CN116271051B - Phactr4抑制剂在制备抑郁症治疗药物中的应用 - Google Patents
Phactr4抑制剂在制备抑郁症治疗药物中的应用 Download PDFInfo
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Abstract
本发明涉及Phactr4抑制剂在制备抑郁症治疗药物中的应用，属于抑郁症靶向治疗领域。现有研究针对Phactr4与精神类疾病的相关性尚不明确，针对该缺陷，本发明发现，Phactr4参与并在抑郁症的发病过程中发挥重要作用，通过靶向调控Phactr4的表达可以有效缓解大鼠的抑郁样表型，改善抑郁大鼠海马CA1区神经元突触结构与功能可塑性损伤，进而发挥抗抑郁治疗效果。基于本发明研究结论，靶向Phactr4的抑制剂有望发挥良好的抗抑郁效果，本发明完善了潜在的抑郁症发病分子机制，为临床抗抑郁药物的研发提供了新靶点。
Description
技术领域
本发明属于抑郁症靶向治疗技术领域，具体涉及Phactr4抑制剂在制备抑郁症治疗药物中的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
抑郁症逐渐成为全球范围内最为普遍的精神障碍类疾病，以长期的心情低落、快感缺失、行为绝望为主要特征，目前抑郁症在全世界范围内具有高致残率和致死率，给患者及其家属和社会都带来了沉重的负担。
近年来对抑郁症的研究表明，抑郁症的发病机制涉及多种高度复杂、相互关联的机制通路，主要包括单胺类递质失衡、HPA轴功能异常、神经营养因子缺乏影响神经网络的形成、线粒体功能障碍、表观遗传学修饰、炎症过程、脱髓鞘化和微生物菌群影响肠-脑轴等。目前使用的一线抗抑郁药存在起效慢、停药易复发、副作用大等诸多问题，严重减弱了它们实际的临床疗效，尚不能达到预期的治疗效果。这些现状表明，临床上缺乏有效的治疗抑郁症的方法与策略。抑郁症发病相关的机制假说尚不能完全揭示抑郁症的病因，抑郁症的发生发展是多种病因机制共同作用的结果。因此亟待进一步探究外界应急刺激与大脑功能障碍之间的作用机制，寻找参与抑郁症发病的关键作用分子，以开发出更好的抑郁症治疗手段、给抑郁症患者及家属带来新的希望。
磷酸酶和肌动蛋白调节因子（Phactr）家族通过与蛋白磷酸酶1（PP1）互作的结构域结合，调节肌动蛋白的解聚和细胞骨架结构进而在多种生物学过程中发挥功能，包括相关基因表达、突触传递和神经元形态，提示Phactr家族可能与神经系统疾病的发生发展有关。发明人认为，Phactr4（磷酸酶和肌动蛋白调控因子4 ，phosphatase and actinregulator 4）目前在抑郁症等神经精神类疾病中的研究较为空白，特别是Phactr4是否参与抑郁症的发病机制，以及抑制其蛋白表达水平是否可缓解抑郁样行为，目前尚无报道；阐明抑郁症潜在的发病机制，寻找更加有效的治疗新靶点，有利于获取新的临床治疗策略、开发疗效更佳的治疗药物，更好地造福人类。
发明内容
本发明首先采用经典的慢性不可预知性温和应激刺激(CUMS)进行抑郁模型的构建，并检测大鼠的抑郁样表现，发现CUMS组大鼠表现出抑郁样表型，并伴随海马区神经元突触的结构与功能可塑性损伤，但具体的病理机制以及在抑郁症发病过程中发挥作用的关键分子靶点需要进一步的研究。基于蛋白组学结果，本发明进行大量文献调研和实验，结果显示，Phactr4在抑郁大鼠海马CA1区表达明显增多，通过与蛋白磷酸酯酶-1（PP1）结合影响下游分子表达及活性，继而引起f-肌动蛋白（F-actin）与球状肌动蛋白（G-actin）的动态平衡紊乱，最终导致神经元突触结构和功能可塑性损伤，诱导动物抑郁样行为的发生。
根据上述研究成果，本发明具体提供如下方案：
本发明第一方面，提供Phactr4抑制剂在制备抑郁症治疗药物中的应用。
优选的，所述Phactr4抑制剂包括但不限于能够刺激受试者体内Phactr4表达含量降低的化合物实体、聚合物、多肽或核酸物质，还包括基于基因工程方式对受试者机体中Phactr4进行抑制的相关试剂，如质粒、慢病毒、腺相关病毒等。
进一步的，本发明提供的一种实施方式中，所述Phactr4抑制剂为靶向抑制Phactr4的腺相关病毒，所述腺相关病毒中具有Phactr4 shRNA序列，由于Phactr4为已知序列，获得相应的shRNA序列对于本领域技术人员来说不具有技术难度，可行的方法如，委托试剂公司合成等。
进一步的，所述抑制剂能够降低受试者脑内海马回结构内Phactr4表达含量，包括海马CA1~4区等；在本发明验证的实例中，研究对象为海马CA1区。
优选的，所述抑郁症包括内源性抑郁、反应性抑郁、药物继发性抑郁、产后抑郁、更年期抑郁、老年期抑郁；本发明通过慢性不可预见性温和应激方法构建抑郁模型，本领域技术人员基于上述结论预期Phactr4抑制剂可能在反应性抑郁患者中能够发挥更好的治疗效果。
本发明第二方面，提供Phactr4检测试剂在制备抑郁症诊断产品中的应用。
优选的，所述Phactr4检测试剂包括能够实现上述标志物检测的各种试剂，如酶联免疫吸附剂测定（ELISA）试剂、Western免疫印迹、Northern印迹、实时荧光定量PCR技术、荧光原位杂交、RNA干扰和RNA结合蛋白免疫沉淀。
优选的，所述抑郁症诊断产品为用于对抑郁症筛查、诊断、辅助诊断或预后评估的产品，包括诊断试剂及诊断器械，具体的实例如，诊断试剂盒。
优选的，所述诊断样本为人源样本，包括但不限于血清(浆)、全血、分泌物、棉拭子、脓液、体液、组织、器官、石蜡切片中的一种或几种等。
本发明第三方面，提供一种药物组合物，所述药物组合物中包括活性剂量的Phactr4抑制剂。
优选的，所述药物组合物中，还包括药学上所述必须的载体。
上述药物组合物中，所述Phactr4抑制剂添加量属于本领域技术人员依据抑制剂种类、施用目的、受试对象、给药方式等因素可以常规确定的技术内容。
所述药物组合物的剂型没有特别的限制，优选的方式为固体制剂或液体制剂；固体制剂的情形下，所述载体包括但不限于赋形剂、润滑剂、粘合剂、崩解剂等；液体制剂的情形下，所述载体包括但不限于溶剂、增溶剂、悬浮剂、等渗剂、缓冲剂、舒缓剂等，还可以根据需要可以适当地加入防腐剂、抗氧化剂、着色剂、甜味剂和其他制剂添加剂。
作为赋形剂的优选实例，例如，可以使用乳糖、蔗糖、D-甘露糖醇、淀粉、结晶纤维素或轻质无水硅酸等。
作为润滑剂的优选实例，例如，可以使用硬脂酸镁、硬脂酸钙、滑石粉或胶体二氧化硅等。
作为粘合剂的优选实例，例如，可以使用结晶纤维素、蔗糖、D-甘露糖醇、糊精、羟丙基纤维素、羟丙基甲基纤维素或聚乙烯吡咯烷酮等。
作为崩解剂的优选实例，例如，可以使用淀粉、羧甲基纤维素、羧甲基纤维素钙、交联羧甲基纤维素钠或羧甲基淀粉钠等。
作为溶剂的优选实例，可以使用注射用水、醇、丙二醇、聚乙二醇、芝麻油或玉米油等。
作为增溶剂的优选实例，例如，使用聚乙二醇、丙二醇、D-甘露醇、苯甲酸苄酯、乙醇、三氨基甲烷、胆固醇、三乙醇胺、碳酸钠或柠檬酸钠等。
作为缓冲剂的优选实例，例如，可以使用磷酸盐、乙酸盐、碳酸盐或柠檬酸盐等的缓冲溶液。
作为舒缓剂的优选实例，例如，可以使用苯甲醇等。
作为防腐剂的优选实例，例如，可以使用对氧苯甲酸酯、氯丁醇、苯甲醇、苯乙醇、脱氢乙酸或山梨酸等。
作为抗氧化剂的优选实例，例如，可以使用亚硫酸盐或抗坏血酸等。
本发明第四方面，提供第三方面所述药物组合物在制备抗抑郁症产品中的应用。
优选的，所述抗抑郁症产品包括诊断、预防、改善或治疗抑郁症的药物制剂。
本发明第五方面，提供一种抑郁症治疗方法，所述治疗方法包括但不限于通过口服、注射或介入方式将Phactr4抑制剂递送至病灶部位，并降低病灶部位的Phactr4表达。
以上一个或多个技术方案的有益效果是：
基于此，本发明对Phactr4蛋白在抑郁症发病中的关键作用进行了更为深入的解析，为完善抑郁症的发病机制和治疗抑郁症提供了新的思路，同时证明了Phactr4可作为抗抑郁治疗的新靶点，具有可观的潜力，确定了通过靶向Phactr4的基因调控可以作为一种治疗抑郁症的新方案。本发明为抗抑郁药物的研发提供了新方向与潜在的靶点，为临床对抑郁症患者治疗提供了具有科学依据的新思路，为抑郁症患者带来了新的希望。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为Phactr4参与应激刺激诱导大鼠抑郁样行为的机制图；
图2为CUMS组抑郁大鼠海马CA1区Phactr4表达显著增高的示意图；
(A)构建慢性不可预知性温和刺激CUMS抑郁模型的示意图；
(B)强迫游泳实验中大鼠静止时间统计结果；
(C)强迫游泳实验中大鼠游泳时间统计结果；
(D)糖水偏好实验结果；
(E)电镜结果；
(F)高尔基染色结果
(G)树突棘密度统计图；
(H) Sholl analysis树形图；
(I)Sholl analysis统计结果；
(J) F-actin蛋白免疫印迹结果；
图3为蛋白组学分析结果及验证；
(A)差异性表达蛋白质火山图；
(B)差异性表达蛋白质热图；
(C) Phactr4的蛋白免疫印迹结果；
(D)PP1蛋白免疫印迹结果；
(E) P-cofilin蛋白免疫印迹结果；
图4为敲低抑郁大鼠海马区Phactr4缓解动物的抑郁样行为及改善突触可塑性损伤的示意图；
(A)敲低Phactr4的AAV腺相关病毒载体的构建；
(B)在CUMS抑郁大鼠海马CA1区通过脑立体定位注射敲低Phactr4的腺相关病毒实验流程；
(C)双侧海马CA1区病毒感染效果图；
(D)为敲低Phactr4的大鼠强迫游泳实验中大鼠静止时间统计结果；
(E)为敲低Phactr4的大鼠强迫游泳实验中大鼠游泳时间统计结果；
(F)敲低Phactr4的大鼠糖水偏好实验结果；
(G)敲低Phactr4的大鼠海马CA1区神经元电生理指标；
(H)敲低Phactr4的大鼠海马CA1区目标分子及下游通路蛋白免疫印迹；
以上附图中，”Ctrl”表示对照组”CUMS”表示造模组。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
本发明选取正常对照组大鼠和抑郁造模组大鼠的海马组织进行蛋白组学分析，结果表明Phactr4蛋白的差异性较为明显，其在抑郁模型组中表达明显上调。进一步的，本发明发现，敲减抑郁模型大鼠神经元Phactr4的蛋白表达水平能够有效改善动物抑郁样表型和可塑性损伤，以上结果提示，Phactr4可作为一个抗抑郁治疗的新的有效靶点。为了使得本领域技术人员能够更加清楚地了解本发明的技术方案，以下将结合具体的实施例详细说明本发明的技术方案。
实施例
1. 抑郁动物模型的构建及行为学测试
从山东大学动物中心购入160-180克体重的雄性Wistar大鼠，单笼饲养，第一周给予正常饮食和光照，让动物适应1周。第二周开始进行慢性不可预知性温和刺激(CUMS)造模，实验开始前随机分组，每天随机给予CUMS组大鼠剥夺食物24小时、剥夺水24小时、足底电击、笼子倾斜45°、湿笼24小时、昼夜颠倒各12小时、夹尾3分钟、冰水游泳5分钟和身体束缚2小时其中一种刺激。对照组不做任何处理。连续5周的CUMS刺激后，对各种大鼠进行行为学测试，包括糖水偏好（SPT）、强迫游泳（FST）、高架十字迷宫（EPM）以及旷场实验（OFT），随后取材，进行后续实验（图2A）。图2B和图2C证实：强迫游泳实验中CUMS组大鼠静止时间升高，游动时间减少；图2D证实，糖水偏好实验中CUMS组大鼠对糖水的偏好度下降。慢性不可预知性温和刺激诱导动物行为绝望和快感缺失等抑郁症的核心症状。按照FD快速高尔基染色试剂盒(货号PK401, FD NeuroTechnologies公司)说明书对新鲜取材的海马组织进行进一步的高尔基染色处理，并通过高倍物镜观察并拍摄树突棘的变化，使用Sholl analysis分析神经元树突的复杂性，观察结构可塑性改变。图2E-图2G表明：CUMS组突触密度和树突棘密度均降低。Sholl analysis发现，CUMS组海马区树突复杂性降低（图2H、图2I）。Westernblot实验结果证实，CUMS组海马区F-actin降低（图2J）。
2. 将正常的大鼠和具有抑郁样表型的大鼠海马区进行蛋白组学分析
样本间蛋白差异性表达分析发现， Phactr4的蛋白表达差异倍数较为明显，接着对Phactr4进行GO分析发现，其与调节细胞骨架重要组成成分-肌动蛋白的蛋白磷酸酶1（PP1）及Rho蛋白信号转导密切相关。将行为学测试后的大鼠进行处死取材，取出大脑海马CA1区组织并加入RIPA裂解液，磷酸酶抑制剂与蛋白酶抑制剂，振荡混匀后进行低温匀浆，然后在4℃条件下使用超高速离心机进行12000 rpm离心20 min，收集上清液，加入等比例的1×Loading Buffer，将混合液在100℃煮沸5 min，将煮好的样本加入提前预制好的10%的SDS-PAGE凝胶微孔中进行电泳，然后转膜到聚偏二氟乙烯膜（polyvinylidenefluoride, PVDF）上，通过一抗与二抗结合的Western blot实验原理验证蛋白组学结果中目标蛋白Phactr4及下游分子（P-cofilin等）的表达。
蛋白组学分析结果如附图3A及图3B所示，蛋白组学分析发现Phactr4在抑郁模型组海马区中蛋白水平明显增高。Western Blot（图3C-3E）验证Phactr4、PP1和P-cofilin蛋白表达水平。
3. 在动物模型上干预Phactr4的表达，检测动物行为学、电生理指标
从上海吉凯公司构建敲低Phactr4的腺相关病毒（图4A），并通过脑立体定位注射在海马CA1区下调Phactr4蛋白表达水平，待动物感染3周后，对各组大鼠进行行为学测试和取材（图4B）。荧光结果显示，病毒感染情况达到了预期（图4C）。采用糖水偏好实验、强迫游泳实验等检测干预Phactr4后动物行为学变化，强迫游泳实验结果显示，在抑郁大鼠海马CA1区敲低Phactr4蛋白表达改善了动物的行为绝望状态（图4D和图4E）；糖水偏好实验结果显示，在抑郁大鼠海马CA1区敲低Phactr4蛋白表达改善了动物的快感缺失现象（图4F）。在氧饱和的切片液中制备新鲜的大鼠脑切片，并转移至37℃的恒温的孵育液中进行活性的恢复，最后在人工脑脊液的灌流中，进行神经元的钳制与破膜，进行全细胞记录各种电生理指标如神经元兴奋性，观察干预Phactr4后突触功能可塑性变化。电生理记录结果显示，敲低Phactr4的抑郁模型大鼠海马区神经元自发性兴奋性突触后电流sEPSC的频率和幅度增高（图4G）。提取各组大鼠海马CA1区的蛋白，通过Western blot实验进行Phactr4及下游信号通路分子表达水平检测，蛋白免疫印迹结果显示，在抑郁大鼠海马CA1区敲低Phactr4蛋白表达逆转了抑郁大鼠Phactr4及下游分子的表达水平（图4H）。
基于上述研究方法，本发明获得以下研究结果：
（1）Phactr4/PP1-RhoA-Limk-cofilin（丝切蛋白）-actin肌动蛋白信号通路首次被证明参与调控大鼠海马突触可塑性及抑郁样行为的发生；
（2）使用腺相关病毒下调Phactr4的蛋白表达水平可有效改善模拟人在社会中遭受各种打击的CUMS动物实验模型的抑郁样行为；
（3）针对Phactr4及下游作用分子在细胞及动物实验中所设计的腺相关病毒或者慢病毒均未出现致死性等不良效果；
（4）对Phactr4下游分子的调控同样可以改善动物的抑郁样表型，对临床开发药物具有更大的选择空间和实际价值；
（5）首次靶向Phactr4蛋白，对其进行基因和蛋白水平调控，证实下调Phactr4与PP1的互相作用能够调控RhoA-Limk-cofilin-actin下游信号通路继而在抑郁症治疗中发挥有益效果。
综合上述研究结果，靶向基因水平对Phactr4的蛋白表达进行下调处理可有效缓解CUMS动物模型的抑郁样行为，发挥抗抑郁治疗的保护作用，而靶向基因水平对Phactr4的蛋白水平过表达处理则可诱导出动物对应激刺激的易感性。因此，Phactr4是一种具有光明前景的抑郁症治疗药物靶点。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (2)
1. Phactr4抑制剂在制备抑郁症治疗药物中的应用，其特征在于，所述Phactr4抑制剂为靶向抑制Phactr4的腺相关病毒，所述抑郁症为反应性抑郁；
所述腺相关病毒中具有Phactr4 shRNA。
2.如权利要求1所述Phactr4抑制剂在制备抑郁症治疗药物中的应用，其特征在于，所述抑制剂用于降低受试者脑内海马CA1区Phactr4表达含量。
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| CN114540460A (zh) * | 2022-02-21 | 2022-05-27 | 浙江大学 | Hdca9抑制剂在制备治疗抑郁症药物中的应用 |
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| CN107012203A (zh) * | 2017-02-22 | 2017-08-04 | 山东大学 | Pdcd4作为抗抑郁症和/抗焦虑症药物治疗靶点的应用 |
| CN114540460A (zh) * | 2022-02-21 | 2022-05-27 | 浙江大学 | Hdca9抑制剂在制备治疗抑郁症药物中的应用 |
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