【作者】 韩勇；

【导师】 曾庆华；

【作者基本信息】 东北师范大学 ， 生理学， 2013， 博士

【摘要】 20-羟二十烷四烯酸（20-hydroxyeicosatetraenoic acid,20-HETE）是由细胞色素P-450（CYP450）ω-羟化酶催化花生四烯酸生成的产物。研究认为，20-HETE可通过激活L-型钙通道并抑制钙依赖钾通道从而增强血管平滑肌的收缩，有效地调节肾、脑等小动脉血管的紧张度，被认为具有很强的收缩血管作用。20-HETE在血管平滑肌方面的研究已经得到广泛开展，但对心脏的作用及机制的研究少有报道。本文从如下几个方面探究了20-HETE对心脏功能的影响以及机制：第一部分20-HETE对大鼠心肌细胞L-型钙通道作用机制研究有研究表明，心肌缺血再灌注中20-HETE含量增加，抑制其生成可减少心肌损伤，但分子细胞机制尚不清楚。该部分研究的目的是在细胞水平揭示20-HETE对心肌的作用机制。首先，应用膜片钳技术研究了20-HETE对大鼠心肌细胞L-型钙通道的作用，发现20-HETE浓度依赖式的增强L-型钙电流，这种作用可被NADPH氧化酶特异性抑制剂gp91ds-tat或超氧化物歧化酶所抑制，提示NADPH氧化酶诱导生成的超氧化物参与了20-HETE激活L-型钙通道的作用中；另外，使用20-HETE孵育心肌细胞增强了NADPH氧化酶的活性并且使活性氧簇（ROS）的生成增加接近2倍；为了探究20-HETE诱导的NADPH氧化酶活性增加的分子机制，我们检测了心肌细胞的蛋白激酶C（PKC）活性，结果发现使用20-HETE孵育细胞显著增加了PKC的活性，而使用PKC的抑制剂GF109203处理后，减弱了20-HETE诱导的增强L-型钙电流效应，同时也减弱了NADPH氧化酶的活性。通过该部分的研究，我们首次在细胞水平发现，20-HETE可以刺激NADPH氧化酶诱导的ROS增多，并通过该途径以PKC依赖的形式最终激活L-型钙通道。如上发现为揭示20-HETE加重心肌缺血再灌注损伤的机制提供了研究线索。（该部分研究成果2010年9月发表于Am J Physiol Heart Circ Physiol, IF=3.71，第二作者）。第二部分20-HETE在心肌缺血再灌注损伤中的作用及机制研究在第一部分的研究中，我们发现了在心肌细胞中，20-HETE可通过PKC依赖机制激活NADHP氧化酶从而导致其诱导ROS生成增多的分子、细胞机制，这可能是20-HETE加重心肌缺血再灌注损伤的重要原因。本部分的研究目的是在大鼠离体心肌缺血再灌注模型中，器官水平上探究并验证如上在细胞水平发现的机制。应用Langendorff装置建立大鼠离体心脏缺血再灌注模型，缺血35分钟再灌注40分钟后，检测心肌力学、生化指标以及ROS生成、NADPH氧化酶活性、表达。首先发现，20-HETE可引起再灌注后心肌细胞凋亡显著增加、心肌功能明显下降、心梗面积加大；而灌流液中加入HET0016抑制内源性20-HETE生成后，显著改善了心肌功能，有效抑制细胞凋亡；此外，HET0016明显减少再灌注后ROS的生成，而外源20-HETE则显著增加了ROS生成水平，并且加重了蛋白质过氧化损伤。如上研究结果表明，20-HETE有明显的加重心肌缺血再灌注损伤的作用。然而，当在灌流液中加入NADPH氧化酶抑制剂加拿大麻素（apocynin），阻断了该效应，表明在该进程中NADPH氧化酶与20-HETE的作用发挥密切相关。为了探究其中的机制，通过检测NADPH氧化酶的表达及活性改变发现，20-HETE显著增加了NADPH氧化酶gp91phox和p22phox亚基表达，并提高了其活性；另外，应用PKC抑制剂GF-109203显著降低了20-HETE诱导的NADPH氧化酶激活效应；最后，灌流液中加入ROS清除剂tempol或apocynin均有效阻断了外源20-HETE所导致的心肌功能下降。如上结果提示20-HETE通过PKC依赖机制激活NADPH氧化酶导致其诱导的ROS生成增多，最终加重了心肌缺血再灌注损伤。（该部分研究成果2013年3月发表于Circulation Journal, IF=3.77，第一作者）第三部分20-HETE介导血管紧张素II诱导的心肌细胞凋亡研究通过如上两部分的研究，我们初步探究了20-HETE对心肌缺血再灌注损伤的作用及机制问题。在该进程中，另一个发挥了重要作用的因素是细胞凋亡。而我们实验室先前的研究中发现，20-HETE可通过线粒体依赖途径诱导心肌细胞凋亡，另外更为重要的是在本文第二部分的研究中，发现在心肌缺血再灌注进程中，20-HETE也具有显著的诱导细胞凋亡的效应。有研究表明血管紧张素II（AngII）在心肌细胞凋亡进程中发挥重要作用，但是其与20-HETE之间的交互作用尚不清楚。本研究在培养的乳鼠心肌细胞中，分别使用Ang II，Ang II加HET0016，HET0016或者20-HETE处理细胞后，使用流式细胞仪检测了细胞凋亡。结果显示，使用AngII或者20-HETE处理导致了显著的凋亡，而AngII诱导的凋亡被HET0016所阻断；另外，AngII诱导的caspas3活性增强作用也被HET0016所抑制。实验结果还显示，HET0016可显著削弱AngII诱导的ROS的生成以及心肌线粒体膜电位的下降；再有，Ang II所诱导的细胞核质皱缩作用、染色质浓缩作用以及片段化作用均被HET0016所阻断。最后使用Western blot、Real time RT-PCR和高效液相色谱等方法，发现Ang II孵育心肌细胞后显著增加了CYP4A1的表达和20-HETE的生成。综上结果表明，20-HETE在Ang II所诱导的心肌细胞凋亡进程中发挥了非常重要的作用。20-HETE以及20-HETE合成酶可能作为治疗Ang II介导的心肌病新靶点。（该部分研究成果2013年2月投稿PLOS ONE, IF=4.01，第一作者）第四部分20-HETE通过δPKC对大鼠心肌细胞L-型钙通道和全细胞钾通道电流及心肌力学的作用研究通过第一部分的研究发现20-HETE可通蛋白激酶C（PKC）依赖机制激活NADPH氧化酶活性诱导过量活性氧簇（ROS）生成，从而诱发心肌细胞L-型钙通道电流增强，导致细胞发生钙超载。PKC家族包括12种亚型，其中δPKC在诱导心肌细胞凋亡、加重心肌缺血再灌注损伤等方面发挥重要作用，但何种因子激活δPKC，尚不清楚。那么20-HETE是否通过δPKC介导而对心肌细胞离子通道造成影响的，尚无研究。因此本部分研究主要通过膜片钳研究手段，探究δPKC在20-HETE对心肌细胞L-型钙通道和全细胞钾通道的作用。结果表明，20-HETE具有显著的激活心肌细胞L-型钙通道电流的作用，同时可明显抑制全细胞钾通道电流，这些作用被加入δPKC抑制剂卡马拉素所阻断，表明δPKC参与并介导了20-HETE对这些离子通道电流的影响。另外，采用Langendorrf离体心肌灌流的方法，检测了20-HETE是否通过δPKC对心脏收缩/舒张功能造成影响，结果表明δPKC抑制剂卡马拉素阻断了20-HETE所导致的心肌力学指标下降作用（LVDP,±dp/dtmax and LVEDP）。如上结果表明，20-HETE通过δPKC介导产生激活L-型钙通道电流以及抑制全细胞钾电流的作用，从而导致心肌细胞去极化，加重细胞钙超载，最终导致心肌力学指标下降。总之，通过本文如上四部分研究首次阐明了20-HETE对心肌缺血再灌注损伤以及诱导心肌细胞凋亡的作用机制，全面深入揭示了20-HETE造成心肌损伤的生理、病理学机制，为探寻有效的预处理药物作用靶点来减少由于20-HETE造成的心肌损伤，应用临床治疗缺血性心肌病提供可行的理论依据。更多还原

【Abstract】 20-hydroxyeicosatetraenoic acid （20-HETE） is a metabolite of arachidonic acidcatalyzed by the ω-hydroxylase enzymes of the cytochrome P-450（CYP）.20-HETE plays animportant role in the regulation of vascular tone in the brain, kidney, heart and splanchnicbeds by activation of L-type Ca²⁺channels and inhibition of Ca²⁺sensitive K+channels invascular smooth muscle cells.20-HETE is a strong vascular constrictor in these vascular beds;thus, plays an important role in not only the physiological regulation of blood supply to thoseorgans but also in the development of ischemic diseases.The researches on20-HETE’s effects on vascular smooth muscle have widely beenconducted. However, so far, very few research reports have been made on the role andmechanisms of20-HETE on hearts. This paper studies the effects of20-HETE on cardiacfunction and mechanism from the following perspectives:Part I:20-HETE increases NADPH oxidase-derived ROS production andstimulates the L-type Ca²⁺channel via a PKC-dependent mechanism in cardiomyocytesThe production of20-hydroxyeicosatetraenoic acid （20-HETE） is increased duringischemia-reperfusion, and inhibition of20-HETE production has been shown to reduce infarctsize caused by ischemia. This study was aimed to discover the molecular mechanismunderlying the action of20-HETE in cardiac myocytes. The effect of20-HETE on L-typeCa²⁺currents （ICa,L） was examined in rat isolated cardiomyocytes by patch-clamp recordingin the whole cell mode. Superfusion of cardiomyocytes with20-HETE （10–100nM） resultedin a concentration-dependent increase in ICa,L, and this action of20-HETE was attenuated bya specific NADPH oxidase inhibitor, gp91ds-tat （5M）, or a superoxide scavenger,polyethylene glycol-superoxide dismutase （25U/ml）, suggesting thatNADPH-oxidase-derived superoxide is involved in the stimulatory action of20-HETE onICa,L. Treatment of cardiomyocytes with20-HETE （100nM） increased both NADPH oxidaseactivity and superoxide production by approximately twofold. To study the molecularmechanism mediating the20-HETE-induced increase in NADPH oxidase activity, PKCactivity was measured in cardiomyocytes. Incubation of the cells with20-HETE （100nM）significantly increased PKC activity, and pretreatment of cardiomyocytes with a selectivePKC inhibitor, GF-109203（1M）, attenuated the20-HETE-induced increases in ICa,L and inNADPH oxidase activity. In summary,20-HETE stimulates NADPH oxidase-derivedsuperoxide production, which activates L-type-Ca²⁺channels via a PKC-dependentmechanism in cardiomyocytes.20-HETE and20-HETE-producing enzymes could be noveltargets for the treatment of cardiac ischemic diseases. Part II:20-hydroxyeicosatetraenoic acid mediates the isolated heartischemia-reperfusion injury via increasing NADPH oxidase-derived ROS productionIn the studies of part1, we find that20-HETE increases NADPH oxidase-derived ROSproduction and stimulates the L-type Ca²⁺channel via a PKC-dependent mechanism incardiomyocytes, which can be the important reason for20HETE to aggravate the myocardialischemia reperfusion injury. In this part, we discuss and testify the the aforementioned celllevel discovering mechanisms in isolated rat heart.Experiments were performed in isolated rat heart subjected to35min of ischemiafollowed by40min of reperfusion on Langendorff preparations. Perfusion with HET0016, aninhibitor of20-HETE production, significantly improved I/R-induced reduction in cardiaccontractility, myocardial infarction, and myocardial apoptosis. In contrast, administration of20-HETE aggravated I/R-induced myocardial injury and enhanced apoptosis. I/R significantlyincreased reactive oxygen species （ROS） production and oxidative stresses, which weresignificantly inhibited by HET0016and enhanced by20-HETE administration. Apocynin, aninhibitor of NADPH oxidase, blocked20-HETE-induced ROS production in the I/R hearts.20-HETE increased the expression of gp91phoxand p22phoxthe subunits of NADPH oxidase;and stimulated NADPH oxidase activity. In addition, GF-109203significantly attenuated the20-HETE-induced increases in the NADPH oxidase expression and activity. Finally, in theLangendorff I/R preparation, both apocynin and tempol, a ROS scavenger, significantlyblocked20-HETE-induced myocardial dysfunction.All of the results demonstrate that20-HETE stimulates NADPH oxidase-derivedsuperoxide production, which aggravates I/R-induced myocardial injury via a PKC-dependentmechanism in isolated rat hearts.Part III:20-Hydroxyeicosatetraenoic Acid is involved in Angiotensin II-mediatedapoptosis in rat cardiac myocytesThrough the studies of the previous two parts we have preliminarily discussed the roleand mechanisms of20-HETE on IR. During that process, another important factor isapoptosis. According to our previous studies,20-HETE is able to induces apoptosis throughmitochondrial-dependent pathways. More importantly, this study, in its second part,discovers that during the course of IR,20-HETE obviously induces apoptosis. Some studiesshow that AngII plays an important role in inducing apoptosis. However, the crosstalkbetween20-HETE and Ang II in cardiomyocyte apoptosis process is unclear.In the current study, we examined apoptosis using flow cytometry in primary culturedneonatal rat ventricular myocytes treated with control, Ang II （100nM）, Ang II plus HET0016（a20-HETE formation inhibitor,10μM）, HET0016, or20-HETE （10nM） alone. The resultsdemonstrated that treatment with Ang II or20-HETE significantly increased apoptosis andthat Ang II-induced apoptosis were markedly attenuated by HET0016. In addition, Ang II-induced increases of caspase-3activity were significantly attenuated by20.9±3.4%afterco-treatment with HET0016. Our results also demonstrated that HET0016significantlysuppressed Ang II-induced increases in superoxide production by27.5±2.3%and AngII-induced decreases in mitochondrial membrane potential by64.5±6.3%. Ang II-inducednuclei crenation, chromatin condensation and fractionation were attenuated by73.6±8.5%with HET0016treatment. Finally, treatment cardiomyocytes with Ang II significantlyincreased CYP4A1expression and20-HETE production, measured by Western blot, real-timeRT-PCR, and mass spectrometric analysis.All results suggest that20-HETE may play a key role in Ang II-induce apoptosis incardiomyocytes.20-HETE and20-HETE-producing enzymes could be novel targets for thetreatment of Ang II related cardiac diseases.Part IV: δPKC mediates20-HETE-induced enhancement of ICa,Landdescreasement of IKFrom the results of part1, we demonstrate that20-HETE can stimulate NADPHoxidase-derived superoxide production, which activate L-type Ca²⁺channels via aPKC-dependent mechanism in cardiomyocytes. PKC family includes12subtypes, amongwhich δPKC plays an important role in inducing the cell apoptosis and aggravating themyocardial ischemia reperfusion injuries, however, the specific factors that activate δPKCstill remain unknown. Therefore, the research of this part majorly, through applying theresearch methods of patch clamp techniques, discusses the effects of δPKC in the role of20-HETE on the L-type calcium channels and whole-cell potassium channels.The results show that δPKC inhibitor Rottlerin inhibited the role of20-HETE onenhancement of ICa,Lwhile descreasement of IK. It proves that δPKC mediates andparticipates in the role of20-HETE on the electric currents inside those ion channels. All sucheffects of20-HETE may result in increased calcium influx, lead to intracellular calciumoverload. Moreover,20-HETE led to the decline in cardiac mechanical index bydetermination of myocardial function in vitro, and administration of δPKC inhibitor Rottlerin,attenuated the myocardial dysfunction induced by20-HETE.In summary, it is the first time to elaborate the roles and mechanisms of20-HETE onmyocardial ischemia reperfusion injuries and the induced cell apoptosis, making in-depthanalysis of the physiological and pathological mechanisms of the myocardial cell injuriescaused by20-HETE, providing applicable theoretic foundations for searching the effectivepretreatment medicines as the target spots to reduce the myocardial cell injuries caused by20-HETE, which can be applied to the clinical treatments of ischemic cardiomyopathy.更多还原