2. 遵义医学院基础药理省部共建教育部重点实验室, 贵州 遵义 563000
2. Dept of Pharmacology and Key Laboratory of Basic Pharmacology of Ministry of Education, Zunyi Medical University, Zunyi Guizhou 563000, China
大脑在缺血、缺氧条件下会发生一系列的瀑布级联反应，从而导致神经系统疾病的发生发展，神经保护则是对级联反应的各个环节加以影响和调控，保护及修复受损的神经组织，并使之恢复或部分恢复原有功能[1]。目前，神经保护药物主要包括氧自由基清除剂依达拉奉、钙通道拮抗剂尼莫地平、谷氨酸受体拮抗剂等。近年来[2-3]，研究者在动物及人类的脑中均发现有磷酸二酯酶5(phosphodiesterase 5，PDE5) 的存在，并且发现传统的PDE5抑制剂，如西地那非和他达那非以及新报道的PDE5抑制剂优克那非和淫羊藿苷 (icariin，ICA) 都可透过血脑屏障，在中风、阿尔茨海默病 (Alzheimer’s disease，AD) 等神经系统疾病中发挥保护作用，其机制与抑制PDE5后产生的抗中风、抗氧化、抗神经炎症及改善认知障碍等作用有关[4-5]。本文结合国内外研究结果及所在团队的研究工作，系统综述了PDE5抑制剂的神经保护作用，旨在为更好地开发利用PDE5抑制剂防治中枢神经系统疾病提供依据。
1 PDE5抑制剂对脑中风后神经血管修复的作用中风的治疗主要通过促进血管重构以恢复大脑的供血和阻断神经细胞死亡的级联反应来实现[6]。Zhang等[7]将内皮细胞与PDE5抑制剂西地那非共培养后发现，西地那非可诱导内皮细胞形成类毛细血管，表明西地那非有促进血管发生的潜能。随后，此课题组采用大脑中动脉梗死诱导大鼠中风模型后发现，西地那非可明显促进中风大鼠神经功能的恢复，并增加大鼠脑皮质区域血管密度，促进内皮细胞的增殖以及突触素的免疫活性[8]。甘靖等[9]发现西地那非可改善大鼠脑缺血/再灌注后急性期神经功能，其机制可能与海马前体神经细胞标记物微管相关蛋白阳性细胞及血管内皮生长因子的表达增加有关。同样，他达那非也可促进大鼠脑栓塞模型的神经功能恢复[10]。Li等[11]通过衡量中风大鼠的核磁共振图像和功能表现来评价西地那非对中风的治疗效果，并应用血管内皮屏障抗原染色来进行脑血管的形态学和量化分析，发现西地那非可选择性地增加缺血周边区脑血流量以及血管活性。但无论是西地那非还是他达那非，均不能有效减少中风病灶的大小[9-10]。在大脑发育过程中，脑室内的神经干细胞 (neuron stem cell, NSC) 分化成神经细胞，而后迁移至皮层，聚集形成皮质神经元[12]。Zhang等[13]采用溴脱氧尿苷标记神经元的方法，发现用西地那非处理 (中风后2 h或24 h) 中风大鼠后，可大幅增加其室下区及齿状回的新生神经元，且某些神经元可表现出成熟神经元的特征。免疫荧光染色法表明，西地那非能够增加中年中风小鼠NSC标记物巢蛋白、成熟神经元及少突胶质细胞的表达[14]。体外实验数据表明，西地那非可直接作用于NSC，通过促进干细胞增殖与分化来增强神经发生作用[15]。研究表明，PDE5抑制剂西地那非与他达那非均能在中风后提高脑环磷鸟嘌呤核苷 (cyclic guanosinc monophosphate, cGMP) 的浓度，从而作用于血管内皮细胞、NSC和少突细胞，进而促进中风诱导的血管发生、神经发生及突触再生过程[4]。
2 PDE5抑制剂的抗神经炎症作用体内、体外实验均证实PDE5抑制剂具有抗神经炎症的作用[16-17]。众所周知，小胶质细胞被认为是中枢神经系统炎症发生的主要介导者之一[18]。Moretti等[19]采用大脑中动脉阻塞诱导新生小鼠中风模型后发现，西地那非可明显缓解由小胶质细胞介导的神经炎症反应。亦有研究证实，西地那非能够治疗由脂多糖诱导星形胶质细胞产生的炎性反应，其机制与降低钙离子反应强度有关。Hernandez-Rabaza等[20]通过制备门腔静脉分流大鼠模型模拟神经炎症，结果发现西地那非能够降低大鼠海马中白细胞介素1β、肿瘤坏死因子等炎症细胞因子来发挥抗炎作用。Raposo等[21]进一步证实了西地那非可缓解由双环己酮草酰二腙诱导的诱导型一氧化氮合酶 (induced NO synthase，iNOS) 基因敲除小鼠的神经炎症反应，其机制可能与增加脑中cGMP浓度有关。有趣的是，iNOS基因敲除小鼠较野生型小鼠更易被诱导发生炎症反应，这可能与iNOS/NO/cGMP信号通路参与了神经炎症的发生关系密切[19, 21]。
3 PDE5抑制剂的抗氧化作用氧化应激条件下可产生过多活性氧 (reactive oxygen species, ROS) 产物，从而导致线粒体的功能和结构遭到破坏，最终导致神经细胞死亡[22]。活化的小胶质细胞通过分泌ROS，促使神经退行性病变的发生及发展[23]。然而，Park等[24]认为，活化的小胶质细胞并非ROS产生的主要来源，而大多由NADPH氧化酶产生。有报道称，PDE5抑制剂西地那非通过抑制NADPH氧化酶的表达，从而明显减少了肺动脉上皮中的负氧离子[25-26]。同时，西地那非可下调gp91phox和细胞内ROS水平，gp91phox是NADPH氧化酶的一个具有催化作用的关键亚基[17]。最近，Sung等[27]用晚期糖基化终末产物诱导HT-22海马神经细胞发生氧化应激反应，使其丧失线粒体功能，给予西地那非后可保护其线粒体功能，此外，还能减少因氧化应激引起的细胞凋亡。进一步研究发现，无活性的cGMP类似物Rp-8-Br-cGMP及cGMP抑制剂都可削弱西地那非的保护作用。此外，NO/cGMP轴本身也可通过增高cGMP浓度来抑制NADPH氧化酶的表达[28]。综上，可以推测PDE5抑制剂抗氧化的机制可能与增高cGMP水平，从而抑制NADPH氧化酶的表达有关。
4 PDE5抑制剂可改善认知障碍亨廷顿病 (Huntington′s disease, HD) 早期可出现认知障碍。Saavedra等[29]采用酶免疫分析法检测HD小鼠模型海马中的cGMP，发现其浓度异常降低，但PDE5水平则无明显变化。进一步研究证实了PDE5抑制剂西地那非可通过增加小鼠海马cGMP，从而改善其学习记忆功能，并认为cGMP水平的调节可作为治疗HD认知障碍的靶点[29]。同时，临床研究表明脑脊液cGMP水平与AD的发展也有关，早期AD患者中cGMP的减少可加剧认知障碍及淀粉样蛋白的沉积[30]，但缺乏临床治疗AD的相关研究。动物实验通过Morris水迷宫及新物体识别测试来评价AD动物模型 (Tg2576、TgAPP/PS1、J20小鼠) 接受西地那非治疗后的认知功能改变情况，发现西地那非可明显降低Tg2576与J20小鼠的逃避潜伏期[31-32]，促进TgAPP/PS1小鼠对新颖物体的探索[16]。进一步研究表明，PDE5/cGMP信号通路可能参与改善AD小鼠认知障碍的过程，此外，西地那非还可明显增加Tg2576与TgAPP/PS1小鼠海马的转录因子环磷酸腺苷反应部分结合蛋白 (cAMP-response element binding protein, CREB) 及其下游脑源性神经营养因子 (brain derived neurotrophic factor, BDNF) 及活性调节细胞骨架联合的蛋白 (recombinant activity regulated cytoskeleton associated protein, Arc) 表达[16, 32]。因此，推测PDE5抑制剂可能通过增加cGMP水平从而作用于pCREB/BDNF/Arc通路，进而改善认知障碍。
5 新报道的PDE5抑制剂西地那非类似物优克那非作为一种新报道的PDE5抑制剂，其半抑制浓度 (half maximal inhibitory concentration, IC50) 为2 nmol·L-1，低于西地那非 (IC50=4.5 nmol·L-1)，且不良反应较少[33]。优克那非也具有抗中风、抗神经炎症、促进神经发生及改善认知障碍等神经保护作用[34-36]。
淫羊藿苷 (ICA) 是从中药淫羊藿中提取的黄酮类物质，在它被证实可抑制PDE5之前，就有研究发现其具有保护大脑缺血性损伤的功效，表明ICA具有潜在神经保护作用[37]。廖晖等[38]证明ICA能通过抑制阴茎海绵体的PDE5来激活NO/cGMP信号通路，从而治疗勃起障碍。本课题组近年来致力于对淫羊藿的神经保护作用的研究，发现ICA不仅能够降低AD小鼠海马中PDE5的水平，增加一氧化氮合酶的表达，从而增加脑中NO和cGMP的浓度[39-40]，还可以通过改善兴奋性氨基酸中毒大鼠模型认知障碍，减少神经细胞凋亡来发挥神经保护作用[41]。此外，ICA能够通过对抗神经炎症反应来预防脑缺血/再灌注损伤[42]。体外实验中，我们采用氧糖剥夺/复氧细胞模型证明ICA可通过抗凋亡及调节自噬起到神经保护作用[43]。然而，ICA会在体内代谢为淫羊藿次苷Ⅱ(icariside Ⅱ, ICSⅡ)[44]，则有理由假设ICS Ⅱ也有类似神经保护效应。我们的研究证实了ICSⅡ可通过降低中风大鼠及AD大鼠海马的PDE5蛋白产生，从而改善其神经功能及认知障碍，减少促炎性因子及神经元的死亡，进而发挥神经保护作用[45-46]。
6 展望大量临床前实验证实PDE5抑制剂具有神经保护作用，效果显著，且不良反应较少。本文结合国内外研究结果及所在团队研究报道，系统地综述了PDE5抑制剂抗中风、抗氧化、抗神经炎症及改善认知障碍等神经保护作用及相关机制。这为更好地开发利用PDE5抑制剂在临床防治中枢神经系统疾病的深入研究提供了线索，对开发新型PDE5抑制剂具有现实的指导意义。
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