对不同的底物都有特异性。SIRT1与SIRT6、SIRT7一起定位于细胞核内，而SIRT2主要位于细胞质内。此外，Sirtuin3(SIRT3)、SIRT4和SIRT5主要存在于线粒体中。已在PD、AD、PD伴痴呆和路易体痴呆的死后脑组织中观察到SIRT1活性下调。过表达SIRT1保护了Rot诱导的人神经母细胞瘤(SH-SY5Y)的细胞死亡，其去乙酰化酶活性具有部分的保护作用。SIRT3基因敲除导致超乙酰化和线粒体功能受损，而SIRT4或SIRT5敲除没有类似的后果。因此，SIRT3被认为是一种主要的线粒体去乙酰化酶，在高氧消耗和氧化应激时特别敏感。PD中SIRT3相关的神经保护与线粒体功能、抗氧化能力和线粒体质量控制系统的调节有关，类似于能量代谢。SIRT3已被证明可以通过抑制线粒体通透性过渡孔开放来提高压力下的细胞存活率。SIRT3通过直接去乙酰化ATP合成酶β(ATP Synthaseβ)和超氧化物歧化酶-2(SOD2)，提高活性氧清除能力和线粒体ATP生成能力，保护DA神经元免受氧化损伤。SIRT3活性和表达异常与PD相关的遗传因素有关，包括PTEN诱导的假定激酶1、Leucine Rich Repeat kinase 2、Parkin 和α-synuclein(α-syn)，从而导致DA神经元的退化。在稳定的α-syn过表达模型中，增加SIRT3蛋白的表达可以通过减少α-syn寡聚物和增强线粒体生物能量学来修复α-syn诱导的线粒体功能障碍和细胞缺陷。敲除SIRT3可导致SH-SY5Y细胞在Rot暴露后出现严重损伤和α-syn聚合，这可通过SIRT3过表达逆转。这些证据表明，在PD相关的发病机制中， SIRTs具有明显的保护作用。
罗汉果(Siraitia grosvenorii)是一种发现于中国南部地区著名的食药用植物。罗汉果中的总苷为葫芦烷型三萜苷类化合物，包括罗汉果苷V(Mogroside V,MV)、罗汉果苷IVe(Mogroside IVe)、罗汉果苷IIIe(Mogroside IIIe)、罗汉果苷II A2(Mogroside IIA2)、罗汉果苷III A1(Mogroside III A1)、罗汉果苷IVa(Mogroside IVa)、罗汉果苷I(Mogroside VI)、赛门苷I(Siamenoside I)、；11-O-罗汉果苷V(11-Oxomogroside V)等，其中的Mogroside I、III、IV和V是从罗汉果中提取的一组具有药理生物活性的三萜类成分，具有抗氧化、抗高血糖、免疫、镇咳和抗细胞毒活性。罗汉果苷V是研究最充分和最常见的形式之一，已被报道可减轻炎症和高反应性，并抑制高血糖诱导的肺癌细胞迁移和侵袭。罗汉果苷V在中枢神经系统疾病中的保护作用日益受到人们的关注。在非竞争性NMDA受体拮抗剂地佐西平 MK801诱导的类精神分裂症小鼠模型中，罗汉果苷V可以通过促进神经突生长、抑制细胞凋亡和[Ca2+]i释放减少来拯救精神分裂症相关的行为表型。在体外卵母细胞衰老过程中，罗汉果苷V能够缓解卵母细胞质量的恶化，可能是通过SIRT1上调来降低氧化应激。
对于罗汉果苷V是否能够通过上调PD中的SIRT3来减轻线粒体功能障碍，从而在神经元损伤中发挥保护作用，进而将罗汉果苷V用于在制备对帕金森病神经元损伤具有保护作用的药物中的应用具有很好对市场前景。
【发明内容】
针对现有研究中，SIRT3是一种关键的线粒体去乙酰酶，在高耗氧和氧化应激中尤其敏感，本发明提供了罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，本申请研究表明，罗汉果苷V(简称MV)能够通过上调PD中的SIRT3来减轻线粒体功能障碍，从而在神经元损伤中发挥保护作用。
本发明的目的通过以下技术方案来实现：
罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用。
进一步的，所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，其中罗汉果苷V通过上调PD中的SIRT3来减轻线粒体功能障碍，从而在神经元损伤中发挥保护作用。
进一步的，所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，所述药物是药物组合物，是以罗汉果苷V为主要活性成分，加上药学中可接受的辅料或辅助性成分制备成临床上可接受的药物制剂，所述的罗汉果苷V在药物组合物中的含量通常为0.01-95.0％(w/w)。
通常而言，作为药物，均是在制备成制剂后才临床应用。本发明所述的药物，作为药物组合物可根据本领域公知的方法制备，可通过将本发明药物与一种或多种药学上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型。
进一步的，所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，所述药物制剂包括口服制剂和注射制剂两种剂型。
进一步的，所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，所述口服制剂为口服胶囊，所述注射制剂为静脉注射液。
本发明所述药物或药物组合物可以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
给药剂型可以是液体剂型、固体剂型或半固体剂型。液体剂型可以是溶液剂(包括真溶液和胶体溶液)、乳剂(包括o/w型、w/o型和复乳)、混悬剂、注射剂(包括水针剂、粉针剂和输液)、滴眼剂、滴鼻剂、洗剂和搽剂等；固体剂型可以是片剂(包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片)、胶囊剂(包括硬胶囊、软胶囊、肠溶胶囊)、颗粒剂、散剂、微丸、滴丸、栓剂、膜剂、贴片、气(粉)雾剂、喷雾剂等；半固体剂型可以是软膏剂、凝胶剂、糊剂等。
本发明药物可以制成普通制剂、也可以制成缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。为了将本发明药物制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；润湿剂可以是水、乙醇、异丙醇等；黏合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将有效成分本发明药物与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分本发明药物先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明药物片剂的各稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明药物的胶囊剂。
为将本发明药物制成注射剂，可以用水、乙醇、异丙醇、丙二醇或它们的混合物作溶剂并加入适量本领域常用的增溶剂、助溶剂、pH调剂剂、渗透压调节剂。增溶剂或助溶剂可以是泊洛沙姆、卵磷脂、羟丙基-β-环糊精等；pH调剂剂可以是磷酸盐、醋酸盐、盐酸、氢氧化钠等；渗透压调节剂可以是氯化钠、甘露醇、葡萄糖、磷酸盐、醋酸盐等。如制备冻干粉针剂，还可加入甘露醇、葡萄糖等作为支撑剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其他添加剂。
和现有技术相比，本发明具有如下优点：
1、本发明所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，研究发现MV能够通过上调PD中的SIRT3来减轻线粒体功能障碍，从而在神经元损伤中发挥保护作用。
2、本发明所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，研究发现罗汉果苷V对鱼藤酮(Rotenone，Rot)诱导的神经毒性起保护作用，通过建立了PD的体内、外模型，然后，检测模型小鼠行为学及神经损伤、细胞活力、ROS水平、线粒体膜电势(Mitochondrial membrane potential，MMP)、线粒体呼吸链功能、早期凋亡和ATP水平，最后，通过上调Sirtuin 3(SIRT3)，探讨了罗汉果苷V在PD神经保护中的作用机制。
【附图说明】
图1是本发明实施例中罗汉果苷V改善Rot引发的运动障碍和神经元损伤的图；
图2是本发明实施例中罗汉果苷V在SH-SY5Y细胞中保护了由Rot引起的细胞损伤的图；
图3是本发明实施例中随着剂量的增加，罗汉果苷V对ROS水平的影响的图；
图4是本发明实施例中罗汉果苷V可以改善SH-SY5Y细胞中Rot引发的线粒体功能障碍的图；
图5是本发明实施例中通过罗汉果苷V抑制线粒体调控的早期凋亡的图；
图6是本发明实施例中罗汉果苷V通过SIRT3/SOD2减少SN中Rot诱导的神经毒性的图；
图7是本发明实施例中通过抑制SIRT3可以消除罗汉果苷V对Rot诱导的神经毒性的保护作用的图。
【具体实施方式】
以下结合实施例对本发明的具体实施方式做进一步说明。
实施例：
1、罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用：
2、材料与方法
2.1.动物和处理
2月龄雄性C57BL/6小鼠购自中国湖南斯莱克景达实验动物有限公司。本申请的所有实验均按照中国桂林医学院实验动物伦理委员会(批准号2019-0011)的指导原则进行。
将30只运动能力相近的小鼠随机分为Con组、Rot组、MV2.5组、MV5组、MV10组。磷酸缓冲盐(PBS，pH值7.4)溶解MV。基于MV药代动力学参数和优秀的生物利用度，分别以2.5，5，10mg/kg灌胃法连续六天预处理；然后，对MV预处理组和Rot组小鼠以0.25mg/kg的剂量进行右侧纹状体内的Rot定向微量注射，所有的老鼠继续饲养一个月；采用旷场实验检测方法，记录小鼠在处死前15min内的平均运动速度，评价小鼠自主运动能力。
2.2.细胞培养与处理
人神经母细胞瘤细胞(SH-SY5Y细胞)购自中国科学院，使用添加10％胎牛血清的DMEM/F12 培养基(Gibco，New York，USA)培养。然后将细胞转入96孔板进行细胞活力和乳酸脱氢酶(LDH) 检测，24孔板进行细胞染色和免疫细胞化学检测，6孔板进行ROS、caspase-3活性和 oxygraphk-2k检测，10cm培养皿进行SIRT3活性检测和western blotting检测。将Rot在 DMSO中溶解至浓度为100mM，再用培养基稀释至400nM。用CQ10(10μM)作为阳性对照。用 DMSO溶解MV，最终浓度分别为25μM、50μM和100μM。CQ10组和MV组分别在Rot给药前预处理1h。细胞培养24h后，用Rot、CQ10或MV处理细胞24h，再进行后续试验。在加入 Rot和/或MV之前，细胞经SIRT3选择性抑制剂3TYP(50μM)预处理2h。
2.3.细胞活力检测使用细胞计数Kit-8(CCK8，DOJINDO LABORATORISE，KUMAMOTO，日本) 评估细胞活力。在100μL的培养基中加入10μL CCK8试剂，在37℃、5％CO2条件下孵育1h。使用酶标仪(Molecular Devices，California，USA)在450nm处读取吸光度值。
2.4.细胞毒性试验
细胞毒性检测使用CytoTox 
非放射性细胞毒性检测(Promega公司，威斯康星州，美国)。简单地说，将所有样品的50μL培养基转移到检测板上。然后加入50μL的CytoTox
试剂，室温避光孵育30min。加入50μL停止液后，戳破气泡，使用酶标仪(MolecularDevices， California，USA)在490nm处记录吸光度。
2.5.PI/Hoechst染色
PI/Hoechst染色采用Hoechst33342和碘化丙啶(PI，碧云天生物科技，上海，中国)。细胞被接种在先前涂有多聚赖氨酸的玻璃片上。将染液滴在载玻片上。细胞侧玻璃片贴在液滴上，以避免气泡。通过荧光显微镜(尼康公司，东京，日本)的红色和蓝色通道观察细胞。
2.6.ROS实验
活性氧活性测定试剂盒[(碧云天生物科技，上海，中国)。细胞治疗10μM 2',7' -Dichlorodihydrofluorescein二乙酸(DCFH-DA)和无血清培养基稀释和孵化37℃5％CO2洗涤三次后20min。用无血清培养基，细胞通过绿色通道的荧光显微镜观察(尼康公司，东京，日本)。
2.7.Mito-Tracker和Annexin-V染色
采用线粒体膜电位和凋亡检测试剂盒(Mito-Tracker red CMXRos和Annexin-V-FITC)检测MMP和细胞凋亡。首先，细胞在1000×g下离心5min，然后加入188μL Annexin-V-FITC 结合缓冲液、5μL Annexin-V-FITC、2μL Mito-Tracker Red CMXRos染色液和5μLHoechst 33342染色液。在室温下轻轻混合细胞溶液并孵育30min后，使用荧光显微镜(尼康公司，东京，日本)对细胞进行成像。
2.8.Oxygraph-2K实验
线粒体complex I活性采用Oxygraph-2k-FluoRespirometer(OroborosInstruments， Innsbruck，Austria)测定。总而言之，在100μL PBS中收集1×106个细胞。一旦细胞实际注射到反应排放口，记录氧浓度和氧通量每体积值。将细胞消化后，分别加入800mM丙酮酸、 2M苹果酸、2M谷氨酸、500mM ADP、300mM MgCl2、1M琥珀酸、1mM CCCP、1mMRot、5mM抗霉素A。加入每种试剂，直到呼吸频率显示稳定。
2.9.流式细胞术检测Annexin-V
采用FITC-Annexin-V凋亡检测试剂盒(Becton，Dickinson and Company，NewJersey，USA)进行流式细胞术评估细胞凋亡。用预冷PBS洗涤细胞2次后，用1×Bindingbuffer重悬细胞，密度为1×106cells/mL。将十分之一的细胞转入5ml试管中，用5μL PI和5μL FITC Annexin-V在室温暗培养15min。培养液中加入400μL 1×Binding buffer，1h内通过流式细胞术(Becton，Dickinson and Company，New Jersey，USA)进行分析。
2.10.TUNEL染色
原位细胞凋亡使用DeadEndTM荧光TUNEL系统(Promega公司，威斯康星州，美国)进行。在4℃条件下，用4％的甲醛固定细胞25min。然后将细胞用0.2％
-100PBS洗涤通透5min，室温用100μL平衡缓冲液平衡10min后，加入50μL TdT反应混合物，37℃孵育60min。用2×SSC停止反应15min。最后，将细胞装入含有DAPI的培养基中。使用荧光显微镜检测凋亡细胞的局部绿色荧光(尼康公司，东京，日本)。
2.11.Caspase 3活性
采用Caspase 3比色检测试剂盒(Applygen Technologies，北京，中国)检测细胞凋亡。总而言之，将2×106细胞离心，在50μL的裂解缓冲液中裂解。上清在12000×g下离心得到， 85μL反应缓冲液、10μL样品和5μL底物混合，37℃孵育2h。用酶标仪(MolecularDevices， California，USA)在405nm处记录Caspase 3活性；将活性与标准曲线进行比较。
2.12.ATP测定
采用ATP检测试剂盒(ATP Assay Kit，碧海生物科技，上海，中国)检测细胞ATP水平。裂解后收集细胞上清，12000×g离心5min作进一步分析。然后在96孔不透明白板上加入100 μL ATP反应缓冲液消耗背景ATP。然后分别取样品和标准品各20μL加入反应孔。然后，使用多模板阅读器(Molecular Devices，California，USA)的Luminometer模式读取RLU值。
2.13.免疫印迹
按照线粒体和细胞质蛋白制备试剂盒(Applygen，北京，中国)和培养细胞线粒体分离试剂盒(Thermo Fisher Scientific，马萨诸塞州，美国)的说明收集并裂解小鼠黑质和SH-SY5Y 细胞。线粒体和细胞质部分被分离并聚集到不同的管中。使用BCA蛋白定量试剂盒(Thermo Fisher Scientific，Massachusetts，USA)评估蛋白浓度。然后用SDS-PAGE分离各组分20μg 蛋白，将蛋白转移到PVDF膜上。然后用5％脱脂奶粉阻断膜，分别加入抗酪氨酸羟化酶(TH)、抗SOD2k122ac、抗SOD2、抗细胞色素C(Cyto C)、抗SIRT3、抗VDAC1、抗β-actin抗体，孵育过夜。将相应的荧光二抗与一抗室温结合1h。经过四次清洗后，ODYSSEY成像系统(美国内布拉斯加州LI-COR生物科学公司)帮助扫描膜。
2.14.免疫细胞化学(IHC)
简单地说，用0.9％NaCl和4％多聚甲醛(PFA)经左心室灌注。脑用4％PFA固定48h，再用含30％蔗糖的PFA脱水。然后用Cryo Star NX50(Thermo Fisher，马萨诸塞州，美国)冷冻后切成20μm厚的切片。片治疗0.3％
100x-PBS(PBST)两天，1％山羊血清在PBST前1h孵化与TH鼠单克隆抗体(1:1000)或SIRT3兔多克隆抗体(1:50)48h。然后，添加Alexa
488 goat-anti-mouse或兔免疫球蛋白(h+L)在室温下孵育1h。最后，将切片倒扣在含有DAPI的封片剂上，并使用荧光显微镜(尼康公司，东京，日本)进行观察。
用4％PFA对细胞进行免疫组化，在4℃下固定细胞25min。然后，用0.2％PBST洗涤细胞并渗透5分钟，然后用5％胎牛血清在0.2％PBST中4℃封闭细胞1h。SIRT3兔多克隆抗体用0.2％PBST(1:50)稀释，室温孵育2h。然后加入Alexa
488山羊抗兔IgG(H+L)，室温孵育1h。最后，将细胞爬片倒扣在含有DAPI的封片剂上，并使用荧光显微镜(尼康公司，东京，日本)进行观察。
2.15.SIRT3活性检测
使用SIRT3活性荧光定量检测试剂盒(GENMED scientific，马萨诸塞州，美国)评估SIRT3 活性。简而言之，1×107细胞用0.01M PBS洗涤，并用细胞刮板收集。然后加入1mLGENMED 裂解缓冲液，Dounce均质器裂解细胞。将匀浆在4℃下1500×g离心10min，收集上清在4℃下10000×g离心10min。将沉淀用200μL GENMED裂解缓冲液重悬，按照厂家说明书进行后续活性测定。使用酶标仪(Molecular Devices，California，USA)读取相对荧光单位。
2.16.统计分析
数据以均数±标准差表示。使用GraphPad Prism软件8.0(GraphPad，California，USA) 进行统计分析。采用单因素方差分析(One-way ANOVA)进行组间差异评估，然后采用Tukey的多重比较检验。P<0.05认为有统计学意义。
3.结果
3.1.MV能有效改善Rot诱导的运动损伤和神经元损伤
单侧纹状体立体定向注射Rot引起一些运动症状。采用旷场实验评估动物的自发运动。与对照组相比，Rot组小鼠的运动轨迹在边缘处明显稀疏而集中，在记录时间内平均运动速度下降。将MV通过灌胃法处理Rot作用的小鼠增加了小鼠运动的平均速度，伴随着旷场中心运动的增加(图1A、C)。为了确定这些帕金森样运动障碍与Rot引起的SN多巴胺能神经元的损伤导致的DA水平降低有关，我们测量了SN中TH阳性神经元的荧光强度(图1B，D)和TH 蛋白的水平(图1E，F)。与之前的研究一致，Rot导致TH阳性神经元染色和SN中的TH蛋白水平严重降低，表明多巴胺能神经元的损伤和丢失。在接受10mg/kg MV治疗的小鼠中，这种Rot诱导的多巴胺能神经元损伤显著减轻，并逆转TH阳性黑质神经元的减少和TH蛋白表达的部分恢复(图1)。
图1：MV改善Rot引发的运动障碍和神经元损伤((A)0.25mg/kg Rot和MV联合处理(2.5mg/kg、5mg/kg、10mg/kg)小鼠的运动轨迹；(B)Rot和MV联合处理小鼠SN中TH的阳性染色；(C)旷场实验小鼠的平均运动速度；(D)TH的平均荧光强度(MFI)统计图；(E，F)SN中 TH蛋白表达的Western blot和光密度分析；采用单因素方差分析进行多重比较，所有数据以均数±标准差表示(n＝3)；*p<0.05，**p<0.01，***p<0.001)。
3.2.MV能有效地挽救Rot引起的细胞毒性
用0.1μM到0.5μM的Rot处理SH-SY5Y细胞24h，通过CCK8试验评估Rot对细胞活力的剂量依赖性影响(图2A)；与0.5μM时相比，0.4μM时细胞活力差异不显著。结果表明， 0.4μM为最佳浓度。我们检测了25、50和100μM MV干预对0.4μM Rot处理的影响。随着 MV浓度的增加，细胞活力增加。CCK8和LDH检测的结果表明，Rot处理后，细胞活力降低了 65.53％，随后LDH释放量增加到159.22％。100μM的MV处理后，特别是细胞生存能力被发现从65.53％提高到80.41％，而LDH释放从159.22％减少到127.65％，这是类似于CQ10的影响(图 2B，C)。对照组细胞具有树突状突起的多边形形状。然而，0.4μM Rot处理24h后，细胞变得更圆，轴突明显减少(图2E，箭头)。PI和Hoechst 33342双染色可以区分死细胞和活细胞。 MV处理后PI阳性水平存在差异，MV-100μM组的PI阳性水平显著低于Rot组(图2D，E)。这种转变伴随着显著的细胞形态变化。SH-SY5Y细胞的形态受到Rot的强烈影响，并在很大程度上通过MV的处理得到恢复(图2E)。上述实验结果表明，MV在体外对Rot毒性引起的细胞损伤具有有效的保护作用。
图2：MV在SH-SY5Y细胞中保护了由Rot引起的细胞损伤((A)Rot(0.1～0.5μM)或0.1％DMSO处理SH-SY5Y细胞24h的活力；0.4μM Rot和MV(25μM、50μM、100μM)或0.1％DMSO共处理细胞24h，用CCK8(B)和LDH(C)检测细胞活力和细胞毒性；(D)用PI/hoechst阳性细胞评价细胞死亡率；(E)亮视野细胞显微图像，PI/Hoechst33342染色，明场视野(上)，染色视野(中)，合并(下)，箭头显示典型细胞(比例尺＝100μm)；采用单因素方差分析进行多重比较，所有数据以均数±标准差表示(n＝3)；*p<0.05，**p<0.01，***p<0.001)。
3.3.MV呈剂量依赖性降低ROS水平。使用DCFH-DA探针检测ROS水平，以评估细胞的氧化应激水平。结果发现，随着Rot处理，ROS水平增加，而MV处理显著降低(图3)。此外，DCFH-DA荧光强度随着MV浓度的增加而降低。当MV浓度为100μM时，ROS水平恢复到阳性对照(CQ10)水平。
图3：随着剂量的增加，MV对ROS水平的影响((A)显微镜图像显示ROS敏感荧光DCFH-DA 染色的细胞；(B)ROS的产生是通过相对荧光强度表达的；采用单因素方差分析进行多重比较，所有数据以均数±标准差表示(n＝3)；*p<0.05，**p<0.01，***p<0.001)。
3.4.MV逆转线粒体功能障碍
鉴于线粒体是细胞内ROS的主要来源，我们评估了线粒体的功能。MMP是线粒体功能的一个主要指标，使用可在正常线粒体中快速积累的Mito-Tracker Red CMXRos荧光探针进行检测。与MV剂量相比，单纯Rot组的荧光强度显著降低，表明MMP去极化，然后逐步恢复(图 4A，B)。为了评估线粒体呼吸，使用Oroboros oxygraphk-2k系统评估氧耗率。MV逆转了Rot 降低的耗氧率(图4C)。由于线粒体的主要功能是生成ATP以维持细胞代谢，我们进一步检测了细胞内ATP水平。结果表明，MV的添加使大部分ATP含量得到恢复(图4D)。
图4：MV可以改善SH-SY5Y细胞中Rot引发的线粒体功能障碍((A)MMP使用MitoTracker Red CMXRos进行荧光评估，比例尺＝100μm；(B)MitoTracker Red CMXRos荧光检测结果；(C) 使用Oxygraph-2k系统定量细胞匀浆中的耗氧量；(D)ATP含量的检测，单因素方差分析用于多重比较，所有数据以均数±标准差表示(n＝3)；*p<0.05，**p<0.01，***p<0.001)。
3.5.MV抑制了Rot诱导的早期线粒体依赖的凋亡。
采用TUNEL法检测细胞凋亡水平，采用Annexin-V/PI双染色法检测细胞凋亡水平。TUNEL 阳性细胞数量随着Rot暴露而增加。0.4μM组和MV组共处理后，TUNEL阳性细胞数量逐渐减少。TUNEL阳性细胞比例的逐渐下降清楚地表明，使用MV逆转了Rot诱导的细胞凋亡(图5A)。 Annexin-V/PI染色进一步证实了这一趋势(图5B，C)。此外，通过检测细胞裂解液中的caspase 3活性来评估细胞凋亡，这是一种线粒体依赖的凋亡相关因子。不同剂量的MV处理后，caspase 3活性明显减少，存在剂量依赖性(图5D)。此外，western blot检测结果显示，MV显著减少了Rot引起的Cyto C释放(图5E，F)。总的来说，我们的数据表明，MV通过线粒体途径抑制细胞凋亡。
图5：通过MV抑制线粒体调控的早期凋亡((A)TUNEL染色检测TUNEL阳性细胞(绿色)，评估凋亡情况。比例尺＝100μm；(B)通过流式细胞术定量活细胞(PI-/Annexin-V-，Q4)、早期凋亡细胞(PI-/Annexin-V+，Q3)、晚期凋亡细胞(PI+/Annexin-V+，Q2)和死亡细胞(PI+/Annexin-V-，Q1)；(C)早期凋亡水平统计直方图；(D)通过caspase 3检测试剂盒检测caspase 3活性。(E，F)线粒体和细胞质中Cyto C蛋白表达的Western blot和光密度分析；采用单因素方差分析进行多重比较，所有数据以均数±标准差表示(n＝3)；*p<0.05，**p<0.01，***p<0.001)。
3.6.MV通过增强SIRT3/SOD2来减轻细胞损伤。
采用免疫荧光法检测SN和细胞中SIRT3的水平。Rot处理后，SIRT3表达减少，在某种程度上，10mg/kg MV治疗小鼠(图6A、B)和100μM MV处理细胞(图7A、B)可以逆转这个现象。免疫荧光结果也得到了SN(图6C，D)和细胞(图7)中线粒体的western blot结果的验证。SIRT3缺失可导致SOD2乙酰化失活。western blot结果显示Rot处理后，SOD2出现明显的乙酰化增加，而MV治疗可以明显翻转在SN(图6C，E)和细胞(图7D)中线粒体提取物中SOD2 的乙酰化增加。这些结果表明，MV可以部分增加SIRT3表达和活性。为了进一步证实SIRT3 在MV对抗Rot中的作用，我们使用SIRT3特异性抑制剂3TYP和MV共同处理细胞。结果表明，SIRT3特异性抑制剂处理后，细胞的活力减少，即使是在100μM MV治疗后(图7E)。此外，3TYP处理导致SIRT3活性降低(图7J)和MPP(图7H，I)降低，伴随着SOD2乙酰化水平增加(图7D)，ROS水平增加(图7F，G)和早期凋亡细胞数量增加(图7K，L)。SIRT3活性在Rot组显著降低，而在MV处理组显著升高。因此，我们的结果表明，MV通过增强SOD2依赖的SIRT3活性减轻了细胞损伤。
图6：MV通过SIRT3/SOD2减少SN中Rot诱导的神经毒性((A)0.25mg/kg Rot和10mg/kg MV处理胡SN中SIRT3的阳性染色，比例尺＝200μm；(B)SN中SIRT3 MFI的统计直方图；(C)SN 线粒体中SIRT3、SOD2和乙酰化SOD2表达的Western blot分析结果；(D)SN中SIRT3水平的光密度分析；(E)SN中SOD2和乙酰化SOD2表达的光密度分析；采用单因素方差分析进行多重比较，所有数据以均数±标准差表示(n＝3)；*p<0.05，**p<0.01，***p<0.001)。
图7：通过抑制SIRT3可以消除MV对Rot诱导的神经毒性的保护作用((A)0.25mg/kgRot 和10mg/kg MV处理SH-SY5Y细胞中SIRT3的阳性染色结果，比例尺＝50μm；(B)SH-SY5Y细胞中SIRT3 MFI测量的统计直方图；(C)SH-SY5Y细胞线粒体中SIRT3表达的Western blot和光密度分析；(D)Western blot和光密度分析Rot、MV和3TYP处理SH-SY5Y细胞线粒体中SOD2和乙酰化SOD2的表达水平；(E)SH-SY5Y细胞活力和细胞毒性；(F，G)细胞内ROS水平的荧光染色和相对荧光强度，比例尺＝100μm；(H，I)使用MitoTracker Red CMXRos进行荧光评估MMP，并对强度进行分析，比例尺＝100μm；(J)SH-SY5Y细胞中SIRT3活性；(K)早期凋亡水平(PI-/Annexin-V+，Q3)；(L)流式细胞术和Annexin-V-FITC/PI染色分析细胞凋亡；采用单因素方差分析进行多重比较，所有数据以均数±标准差表示)。
上述说明是针对本发明较佳可行实施例的详细说明，但实施例并非用以限定本发明的专利申请范围，凡本发明所提示的技术精神下所完成的同等变化或修饰变更，均应属于本发明所涵盖专利范围。
Claims (5)
1.罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用。
2.罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，其特征在于：其中罗汉果苷V通过上调PD中的SIRT3来减轻线粒体功能障碍，从而在神经元损伤中发挥保护作用。
3.根据权利要求1所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，其特征在于：所述的药物是药物组合物，是以罗汉果苷V为主要活性成分，加上药学中可接受的辅料或辅助性成分制备成临床上可接受的药物制剂，所述的罗汉果苷V在药物组合物中的含量通常为0.01-95.0％w/w。
4.根据权利要求3所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，其特征在于：所述药物制剂包括口服制剂和注射制剂两种剂型。
5.根据权利要求4所述的罗汉果苷V在制备对帕金森病神经元损伤具有保护作用的药物中的应用，其特征在于：所述口服制剂为口服胶囊，所述注射制剂为静脉注射液。
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