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·综述· 环氧二十碳三烯酸在心肌缺血/心肌缺血再灌注损伤中的保护性作用机制研究进展*宋其泰1 综述 钟武2 审校 (西南医科大学 1.临床医学院,2.附属第一医院急诊医学部, 四川 泸州 646000) 【摘要】环氧二十碳三烯酸(epoxyeicosatrienoic acid,EETs)是花生四烯酸(arachidonic acid，AA)在细胞色素P450表氧化酶(Cytochrome P450 epoxygenases)作用下生成的一类化合物。在包括人在内的诸多哺乳动物多个器官都发挥重要的生物学作用。在心脏和冠脉，EETs通过调节离子通道、调节基因表达、激活信号转导、与受体结合以及作用于细胞特定位置等诸多方式，发挥着舒张血管、减小冠脉循环阻力、促进心肌细胞缺血后功能恢复、抗凋亡等诸多生物学效应。本文主要就EETs在心肌缺血/缺血再灌注损伤中产生的保护性作用机制及研究进展综述如下。 【关键词】环氧二十碳三烯酸； 心肌缺血/缺血再灌注损伤； 细胞色素P450途径； K+通道； 活性氧簇； 信号通路 花生四烯酸(arachidonic acid，AA)是一种多元不饱和脂肪酸，广泛存在于人和动物的体内。通常情况下以酯化的甘油酰磷脂(glycerophospholipids)存在于细胞膜上，在受到一定刺激(比如缺血或缺血再灌注刺激)后，通过细胞膜结合磷脂酶(phospholipase)，主要是磷脂酶A2的作用而生成未酯化的花生四烯酸[1]。AA在体内的代谢途径主要有3种，即环氧化酶途径(cyclooxygenase pathway,COX)、脂氧合酶途径( lipoxygenase pathways，LOX)和细胞色素P450途径(CYP)。CYP途径则包括表氧化酶(epoxygenases)和羟化酶(-hydroxylases)的作用，前者代谢AA生成4种异构体(5,6-，8,9-，11,12-和14,15-EET)，统称环氧二十炭三稀酸(epexyeicosatrienoicaid,EETs)；后者代谢AA生成包括19-和20-HETE在内的多种二十碳四烯酸(HETEs)[2]。所有的EETs都已证实具有生物活性[3-4]，其中5,6-EET是一种较弱的酶作用底物，11,12-EET和14,15-EET则是多数细胞和血管中EETs存在的主要形式[5]。活化的EETs在被环氧化物水解酶(epoxide hydrolase)作用后转化为对应的、并相对稳定的碳三烯酸(DHETs)[1]。在哺乳动物组织中发现的两个主要环氧化物水解酶分别是微粒体环氧化物水解酶(microsomal epoxide hydrolase，mEH)和可溶性环氧化物水解酶(soluble epoxide hydrolase，sEHor EPHX2)[3]，后者更是在体内参与EETs水解的主要酶[4,6]。很多研究都表明，AA经过不同的代谢途径生成的代谢产物在不同的物种、不同的器官都具有各种不同的生物活性并带来相应的效应，比如20-HETE的强烈缩血管效应[7]；EETs通过激活血管平滑肌细胞Ca+-K+通道(BKCa)而使静息电位发生超极化而舒张冠状循环[8-10]；在血管系统内发挥抗炎、溶栓和促进血管新生的作用[11-13]；调节葡萄糖代谢、胰岛细胞增殖与凋亡、胰岛素的合成与分泌[14]；促进细胞有丝分裂和细胞增殖[15]等。在这些化合物众多的生物作用中，在心肌缺血/心肌缺血再灌注损伤中的作用是近十年来的研究重点和热点，本文主要就EETs在心肌缺血或心肌缺血再灌注损伤中的保护性作用及作用途径、机制进行综述。 1 EETs和CYP表氧化酶途径与心肌缺血/缺血再灌注损伤EETs和CYP表氧化酶途径在心肌缺血/缺血再灌注损伤中发挥着重要的保护性作用。EETs通过激活血管平滑肌细胞Ca2+-K+通道(BKCa)使静息电位发生超极化而舒张冠状循环[8-10]；在血管系统内发挥抗炎、溶栓和促进血管新生的作用[11-13]；在血管内皮细胞中，EETs通过激活丝裂原蛋白激酶(mitogen activated protein kinase，MAPK)和磷脂酰肌醇-3激酶(phosphatidylinositol-3 kinase，PI3K)-Akt信号通路[13]以提高细胞内环磷酸腺苷(cyclic Adenosine monophosphate,cAMP)水平[12]，然后上调一氧化氮合成酶的表达[16]而保护细胞缺氧再复氧带来的损伤[17]。EETs通过激活心肌ATP-K+(KATP)通道[18-21]、增加L型钙通道电流[22-23]、改善缺血后期左室功能的恢复而起到保护作用[21, 24-26]。最近研究发现了脂质介质-3PUFA环氧化合物在某些生理病理过程中也具有保护作用，包括在心肌缺血再灌注损伤中具有较强的抗心律失常作用、在炎症反应中的镇痛作用和减轻神经性疼痛作用等；并且在抗心律失常、抗炎症反应和减轻疼痛等方面的作用甚至比EETs更强[27-29]。-3PUFA是由CYP表氧化酶氧化18-C-6PUFA产生的，同时CYP表氧化酶还能氧化亚油酸(LA)生成EpOME，氧化二碳六烯酸(DHA)生成EpETE和EpDPE[30]。 上述所有这些保护性作用都是CYP表氧化酶途径代谢AA或者其他脂质所产生的。由此可见，CYP表氧化酶途径在心内起着重要的保护作用。CYP表氧化酶最早是从人的心脏和肝脏中提取到的[31]。随后，很多其他的CYP家族在不同种属的各个器官分别被证实[26]。其中在心脏高表达的CYP2J2和它介导的途径在心肌缺血/缺血再灌注损伤中发挥着重要的保护性作用。有研究者也通过实验证实了这一观点。Seubert J等[21]人采用过表达人CYP2J2转基因小鼠和野生型基因小鼠离体心脏模拟心肌缺血再灌注损伤，通过在缺血前20min给予选择性P450表氧化酶抑制剂MS-PPOH灌注，观察结果发现，野生型基因小鼠模型中缺血后左室形成压(LVDP)恢复减弱,而过表达CYP2J2转基因小鼠模型的缺血后LVDP恢复效应也被完全消除。他们认为，过表达CYP2J2转基因小鼠产生的心肌保护效应涉及到p450表氧化酶代谢物，并且p450表氧化酶在缺血后心肌的功能恢复过程中发挥着重要作用[21]，其中涉及到的机制则可能是提高了KATP通道的活性。同时过表达人CYP2J2转基因小鼠在缺血后p42/p44丝裂原活化蛋白激酶(MAPK)表达也增高，当复灌期间给予p42/p44 MAPK抑制剂PD98059灌注后，转基因小鼠的心肌保护效应则被消除。所以，心肌缺血后CYP2J2衍生物所产生的心肌保护效应的机制可能涉及到KATP通道的活化和 p42/p44 MAPK。但是CYP酶是一个复杂的酶系统，并且很多CYP酶抑制剂都缺乏针对某个亚型的特异性，所以这些抑制剂也有可能作用于其他的信号通路[32]。在大鼠和兔心肌缺血模型中的保护效应则可能涉及到CYP依赖性线粒体释放活性氧簇(CYP-dependent reactive oxygen species，ROS )产物的产生减少[33]，这一假说在后来Fichtlscherer等[34]的试验中得到了证实，他们推断可以通过阻断 内皮细胞CYP2C9介导的超氧化物生成，而减少ROS的生成来提高NO的生物活性[35]。 2 EETs在心肌缺血/缺血再灌注损伤中的作用机制2.1 EETs与K+通道 心肌细胞是永久性细胞，所以保护缺血心肌组织中细胞的存活是最主要目标。心肌保护机制要么是直接通过信号途径，要么是通过对关键蛋白的翻译后修饰，要么是通过新的转录和翻译来调节细胞代谢[36-37]。几乎所有信号都会通过作用于共同的终效应器来维持细胞膜的完整性和阻止细胞死亡[38]。线粒体则因为生成ATP以及在细胞凋亡中发挥关乎细胞存亡的关键作用而成为这些终效应器之一[36-38]。线粒体上的关键蛋白，如K+ 通道[39-43]和线粒体通透性转换孔(mitochondrial permeability transition pore，mPTP)[38, 44-46]将那些上游信号整合起来，进而引起心肌保护效应。在已知的很多涉及怎样增强缺血后心脏功能恢复的机制中，KATP通道的作用似乎成为了焦点。在心肌细胞存在两种药理学效应完全不同的KATP通道，即sarcKATP 和 mitoKATP，从结构上讲，sarcKATP是两种亚基(Kir6.2 and SUR2A)构成的八聚体，而mitoKATP通道的结构则有研究者认为是包含有琥珀酸脱氢酶的蛋白复合体[47]。之前的多篇文章都认为，EETs是较强的ATP敏感型膜钾离子通道(sarcKATP)活化剂[19-20]。Lu等[19]采用膜片钳技术来观察EETs对大鼠心肌细胞 除EETs通过对 2.2 EETs与ROS 活性氧簇(reactive oxygen species，ROS)被认为是造成缺血再灌注损伤，尤其是再灌注后损伤的重要因素之一。随着EETs在各种动物心肌缺血再灌注损伤模型中的研究，研究者也开始通过实验探讨EETs对ROS的作用。Garrett J Gross等[35]使用自由基清除剂2-巯丙酰甘氨酸(2-mercaptopropionyl glycine，2-MPG)来研究ROS是否在EETs引发的心肌保护效应中发挥作用。对照组按20mg/kg的浓度在结扎冠状动脉或者复灌前20min静脉给药；实验组在结扎前15min分别给于11,12-EET和14,15-EET，并在结扎前5min静脉注射相同浓度的2-MPG。观察结果得出，2-MPG自身并不引起梗死范围(Myocardial infarct size,IS)与缺血总面积(area at risk，AAR)之比(IS/AAR)，但是11,12-EET组[(58.2±1.6)%]和14,15-EET组[(61.4±1.0)%]所产生的心肌保护效应则完全被消除。所以推论，EETs在大鼠模型中的部分心肌保护效应需要通过ROS的“爆发”而触发。Cohen等[55]的研究也使用2-MPG来评估ROS在EETs介导的心肌保护效应中的“扳机”作用，并得出和Garrett J Gross等人一致的结论。不过有实验证实，在大鼠模型中，EETs在马上要开始复灌的时候就能起到保护性作用[56]，在这样的情况下，ROS的“爆发”是否还是EETs介导的心肌保护效应所必须的呢？ 2.3 EETs与信号通路 之前的多个研究已经发现，EETs转导信号能激活包括PI3K、Akt、PKC和PKA在内的多种蛋白激酶，并且EETs所介导的效应会被这些激酶抑制剂显著削弱[21, 57-59]。Anuradha Dhanasekaran 等[59]通过实验发现，EETs可以通过作用于PI3K/Akt通路的多个靶蛋白而发挥作用，包括激活PI3K、诱导Akt磷酸化、启动BAD磷酸化、升高细胞内X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)水平和削弱细胞凋亡蛋白酶9(caspase 9)的活性等。其他的关于EETs和信号通路的研究还包括：通过ADP糖基化激活鸟嘌呤核苷酸结合蛋白Gs[60]； Spector 等[61]证实血管平滑肌中，活化的蛋白激酶A(PKA)可能是EET信号途径的另一个分支点，PKA通过一边转位到细胞核并改变基因转录，一边通过磷酸化丝氨酸残基活化Bkca通道发挥作用。在 分离的猪冠状动脉内皮细胞中，EETs通过酪氨酸激酶作用，还可以引起表皮生长因子受体(EGFR)、3-磷脂酰肌醇激酶(PI3K)、cSrc和细胞外信号调节激酶(ERK1/2)等磷酸化[62]；Seubert等[21]证实在过表达CYP2J2转基因小鼠模型中，ERK的表达被上调了，并且给予MEK1,2抑制剂干预后，复灌过程中的心肌保护效应被阻断，说明EETs激活ERK1和2是一条相当重要的途径。随着近期发现表皮生长因子受体(epidermal growth factor receptor，EGFR)参与受体介导阿片类效应，所以EETs磷酸化EGFR也可能是另一条潜在的重要途径[63]。Spector等[61]认为，EETs可以通过上游调节金属蛋白酶而调节EGFR。 不过，近几年研究者把更多的注意力放在了EETs对蛋白磷酸酶2A(Protein phosphatase 2A，PP2A)的活化作用上。Ketul R Chaudhary等[64]通过实验研究PP2A活性与EETs介导的心肌保护效应之间的关系。通过对比新生和成年CYP2J2转基因小鼠、新生和成年sEH敲除小鼠和WT基因小鼠心脏模型中PP2A的活性得出，新生CYP2J2转基因小鼠和sEH敲除小鼠模型中PP2A的活性相对成年WT基因小鼠模型显著升高；成年CYP2J2转基因小鼠模型中PP2A的活性并没有比成年WT基因小鼠模型增高。但当用 sEH 抑制剂t-AUCB处理成年CYP2J2转基因小鼠和成年sEH敲除小鼠后，模型中的PP2A活性与对照组相比显著升高。当使用sEH 抑制剂t-AUCB和CYP表氧化酶抑制剂(MS-PPOH)共同处理成年CYP2J2转基因小鼠后，PP2A活性又降低。为了进一步地了解PP2A的活性变化，他们通过分析PP2A的其中一个靶蛋白-腺苷酸活化蛋白激酶(Adenosine Monophosphate Activated Protein Kinase,MAPK)在这些模型中的表达发现，新生CYP2J2转基因小鼠中pThr172-AMPK的表达显著降低，而成年CYP2J2转基因小鼠模型中pThr172-AMPK则明显升高。在成年CYP2J2转基因大鼠模型中PP2A失活则EET介导的心肌保护效应消失，所以他们认为成年CYP2J2转基因小鼠模型中EETs介导的心肌保护效应消除可能与PP2A的活性降低有关。在血管平滑肌细胞EETs和PP2A之间的关系则研究的相对少一些，目前还没有获得实质性的成果[65-66]。 2.4 EETs与其他途径 关于EETs与缓激肽(bradykinin)作用关系的描述最早可以追溯到1999年，有几篇文章认为他们之间的关系是通过缓激肽和乙酰胆碱(acetylcholine，ACh)等舒血管因子或者剪切力刺激EETs进而使内皮细胞表达CYP2J2和CYP2C9[67-68]。直到近几年，似乎才再被提起，Gross等[69-70]在犬类和兔模型中研究了CYP450表氧化酶在RPCT(remote preconditioning of trauma)模型中的心肌保护作用时发现，经过β2受体阻断剂(HOE 140)、EET受体阻断剂(14,15-EEZE)或EETs抑制剂(MSPPOH)处理后，RPCT模型梗死面积减小这个效应明显减弱。外源性地给于缓激肽(60ug/kg iv)也能起到类似的效果，并且缓激肽诱导的心肌保护效应也能被EETs拮抗剂减弱，这说明了RPCT可能触发了缓激肽的释放，而间接地通过EETs产生心肌保护效应[71]。EETs还被证实在内皮细胞以自分泌的方式减轻炎症反应。Gross ER 等[56]认为，EETs可以减少血管内壁白细胞的粘附以及抑制NF- B的活性。EETs还能通过激活血管平滑肌细胞Ca+-K+通道(BKCa)而使静息电位发生超极化而舒张冠状循环[8-10]。 最近，Ket ul R Chaudhary等[73]通过实验发现了又一个潜在的EETs介导的心肌保护效应机制。他们采用可溶性环氧化物(sEH)水解酶敲除(knockout,KO)小鼠和同窝的野生型(wild type,WT)小鼠离体心脏给于20min的缺血40min复灌建立模型，通过免疫组化、免疫印迹和电子显微镜观察小窝蛋白[75]结合位点和超微结构的变化得出：在WT小鼠心肌细胞，Cav-1和Cav-3都有表达，在心肌缺血后，Cav-1不表达，而Cav-3却表达；并且当细胞线粒体嵴被破坏后，小窝蛋白也不再表达。当给于11,12-EET干预后，不管是KO小鼠还是WT小鼠心肌细胞中Cav-1的表达都增高，质膜“小窝结构”得以保存并且缺血后期细胞功能的恢复也得到改善。所以他们认为，WT小鼠心肌缺血再灌注损伤后“小窝结构”和Cav-1“消失”而Cav-3依旧表达，提示心肌细胞缺血后不仅仅靠Cav-3来维持质膜“小窝结构”；缺血后线粒体和肌丝超微结构的改变则表明缺血再灌注损伤后，质膜“小窝结构”和Cav-1的“消失”可能会影响心肌细胞的形态发生改变；选择性清除sEH，从而阻止质膜和线粒体上Cav-1的“消失”，可能是EETs介导的心肌保护效应的一个新机制。 3 小结与展望随着人们对EETs、CYP代谢途径和代谢产物在心血管系统内生物作用机制的研究，越来越多的生物学效应包括正性作用、负性作用被发现，比如EETs及其经水溶性环氧化物酶代谢后的产物二羟基二十碳三烯酸(DHETs)具有强大的促进血管新生、抗炎、抗心律失常等作用，EETs在心肌缺血后通过维持梗死区域内心肌细胞的存活、改善缺血后左室收缩功能、促进细胞功能的恢复、减少ROS的产生等，EETs及其代谢途径、代谢产物对心血管系统的作用显得愈发重要。但还需要更多的动物实验和药理学实验去研究EETs某些更精确的机制，比如潜在的EETs受体；EETs介导的潜在信号转导途径；EETs作用于K+通道的精确位点和调节机制；比如如何在心肌缺血早期维持心肌组织内较高浓度的EETs水平等。本文只针对EETs和它的代谢途径、代谢产物在心肌缺血/缺血再灌注动物模型中的正性作用和机制进行了探讨，而EETs经过其他的代谢途径、所生成的其他代谢产物在该疾病模型中的负性作用同样值得我们去认识和研究，针对某些典型负性作用的关键因子的抑制剂或许可以成为治疗的重要方法。 【参考文献】 [1]Morisseau C,Hammock BD. 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Annual review of pharmacology and toxicology,2008, 48:359-391. Progress of mechanisms of cardioprotective effect of epoxyeicosatrienoic acids on myocardium after ischemia/reperfusion injury SONG Qitai1reviewing Zhong Wu2checking (1. Clinical College, The First Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan, China; 2. Department of Emergency, The 1st Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan, China) 【Abstract】Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. They play an important role in mammal organs including human especially in hearts by modulating ion transportation, gene expression, activating signal transduction pathways and binding to putative EETs receptors which result in the resistance of coronary circulation reduced, antiapoptotic and the recovery of functional in postischemia improved, etc. The EETs mediated myocardial protection during myocardial ischemia/ reperfusion injury and the mechanisms are reviewed in this paper. 【Key words】Epoxyeicosatrienoic acid； Myocardial ischemia/ reperfusion injury; Cytochrome P450 epoxygenases； Myocardial KATP channels; Reactive oxygen species; Signal pathways 基金项目：四川省科学技术厅课题(14JC0143) 通讯作者：钟武, E-mail:zhongwu2876@sina.com 【中图分类号】R 54; R 34 【文献标志码】A doi：10.3969/j.issn.1672-3511.2017.03.033 (收稿日期：2016-05-20； 修回日期：2016-09-22； 编辑： 母存培) |
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通道的作用，发现在ATP为100μM的条件下，加入0.1μ
通道开放的几率增加240%；而加入5 μM 11,12-EET则开放的几率增加400%。8,9-EET具有和11,12-EET相同的效应，而AA和11,12-DHET则完全不会对sarcK
通道的开放。Lu等
通道，但是其同分异构体11(R),12(S)-EET则不能激活。只有11(S),12(R)-EET能超极化膜静息电位而缩短动作电位。所有11(S),12(R)-EET衍生物都因为它们能降低
和mitoK
