2015, Vol. 50
2. 安徽中医药大学 药物代谢动力学研究室, 安徽 合肥 230031
, WANG Chen-yin1, YANG Li-li1, CHEN Jin-pei1
2. Laboratory of Drug Metabolism and Pharmacokinetics, Anhui University of Chinese Medicine, Hefei 230031, China
慢性阻塞性肺疾病 (chronic obstructive pulmonary disease,COPD) 是一种可预防和治疗的疾病,与肺脏对烟草烟雾等有害气体或颗粒的异常炎症反应 有关。COPD影响全球超过6 400万人[1],据估计,到2020年COPD将成为继缺血性心脏疾病和脑血管疾病之后,死亡率在全球排名第3的疾病[2]。其发病机制主要涉及炎症反应、黏液分泌、氧化应激和上皮细胞凋亡等方面[3, 4, 5]。肺部上皮细胞基因的表达和功能改变会引起永久性气道损伤和肺基质损坏。研究表 明,COPD患者肺部组织、气道的炎症以及细胞凋亡均与丝裂原活化蛋白激酶 (mitogen-activated protein kinases,MAPKs) 信号转导通路有关[6]。另一方面,COPD患者与正常人体及非COPD患者的吸烟人群相比,其肺部表现出氧化/抗氧化系统失衡[7],使机体处于氧化应激状态。而氧化应激可激活Kelch样环氧氯丙烷相关蛋白-1 (Kelch-like epichlorohydrin-associated protein 1,Keap1)-核转录因子E2相关因子2 (nuclear factor erythroid 2-related factor 2,Nrf2)-抗氧化反应元件 (antioxidant response element,ARE) 通路,从而转录下游靶蛋白。通过动物模型和临床试验研究,已经明确Nrf2及其靶基因在引起COPD的主要致病因素,如香烟烟雾引起的炎症和氧化应激中扮演保护作用[8]。随着对COPD病理生理分子机制的深入研究,越来越多文献[9]报道,COPD不可能只受单一信号通路影响。MAPKs及Keap1-Nrf2-ARE信号通路与COPD的典型病理生理特征慢性炎症反应及氧化/抗氧化失衡密切相关[6, 10],因此,本文以MAPKs及Keap1-Nrf2- ARE信号通路的窜扰 (cross talk) 为切入点,重点探讨其对COPD特征病理生理过程的影响,并综述与之相关药物的最新进展,以期阐明MAPKs和Keap1- Nrf2-ARE信号通路在调控COPD在病理生理中的重要作用和意义,同时为防治COPD的药物研究和开发提供理论基础。
1 COPD的主要发病机制COPD是一种以呼吸气流受限为特点的常见疾病[11, 12],核心病理为呼吸道的慢性炎症,主要表现为杯状细胞数量增加,黏液分泌腺增生,小气道的纤维化、狭小。同时由于肺气肿引起肺泡壁破坏,呼吸道间的牵连减少导致气道衰竭[13]。黏液分泌过量使病情加重,同时炎症会扩散至病变组织周围黏膜下层腺体,从而使气道炎症进一步加重[14]。在COPD的病理发展中占主导地位还有持续的氧化应激。据报道,COPD患者体内氧化应激的增强与长期暴露于香烟烟雾、受污染环境和在慢性炎症过程中产生的內源性氧化物等有关[10]。活性氧自由基 (reactive oxygen species,ROS) 超过抗氧化防御体系的清除能力时,氧化应激随即产生从而引起体内脂质、蛋白质 (扰乱蛋白酶-抗蛋白酶平衡) 和细胞DNA的严重损坏[15, 16]。除炎症、氧化应激和体内蛋白酶/抗蛋白酶失衡外,肺部细胞凋亡增多也是COPD重要的发病机制之一[17]。研究表明COPD患者和普通吸烟者相比,前者肺部细胞凋亡情况更严重[17, 18]。
2 MAPKs及Keap1-Nrf2-ARE通路的组成及两者的窜扰作用 2.1 MAPKs信号通路及其作用MAPKs信号通路在调控真核细胞基因的表达中起着重要作用: 连接胞外信号传递入细胞内,通过磷酸化下游蛋白激酶、细胞内底物和转录因子[19],调控基本的细胞生理过程如生长、增殖、分化、迁移和凋亡过程[20, 21, 22]。MAPKs通路可被多种因素激活,包括渗透压的改变、氧化应激、细胞因子、生长因子、抗原、毒素和药物[23, 24]。研究表明,COPD患者肺部组织、气道的炎症以及细胞凋亡均与MAPKs信号转导通路有关,虽然在不同类型的细胞中,COPD状态下MAPKs受到激活或抑制的作用没有充分明确,但可以肯定的是MAPKs信号通路在COPD的病理过程中发挥重要的作用[6]。
MAPKs通路主要包括3种信号途径: 细胞外调节蛋白激酶 (extracellular signal-regulated kinase,ERK) 途径; p38 MAP激酶 (p38 MAP kinase) 途径和c-Jun氨基末端激酶 (c-Jun N-terminal kinase,JNK) 途径。不同的MAPKs途径其MAPKK激酶 (MAP3K或MKKK) 和MAPK激酶 (MAP2K或MKK) 的底物、分子间的相互连接以及支架蛋白不同[25, 26]。促有丝分裂刺激物,如生长因子、细胞因子等可激活ERK通路,其参与调节细胞生长、存活和分化等重要的细胞生理活动。相反,JNK和p38 MAPK通路对生长因子刺激较不敏感,而对于肿瘤坏死因子 (tumor necrosis factor,TNF)、白介素-1、电离和紫外线辐射、高渗应激和化疗药物,可引起强烈的应答。同时,应激刺激引起JNK和p38 MAPK通路的活化与细胞凋亡密切相关[24]。
2.2 Keap1-Nrf2-ARE通路及组成研究表明,氧化应激、亲电应激、有害物质及其代谢产物的解毒和消除过程可激活Nrf2与ARE结 合[10, 27],其诱导生成的下游靶蛋白包括NADPH奎宁氧化还原酶-1 (NQO-1)、血红素加氧酶-1 (HO-1)、谷胱苷肽硫转移酶 (GSTs)、硫氧还蛋白[28, 29]和多药耐药家族(MRP1[30]、MRP2[31])。由此可知,Nrf2调控的蛋白有参与正常生理、解毒[32]、抗癌[33],恢复细胞内稳态[34]的作用。因此,Nrf2可视为具有细胞保护作用的转录因子,对于疾病的预防及治疗有重要作用。与此同时,临床研究发现,在大量烟雾暴露情况下,可激活人巨噬细胞中的Nrf2,并且吸烟者及COPD患者体内肺巨噬细胞中的Nrf2表达下降[35]。Nrf2 基因敲除小鼠不仅对氧化应激/亲电应激敏感性增 强,也增强了对蛋白酶/抗蛋白酶失衡和炎症组织损伤的敏感性,表现出复杂病理情况[36],如在弹性蛋白酶诱导[37]和CS诱发肺气肿的Nrf2缺陷小鼠都有表现[38]。以上均表明Nrf2在COPD病程中有重要的细胞保护作用。
在正常生理条件下,Nrf2在细胞质中和Keap1结合。Keap1负调控Nrf2,因为Keap1与Cullin(Cul)3/ Rbx1蛋白形成一个E3-泛素连接酶环,从而将转录因子通过泛素蛋白酶体降解[39]。在氧化物质或异型物质的刺激下,Nrf2与Keap1解离,易位到细胞核中,与Maf、JunD、c-Jun等结合形成杂化二聚体,进一步与下游Ⅱ相解毒基因和抗氧化酶基因启动子中的ARE位点结合,激活下游启动子,转录大量相关的下游蛋白[40, 41]。
2.3 MAPKs信号通路与Keap1-Nrf2-ARE信号通路的关系MAPKs家族作为细胞信号转导重要的上游调节者,通过影响多种细胞外刺激传入细胞内的活动,控制下游转录因子的活性,这其中涉及细胞凋亡、氧化应激及癌变细胞的增殖、分化等[42, 43]。体内一些激酶可共同调节MAPKs及Nrf2,继而两者共同调节下游靶蛋白。有研究[44]报道在人肝癌HepG2细胞上,MAPK激酶激酶1 (mitogen-activated protein kinase kinase kinase 1,MEKK1)、转化生长因子-β激活的蛋白激酶 (transforming growth factor-β-activated kinase,TAK1) 和凋亡信号调控激酶 (apoptosis signal-regulating kinase,ASK1) 可激活MAPK通路中MAPK激酶(MAP kinase kinase,MKK) 4、MKK6和JNK并共同作用于Nrf2,促进ARE的活化。另有研究[45]通过分析氨基酸序列发现,MAPKs与Nrf2两者间的作用可能与Nrf2的反式激活域中存在MAPK蛋白磷酸位点有关 (图 1)。大量文献[46, 47]报道MAPKs对Nrf2的活性有影响,但在不同细胞中对于ERK、JNK、p38调控Nrf2活性的结果存在矛盾,原因可能为MAPKs对Nrf2的调控是间接的,因为MAPKs直接磷酸化Nrf2,并不引起显著的Nrf2激活[43]。同时,体外实验表明MAPKs的过表达及Nrf2磷酸化缺陷体不会影响Keap1与Nrf2间的相互作用[43]。另一方面Nrf2被激活进入细胞核后,Nrf2需与c-Jun结合形成异二聚体,可以激活依赖亲电反应元件的靶基因转录。Levy等[48]在此基础上进一步发现,在人支气管上皮细胞中,只有被JNK通路磷酸化的c-Jun与Nrf2结合才可增加下游基因的表达; 然而,在肝癌细胞系HepG2中,c-Jun不通过JNK通路磷酸化即可与Nrf2相结合以转录靶基因。由此可知,不同细胞类型、外界刺激及化学异物刺激都可能对MAPKs通路与Nrf2间的作用机制产生影响。
图1 Nrf2与经典MAPKs信号通路的激活机制以及MAPKs对Nrf2影响的示意图。橘色虚线箭头表示,Nrf2的反式激活域中存在一些MAPK蛋白磷酸位点[40],从而影响Nrf2的磷酸化
3 MAPKs通路与Keap1-Nrf2-ARE通路对4种病理生理环境的作用目前,在COPD病情的发生与发展进程中,有关MAPKs与Keap1-Nrf2-ARE信号通路的报道较少。炎症、黏液分泌过量、细胞凋亡及氧化应激等是COPD主要的病理表现。下面通过探讨MAPKs与Keap1-Nrf2- ARE信号通路分别在上述四种病理状态下可能存在的相互联系,为研究其在COPD病理下的作用提供依据。
3.1 炎症反应炎症细胞浸润会引起肺损伤,并参与COPD的肺部重构[49]。调控炎症反应过程中,JNK通路可影响Nrf2-ARE通路; 丙烯醛可减弱卵清蛋白诱导引起C57BL/6小鼠的肺部炎症,进一步研究发现,其机制为激活Nrf2、抑制JNK信号通路[50]。Hristova等[51]发现在人肺巨噬细胞中,丙烯醛的免疫抑制作用机制为激活Nrf2、诱导抗炎和抗氧化基因,并与核因子κB (nuclear factor-κB,NF-κB) 和JNK2相关。抑制MAPKs通路的同时激活Nrf2通路,可以促进抗炎反应: 内源性H2S参与哮喘发病过程的抗炎和抗气道重塑作用,研究其机制发现,H2S诱导肺组织中Nrf2的核积累,抑制ERK1/2、JNK、p38 MAPK的磷酸化以降低香烟引起的炎症[52]。p38通路对Nrf2具有调节作用: 文献[53]研究表明,L-2-氧代硫氮杂戊环烷-4-羧酸 (OTC) 和α硫辛酸 (LA) 可能通过抑制PI3K/ Akt和p38蛋白激酶途径从而抑制Nrf2和NF-κB 的激活,减少参与气道重塑的各种分子表达,以改善C57BL/6小鼠支气管哮喘气道变应性炎症及气道重构。
3.2 细胞凋亡MAPKs途径参与Nrf2的激活。Hsu等[54]报道,银杏提取物 (EGb) 可保护人肺动脉内皮细胞 (HPAECs),降低香烟烟雾引起的氧化应激及细胞凋亡,机制为促进HO-1的表达,进一步研究发现,EGb可激活ERK、JNK、p38及Nrf2通路,当给予MAPKs抑制剂时,Nrf2的核转移减少且HO-1的表达下降。ERK通路参与调控Nrf2途径。Kweon等[55]发现,没食子儿茶素3-没食子酸酯 (EGCG) 可显著诱导人肺癌A549细胞的抗凋亡作用,这与细胞中作为增强细胞增殖、促进耐药性产生的HO-1过表达相关; 进一步研究发现,当给予细胞特异性ERK抑制剂时,HO-1及与其相关的Nrf2表达下降。同时,从实验动物模型中得知,当组织处于高氧情况可导致肺损伤、炎症、水肿、上皮细胞和内皮细胞死亡,而Nrf2可保护细胞抵抗高氧或其他刺激诱导的细胞凋亡。Papaiahgari等[56]表明在 小鼠肺上皮细胞系C10中,通过抑制表皮生长因子受体 (epidermal growth factor receptor,EGFR)-PI3K- Akt/ERK MAPK途径可以减弱高氧诱导的Nrf2向核易位。
3.3 黏液分泌过度黏液产生及分泌过多是COPD的重要病理表现之一。黏液产生及分泌受到MAPKs和Nrf2的调控,然而MAPKs与Nrf2在调控黏液分泌中的相互作用尚不十分明确。
3.3.1 Nrf2参与机体黏液分泌的调节Cho等[57]研究发现给予臭氧 (O3) 暴露后,Nrf2-/-与Nrf2+/+小鼠相比,前者的代偿性上皮增生、支气管黏膜细胞增生,黏液分泌更严重,表明Nrf2缺乏会加重O3刺激的黏液分泌。同时Nrf2也影响COPD患者的黏液分泌。已知羧甲司坦主要用于治疗慢性支气管炎、支气管哮喘等疾病引起的痰液黏稠,可防止COPD的急性加重。有研究[58]将感染流感的小鼠暴露于香烟烟雾中,观察羧甲司坦对其巨噬细胞Nrf2的影响。结果显示羧甲司坦通过诱导Nrf2的活化,减少暴露在香烟烟雾的患流感小鼠肺部黏液分泌量。
3.3.2 MAPK通路参与机体黏液分泌的调节最近,研究人员发现香烟烟雾可以诱导激活气道上皮细胞ErbB3受体的表达,神经调节蛋白 (NRG) 1 β是ErbB3的配体。Yu等[59]研究表明,香烟烟雾引起原代人支气管上皮细胞及人肺癌NCI-H292细胞系中NRG1β/ ErbB3的激活,诱发黏液产生过量,与MAPKs和PI3K信号通路有关。同时有研究表明,表皮生长因子受体 (EGFR)-p38 MAPK/JNK信号通路参与脂多糖诱导黏蛋白MUC5AC的生成[60]。
3.4 氧化应激氧化应激在COPD的病因中起核心作用,除了直接造成呼吸道损伤,氧化应激还触发和加剧了以上提及的炎症、凋亡及黏液分泌[61, 62, 63]。HO-1是一种氧化应激蛋白,具有抗炎、抗氧化、抗凋亡、抗增生效应,其表达量受多种刺激影响,转录及蛋白表达与MAPKs信号通路和Nrf2的调控有关。香烟颗粒固体提取物 (CSPE) 已被证明可引起呼吸障碍,表现为肺部炎症,且与COPD加重有关。有报道表明,CSPE通过不同的信号通路诱导HO-1的表达且不同刺激及作用时间也会产生不同的影响。Cheng等[64]研究表 明CSPE诱导人气管平滑肌细胞ROS生成,是通过c-Src/NADPH氧化/MAPKs通路,依次激活Nrf2,最终诱导HO-1表达。研究提示MAPKs和Nrf2在CSPE引起的HO-1表达改变中存在相互作用。ERK1/2和JNK通路被证明诱导Nrf2的核转位,增加HO-1的表达,以抵抗氧化应激。Goven等[65]报道,人单核细胞/巨噬细胞系 (THP-1) 经长期的香烟烟雾冷凝液 (CS) 孵育后,细胞质中Keap1与Nrf2积累,HO-1蛋白量降低; 但抑制ERK1/2和JNK通路后,CS对HO-1表达和Nrf2/Bach1转位的影响消失,结果显示ERK1/2和JNK通路通过调节Nrf2/Keap1-Bach1参与CS诱导的HO-1表达。7,8-二羟黄酮上调仓鼠肺成纤维细胞 (V79-4) 中Nrf2介导的HO-1表达可被ERK特异性抑制剂抑制,再次证明Nrf2与HO-1表达的调控依赖ERK通路[66]。
4 与MAPKs、Keap1-Nrf2-ARE信号通路相关药物研究通过长期临床用药发现,用于治疗COPD的传统抗炎药物治疗效果不尽理想,如吸入型皮质类固醇类不能改善COPD患者持续下降的肺功能及肺细胞的死亡率,并可引起肺炎、骨质疏松症等不良反应[67]。近年来,激酶抑制剂是抗炎药物研发的一个重要发展方向,在涉及治疗COPD的MAPKs通路中,其抑制剂的开发目前主要集中在p38 MAPK途径上。p38 MAPK可被氧化应激、病毒以及其他炎症信号激活,在调控炎症反应中有重要的作用。因此,p38抑制剂是研究抗炎药物的方向之一。目前有多个用于治疗COPD的p38 MAPK抑制剂正处于临床研究阶段,其中losmapimod药理作用表明,在服药12周后可显著减少血浆纤维蛋白原,并改善肺部过度膨胀,但未见其对痰中性粒细胞或肺功能产生影响[68]。p38 MAPK抑制剂SB681323的临床药理作用显示,相比于安慰剂其可减少痰嗜中性粒细胞和血浆纤维蛋白原,改善用力肺活量 (forced vital capacity,FVC)。另有p38 MAPK抑制剂PH797804可改善中度及重度COPD患者的 一秒用力呼气容积 (forced expiratory volume in one second,FEV1)[69],研究[70, 71, 72]进一步发现,PH797804 (3 mg/天和6 mg/天,服用6周) 治疗成人中度至重 度COPD,相比于安慰剂改善了呼吸困难指数/过渡呼吸困难指数,患者耐受性良好。也有一些p38 MAPK抑制剂存在多种不良反应,如感染和心脏毒性,在临床试验中已经失败。为了减少不良反应,目前发展转向研究吸入型p38蛋白激酶抑制剂,代表药物有ARRY371797、PF03715455、GSK681323和GSK856553,均处于研发阶段[71, 72],但在平行比较治疗COPD的抗炎药物时,一些p38抑制剂也表现出一定的优势。比较磷酸二酯酶IV (PDEIV) 抑制剂西洛司特、类固醇药物布地奈德和p38 MAPK抑制剂BIRB-796对原代COPD患者肺泡巨噬细胞的作用,BIRB-796较西洛司特与布地奈德在抑制TNFα和白细胞介素-6 (IL-6) 时有明显的优势[73]。p38 MAPK抑制剂SB681323可降低COPD患者体内的活化血清热休克蛋白27水平,并减少LPS刺激产生的TNFα释放到血清中,然而氢化泼尼松可降低LPS刺激的TNFα释放入血清,但却对血清热休克蛋白27影响较小,提示有多种炎症信号通路影响COPD,其中p38蛋白激酶抑制剂可减少COPD患者血清中的炎症介质[74]。
围绕调控氧化-抗氧化平衡,降低肺部炎症等机制,不断发现一些药物的作用机制与Nrf2相关。N-乙酰-L-半胱氨酸 (NAC) 是一种强还原剂,它可以减少黏液黏度,从而提高黏液纤毛对黏液的清除功能。此外,NAC是还原型谷胱甘肽 (GSH) 的前体,可作为ROS的清除剂[75],具有抗氧化能力。同时,它可以增加呼吸道上皮的GSH水平[76],因此其作为抗氧化剂/黏液调节剂的药物被广泛应用于治疗COPD。Tse等[77]经临床研究发现,高剂量的NAC (600 mg,每天两次,1年) 可减少中国COPD患者的急性发作,并延长高危患者的第一恶化发作时间。而Nrf2是NAC减弱氧化应激和II型肺泡上皮细胞损伤的关键因素[78]。此外,羧甲司坦 (S-CMC) 治疗COPD,可以降低患者急性发作的频率[79]。Yageta等[58]发现,S-CMC通过激活Nrf2可减少染流感病毒的小鼠暴露于CS后产生的肺部炎症和黏液高分泌。其他Nrf2的活化剂包括植物产品6-HITC (6-methylsulfinylhexyl isothiocyanate)、西兰花、芥末提取物和萝卜硫素对COPD的影响也正在研究中[80]。其中,萝卜硫素可恢复COPD患者肺泡巨噬细胞对细菌的识别和吞噬,缓解细菌引起的急性加重COPD状况[81]; 同时也可恢复COPD患者对地塞米松的敏感性,提示可用于类固醇类激素抵抗的COPD患者[34]。随着药物开发的不断深入,可以推测以Nrf2为靶向的药物研究有望成为一个新的治疗COPD的合理策略。
5 展望近年来分子生物学相关研究表明,MAPKs与Nrf2信号通路参与调控炎症、细胞凋亡、黏液分泌、氧化应激等方面,而这些病理生理在COPD的病程中发挥着重要的作用,提示COPD的病理特征与MAPKs信号通路及转录因子Nrf2密切相关,并且诸多报道提示MAPKs对Nrf2的活性有影响。目前已有部分以MAPKs、Nrf2为靶向的药物进入临床试验阶段,期望可以改善COPD患者的肺功能、提高患者顺应性,此类药物开发前景良好。虽然MAPKs通路与Keap1- Nrf2-ARE通路共同对细胞产生影响的具体作用机制复杂,至今尚不明确,可能与不同的外界刺激及细胞类型有关,仍有待进一步研究,但信号通路间存在交互作用,在药物的研发过程中均得到证实。MAPKs与Nrf2信号通路对COPD的相关性研究将为研发更明确的靶向治疗COPD的新型药物提供更多思路。
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