【作者】 刘柳；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 研究背景和目的：环氧化二十碳三烯酸(epoxyeicosatrienoic acids，EETs)是花生四烯酸(arachidonicacid，AA)的表氧化酶代谢产物。它们由细胞色素P450表氧化酶代谢产生，作为参与体内多种生物活动的重要脂质中介物。EETs对于调节心血管稳态发挥了重要的作用，它能促进内皮型一氧化氮合酶的表达和提高其活性以增加一氧化氮(nitric oxide，NO)的合成从而保护内皮功能；能抑制内皮细胞炎症因子和粘附分子的表达；能促进内皮细胞的增殖和血管生成；能抑制血管平滑肌细胞的增殖和移行；还能促进组织型纤溶酶原激活物的表达促进纤溶而发挥抗凝血的效应，这些都证明了EETs对于心血管系统起广泛的保护作用。氧化应激(oxidative stress)与机体的多种疾病联系紧密，包括神经退行性疾病、糖尿病、肾病以及肿瘤等。近年来的研究发现，活性氧(reactive oxygenspecies，ROS)具有灭活NO从而损害内皮依赖的血管舒张反应、促进炎症细胞的粘附、促进血管平滑肌细胞的增殖和迁移等效应。这些效应使得ROS促进了动脉粥样硬化、高血压、血管形成术后再狭窄、心血管重塑、心衰等心血管疾病的病理生理过程的发生和发展。既然EETs在维持心血管稳态中起到了重要的作用，而氧化应激和活性氧是心血管的“不稳定因素”之一，这二者之间是否有联系?目前还未有研究。探讨EETs对心血管系统氧化应激及ROS产生的影响，可以明确EETs的生理作用、为心血管系统疾病的防治提供靶点和理论依据。同时，我们还首次发现EETs促进人类肿瘤细胞的增殖和转移，并具有抑制肿瘤细胞凋亡的作用。而ROS亦是肿瘤细胞凋亡信号的重要调节分子，在缺氧、药物、受体介导等各种因素诱导的肿瘤细胞凋亡中起到重要作用。ROS升高可以诱发肿瘤细胞凋亡，抗氧化剂可以抑制凋亡。因此，本文还研究了EETs抑制肿瘤细胞凋亡是否和ROS有关，以期完善我们前期的研究成果，深化EETs对肿瘤细胞的作用机制。实验方法：体外培养的牛主动脉内皮细胞(bovine arotic endothelial cells，BAECs)用EETs预处理24小时，接着用血管紧张素Ⅱ(angiotensinⅡ，AngⅡ)处理1小时，使其胞内ROS升高，用流式细胞仪检测细胞内的ROS产生。用western blot的方法检测不同时间EET处理的内皮细胞NAD(P)H氧化酶各亚单位的表达和抗氧化酶超氧化物歧化酶和过氧化氢酶的表达，用生化方法检测其活性。分离细胞胞浆和胞膜蛋白，用westernblot的方法分别检测胞浆和胞膜NAD(P)H氧化酶各亚单位的分布和Racl GTP酶的膜转位。在肿瘤细胞中，用三氧化二砷(arsenic trioxide，ATO)诱导人舌鳞癌细胞Tca-81 13 ROS的产生和细胞凋亡。同样用流式细胞仪检测细胞内的ROS产生和细胞凋亡，用比色法测定caspase活性，JC-1测定线粒体膜电位，western blot检测抗氧化酶的表达和氧化还原敏感的相关信号通路的改变。实验结果：1．EETs对体外培养的BAECs活性氧产生的作用及机制通过流式细胞仪检测发现：用EETs预处理的24h后，能明显抑制AngⅡ诱导的BAECs的ROS生成。通过western blot检测发现这一效应可能是通过抑制NAD(P)H氧化酶的亚单位gp91phox的表达；抑制NAD(P)H氧化酶的胞浆组份p47phox和racl的膜转位和上调抗氧化酶超氧化物歧化酶的表达和活性来实现的。2．EET对肿瘤细胞的活性氧产生的作用及机制EET同样能抑制ATO导致的肿瘤细胞的活性氧产生以及细胞凋亡，它还能抑制ATO导致的线粒体膜电位的丢失、caspase 9和3的激活以及P38、JNK等信号通路的激活。抑制CYP2J2的活性能和ATO起到协同作用，增加肿瘤细胞的ROS产生和促进肿瘤细胞凋亡。结论：本实验通过体外培养的内皮细胞和肿瘤细胞，利用分子生物学和细胞生物学技术，对EET在活性氧的调节及其机制进行了研究，得出以下结论：1．EET抑制AngⅡ诱导的BAECs活性氧产生，其机制可能是抑制了NAD(P)H氧化酶主要亚单位的表达和膜转位以及上调抗氧化酶的表达和活性，AMPK、PPAR-γ、ERK途径可能是EET发挥抗氧化效应的机制。2．EET同时也抑制了肿瘤细胞内由ATO诱导的ROS产生。还能抑制ROS介导的线粒体途径的细胞凋亡，完善了关于EET抑制细胞凋亡的相关机制。同时，抑制CYP2J2能够增强ATO对肿瘤细胞的作用，为临床肿瘤的防治提供了新的策略。更多还原

【Abstract】 BackgroundCytpchrome P450 (CYP) epoxygenases convert arachidonic acid to four epoxyeicosatrienoic acid (EET) regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET, that function as the critical lipid mediators involved in several biological events in human body. EETs play a crucial role in cardiovascular homeostasis that have been considered including mechanisms 1) to preserve endothelial function by increasing NO bioavailability, 2) to inhibit the expression of inflammation and adhesion factors in endothelial cells, 3) to promote endothelial cells proliferation and angiogenesis, 4) to inhibit vascular smooth muscle cells (VSMC) proliferation and migration, 5) to increase fibrolysis. These evidences indicated that EET extensively protect the cardiovascular system. Oxidative stress has been associated with diverse pathophysiological events, including neurodegeneration, diabetes mellitus, renal disease and cancer. Reactive oxygen species (ROS) elicit direct cellular damage and mitogenicity, serve as intracellular second messengers, scavenge vasoprotective NO, increase inflammatory cells adhesion, and promote VSMC proliferation and migration. Recently, it has been verified ROS also play a critical role in the incidence and development of vasculopathies, including atherosclerosis, hypertension and restenosis after angioplasty. Now that the EET maintained cardiovascular homeostasis, whereas ROS has been served as the "unstable factors", whether or not EET reduces ROS injury in vascular walls is not clear. Investigating the action of EET on oxidative stress and ROS generation in cardiovascular system could definite the physiological role of EET and provide the theoretical basis and the target for the prevention and treatment of cardiovascular diseases. At the meantime, we revealed for the first time that EETs promote human tumor cells proliferation and metastasis, and inhibit tumor cells apoptosis. ROS are the critical regulating molecules of apoptosis in tumor cells, and extensive evidence indicates that ROS plays a piotal role in tumor cell apoptosis whether induced by ischemia, drugs, or receptor mediated factors. For this reason, we investigated whether EET inhibits tumor cells apoptosis correlated with ROS generation.MethodsAfter treating with EET for 24 hours, bovine arotic endothelial cells (BAECs) was treated with angiotensin II (Ang II) for 1 hour. The generation of ROS was measured by flowcytometry. The expression of NAD(P)H oxidase subunits, superoxide dismutases (SOD) and catalase (CAT) were detected by western blot. The activity of SOD and CAT were measured by biochemical assays. Following cytoplasmic and membrane protein separation, the NAD(P)H oxidase subunit p47phox and Rac1 GTPase translocation were detected by western blot. ROS and apoptosis were induced by arsenic trioxide (ATO) in Tca-8113 human tumor cells, and were measured by flowcytometry. Caspase activity was measured by colorimetry assay. Mitochondrial membrane potential was measured by JC-1 assay. The involved cell signal molecular changes were detected by western blot.Results1. EETs alleviate reactive oxygen species generation in BAECs and the mechanisms24 hours pretreatment of EETs significantly inhibited Ang II-induced ROS generation in BAECs. This effect may be mediated by inhibiting NAD (P) H oxidase subunits: gp91phox expression, reduced translocation of p47phox and rac1 and by increasing SOD expression and its activity. 2. EETs reduce reactive oxygen production in tumor cells and the mechanismsEETs can inhibit the tumor cells reactive oxygen generation and apoptosis induced by ATO. EETs can also attenuate the loss of mitochondrial membrane potential, caspase 9 and 3 activation as well as P38, JNK signaling pathway activation caused by ATO. Inhibition of CYP2J2 activity cooperated with ATO increased ROS generation and apoptosis in tumor cells.ConclusionUsing molecular and cellular biology techniques, we investigated the regulation of reactive oxygen by EETs and the mechanisms in vitro in cultured endothelial cells and tumor cells. We found that:1. EETs inhibited Ang II-induced reactive oxygen species generation through inhibiting NAD(P)H oxidase subunits expression and translocation and through increasing SOD expression as well as its activity. These effects may dependent on AMPK, PPAR-γ, ERK signaling pathway.2. EETs also inhibited the ROS generation induced by ATO in tumor cells. Moreover, EETs can inhibit ROS-mediated mitochondrial pathway of apoptosis, which enriched the mechanisms of EETs act on tumor cell apoptosis. Meanwhile, inhibition of CYP2J2 enhanced the effect of ATO on tumor cells, which raise a new strategy for the prevention and treatment of clinical oncology.更多还原