5-脂氧合酶及其抑制剂

5-脂氧合酶（5-lipoxygenase, 5-LOX）是生物体内一种重要的双加氧酶.5-LOX是催化花生四烯酸（arachidonic acid,AA）生成白三烯类（leukotrienes, LTs）的关键酶. LTs是重要的炎症介质,并且在许多疾病中发挥着重要作用.近年来,很多学者致力于5 LOX的研究,从而为5-LOX抑制剂的研发提供理论依据.本文对5-LOX的分子结构、细胞内定位、表达与活性调控及其抑制剂的研究进展进行综述.     

慢性炎症与动脉粥样硬化关系的研究进展

动脉粥样硬化（atherosclerosis,AS）的发病机制非常复杂,对其研究经历了一个半世纪,直到1999年Ross提出”动脉粥样硬化是一种炎症性疾病”,各种炎症细胞和炎症因子参与动脉粥样硬化的发生和发展过程。已有众多的基础和临床研究都证实炎症在AS中的重要作用,但仍需要对AS发生发展的深入研究,使我们更准确认识和有效的防治AS。本文就近年来慢性炎症与动脉粥样硬化关系的研究进展作一综述。     

C反应蛋白与动脉粥样硬化

C反应蛋白（C-reactiveprotein,CRP）是人类非特异性急性期蛋白,是判断组织损伤和炎症反应的敏感指标之一。CRP的表达水平与动脉粥样硬化（atherosclerosis,AS）和心血管疾病的发生具有冠著的相关性。但是关于CRP是否是AS的独立危险因素并参与AS的发病机制,目前尚存在很大争议。新近的研究发现,CRP与某些特定的配体结合后,五聚体结构CRP可分离形成单体结构CRP。这一发现为研究CRP蛋白与AS的相互关系提供了新的线索,对CRP及其单体结构的深入研究,将有可能帮助人们找到治疗心血管疾病的有效方法。就炎性反应标志物CRP及其单体（monomeric CRP,mCRP）与动脉粥样硬化的相关研究进展进行综述,以探讨分析CRP在AS中的作用。     

Toll样受体4对动脉粥样硬化发生、发展的研究进展

动脉粥样硬化（atherosclerosis,AS）是多种细胞、炎性介质参与形成的慢性炎症性疾病。Toll样受体家族（Toll like receptors,TLRs）中的TLR4是机体重要的诱导分泌多种炎性因子的模式识别受体。现有证据表明,TLR4不仅产生多种炎性因子诱发血管炎症反应,而且促进AS斑块形成和发展,造成斑块不稳定,甚至破裂,对AS的发生、发展具有重要作用。因此,了解TLR4对AS的影响有助于发现新的治疗靶点和对策。主要对TLR4在AS发病机制和易损斑块发展中的作用进行综述。     

慢性炎症与动脉粥样硬化关系的研究进展

动脉粥样硬化(atherosclerosis,AS)的发病机制非常复杂,对其研究经历了一个半世纪,直到1999年Ross提出"动脉粥样硬化是一种炎症性疾病",各种炎症细胞和炎症因子参与动脉粥样硬化的发生和发展过程。已有众多的基础和临床研究都证实炎症在AS中的重要作用,但仍需要对AS发生发展的深入研究,使我们更准确认识和有效的防治AS。本文就近年来慢性炎症与动脉粥样硬化关系的研究进展作一综述。     

氟灭酸选择性抑制花生四烯酸5-脂氧酶和环氧酶代谢途径

花生四烯酸(AA)是细胞膜磷脂中的重要成份,它在不同酶的作用下可转化成多种代谢产物。继前列腺素(PGs)后,70年代末,人们又发现一类AA5-脂氧酶代谢物,因其发现于白细胞,分子中又含三个共轭双键而被称作白三烯(LTs)。已证明其中LTB_4是一个极强的白细胞趋化剂和聚集剂,LTC_4、LTD_4和LTE_4则为过敏性慢反应物质(SRS-A)的活性成份,它们分别参与多种炎症和过敏性哮喘等病理过程。由于LTs对一些细胞和器官的生物活性超过许多自体活性物质(如组胺、PGs等),对LTs合成抑制剂的研究受到高度重视。附图表示由AA代谢成PGs和LTs的关键步骤。甾     

肠道微生物在动脉粥样硬化发生发展中的作用机制及未来治疗靶点的研究进展

动脉粥样硬化是多种心脑血管疾病的重要病理基础。目前研究发现肠道微生物在动脉粥样硬化的风险因素中起着非常重要的作用。随着研究的深入,人们发现肠道微生物及其代谢产物可能通过影响人体物质代谢、能量的吸收与利用、全身慢性炎症与氧化应激等,参与动脉粥样硬化的发生发展。本文就肠道微生物在动脉粥样硬化的发生发展中作用机制及临床治疗靶点作一简要综述。     

15-脂氧合酶/15-羟廿碳四烯酸在缺氧性肺动脉高压中的作用和机制

肺动脉高压是一种病因复杂的罕见病,以肺动脉阻力增高,引起右心室后负荷增大,最终导致右心室功能衰竭而使患者死亡为特征。肺血管花生四烯酸信号通路异常在肺动脉高压中发挥重要作用。肺动脉高压患者肺动脉内皮细胞、平滑肌细胞和成纤维细胞中15-脂氧合酶(15-lipoxygenase, 15-LO)及其代谢产物15-羟廿碳四烯酸(15-hydroxyeicosatetraenoic acid,15-HETE)水平均升高。在缺氧条件下,15-LO/15-HETE引起肺动脉收缩,促进肺动脉内皮细胞和平滑肌细胞增殖,抑制肺动脉平滑肌细胞凋亡,促进肺血管外膜纤维化,进而导致肺动脉高压的发生。本文主要对15-LO/15-HETE与缺氧性肺动脉高压相关性的研究进行综述,以阐明15-LO/15-HETE在缺氧性肺动脉高压中发挥的核心作用。     

失功能性HDL研究进展

高密度脂蛋白胆固醇(HDL-C)水平与动脉粥样硬化呈负相关,HDL被认为具有抗动脉粥样硬化(AS)作用。但急性期反应、慢性炎症及一些代谢性疾病中,HDL组成成分变化及其功能基团的病理性修饰可造成HDL失功能化,失功能性HDL中蛋白质、脂类、酶类发生特征性改变,具有促动脉粥样硬化作用。随着研究深入,人们逐步认识到HDL的功能比HDL-C水平更重要。microRNAs与失功能性HDL具有相关性,还参与心血管系统及代谢系统疾病的发生发展,HDL受到包括microRNAs在内的一系列信号分子调控。本文主要综述失功能性HDL的结构、特征以及与动脉粥样硬化、microRNAs之间的关系,为动脉粥样硬化防治提供新的思路和方法。     

NLRP3炎症小体及其相关通路与动脉粥样硬化研究进展

动脉粥样硬化所导致的心血管病是人类致死致残的重要原因。炎症因素作为重要危险因素已得到研究者共识,越来越多的证据表明炎症参与了从早期内皮功能障碍到晚期斑块破裂的AS形成的各个阶段。CANTOS研究作为测试AS炎症假说的第一个大规模临床试验,为寻找IL-1β介导的炎症性AS的潜在治疗靶点提供依据。Nod样受体蛋白3炎症小体作为固有免疫系统的一种识别受体,可引起半胱氨酸天冬氨酸特异性蛋白酶1激活,产生和释放成熟的促炎因子白细胞介素1-β,参与炎症反应发生发展。本文对胆固醇结晶,氧化低密度脂蛋白,血流动力学改变,斑块内细胞代谢方式变化等对NLRP3炎症体激活机制以及NLRP3炎症小体的激活途径和临床治疗方法进行综述,以期通过对NLRP3及其相关通路的研究,为临床动脉粥样硬化治疗提供新思路。     

5-Lipoxagenase deficiency attenuates L-NAME-induced hypertension and vascular remodeling

BackgroundAbnormalities of the L-arginine-nitric oxide pathway induce hypertension. 5-Lipoxygenase (5-LO) is the key enzyme involved in synthesis of leukotrienes (LTs). However, whether nitricoxide synthase dysfunction induces hypertensive vascular remodeling by regulating 5-LO activity and its downstream inflammatory metabolites remains unknown.Methods and resultsSix-week L-NAME treatment significantly induced hypertension and vascular remodeling in both wild-type (WT) and 5-LO–knockout (5-LO–KO) mice, and blood pressure in caudal and carotid arteries was lower in 5-LO–KO than WT mice with L-NAME exposure. On histology, L-NAME induced less media thickness, media-to-lumen ratio, and collagen deposition and fewer Ki-67–positive vascular smooth muscle cells (VSMCs) but more elastin expression in thoracic and mesenteric aortas of 5-LO–KO than L-NAME–treated WT mice. L-NAME significantly increased LT content, including LTB4 and cysteinyl LT (CysLTs), in plasma and neutrophil culture supernatants from WT mice. On immunohistochemistry, L-NAME promoted the colocalization of 5-LO and 5-LO–activating protein on the nuclear envelope of cultured neutrophils, which was accompanied by elevated LT content in culture supernatants. In addition, LTs significantly promoted BrdU incorporation, migration and phenotypic modulation in VSMCs.ConclusionL-NAME may activate the 5-LO/LT pathway in immune cells, such as neutrophils, and promote the products of 5-LO metabolites, including LTB4 and CysLTs, which aggravate vascular remodeling in hypertension. 5-LO deficiency may protect against hypertension and vascular remodeling by reducing levels of 5-LO downstream inflammatory metabolites.     

The 5-lipoxygenase pathway in arterial wall biology and atherosclerosis

Leukotrienes (LTs) are powerful inflammatory lipid mediators derived from the 5-lipoxygenase (5-LO) cascade of arachidonic acid. Recent clinical, population genetic, cell biological, and mouse studies indicate participation of the 5-LO pathway in atherogenesis and arterial wall remodeling. 5-LO is expressed by leukocytes including blood monocytes, tissue macrophages, dendritic cells, neutrophils, and mast cells. LTB4 and the cysteinyl LTs LTC4, LTD4, and LTE4, act through two BLT and two cysLT receptors that are differentially expressed on hematopoietic and arterial wall cells. The precise roles of LTs or the LT receptors in cardiovascular physiology remain largely to be explored. In this review, we will discuss what is currently known about the 5-LO atherosclerosis connection. We will attempt to propose strategies to further explore potential links between the 5-LO pathway and blood vessel physiology and disease progression.     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

Leukotriene synthesis by epithelial cells

Leukotrienes (LTs) are intercellular signaling molecules that evoke a variety of responses. They are best known as potent promoters of inflammation. Normally, LTs are produced primarily by leukocytes. As a result, current models regarding the production of LTs in the context of disease focus on the leukocytes as the site of production. Structural cells, including epithelial cells, are typically relegated to supportive roles. It is recognized that epithelial cells normally contain all the components necessary for LT synthesis except the enzyme 5-lipoxygenase (5-LO). There is accumulating evidence that some populations of epithelial cells normally express low levels of 5-LO and can synthesize LTs autonomously. Moreover, certain factors, including bacterial and viral infection, can promote the expression of 5-LO in airway, gastrointestinal and skin epithelial cells. The appearance of active 5-LO enzyme in epithelial cells at these sites may contribute to diseases like cancer, colitis and psoriasis. This paper reviews the state of our knowledge regarding the expression of 5-LO in epithelial cells, the factors that modify that expression, and the implications regarding pathogenesis.     

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.     

Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12

Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.     

Inhibition of 5-lipoxygenase activity in mice during cuprizone-induced demyelination attenuates neuroinflammation, motor dysfunction and axonal damage

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Increased expression of 5-lipoxygenase (5-LO), a key enzyme in the biosynthesis of leukotrienes (LTs), has been reported in MS lesions and LT levels are elevated in the cerebrospinal fluid of MS patients. To determine whether pharmacological inhibition of 5-LO attenuates demyelination, MK886, a 5-LO inhibitor, was given to mice fed with cuprizone. Gene and protein expression of 5-LO were increased at the peak of cuprizone-induced demyelination. Although MK886 did not attenuate cuprizone-induced demyelination in the corpus callosum or in the cortex, it attenuated cuprizone-induced axonal damage and motor deficits and reduced microglial activation and IL-6 production. These data suggest that during cuprizone-induced demyelination, the 5-LO pathway contributes to microglial activation and neuroinflammation and to axonal damage resulting in motor dysfunction. Thus, 5-LO inhibition may be a useful therapeutic treatment in demyelinating diseases of the CNS.     

Eugenol--the active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells

Polymorphonuclear leukocytes (PMNL) play an important role in the modulation of inflammatory conditions in humans. PMNL cells recruited at the site of inflammation, release inflammatory mediators such as leukotrienes, proteolytic enzymes and reactive oxygen species. Among these, leukotrienes are implicated in pathophysiology of allergic and inflammatory disorders like asthma, allergic rhinitis, arthritis, inflammatory bowel disease and psoriasis. 5-lipoxygenase (5-LO) is the key enzyme in biosynthetic pathway of leukotrienes. Our earlier studies showed that spice phenolic active principles significantly inhibit 5-LO enzyme in human PMNLs. In this study we have further characterized the inhibitory mechanism of eugenol, the active principle of spice-clove on 5-LO enzyme and also its effect on leukotriene C((4)) (LTC(4)). Substrate dependent enzyme kinetics showed that the inhibitory effect of eugenol on 5-LO was of a non-competitive nature. Further, eugenol was found to significantly inhibit the formation of LTC(4) in calcium ionophore A23187 and arachidonic acid (AA) stimulated PMNL cells. These data clearly suggest that eugenol inhibits 5-LO by non-competitive mechanism and also inhibits formation of LTC(4) in human PMNL cells and thus may have beneficial role in modulating 5-LO pathway in human PMNL cells.     

Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523

Leukotrienes (LTs) are lipid messengers generated by leukocytes that drive inflammation and modulate neighboring cell function. The synthesis of LTs from arachidonic acid is initiated by the enzyme 5-lipoxygenase (5-LO). We report for the first time that LT synthesis is inhibited by the direct action of protein kinase A (PKA) on 5-LO. The catalytic subunit of PKA directly phosphorylated 5-LO in vivo and in vitro and inhibited activity in intact cells and in vitro. Mutation of Ser-523 on human 5-LO prevented phosphorylation by PKA and restored LT synthesis. Treatment with PKA activators inhibited LTB(4) synthesis in 3T3 cells expressing wild type 5-LO but not in cells expressing the S523A mutant of 5-LO. The mechanism of inhibition of LTB(4) synthesis did not involve either reduced membrane association of activated 5-LO or redistribution of 5-LO from the nucleus to the cytoplasm. Instead, PKA phosphorylation of recombinant 5-LO inhibited in vitro activity, as did co-transfection of cells with 5-LO plus the catalytic subunit of PKA. Also, substitution of Ser-523 with glutamic acid, mimicking phosphorylation, resulted in the total loss of 5-LO activity. These results indicate that PKA phosphorylates 5-LO on Ser-523, which inhibits the catalytic activity of 5-LO and reduces cellular LT generation. Thus, PKA activation, as can occur in response to adenosine, prostaglandin E(2), beta-adrenergic agonists, and other mediators, is a means of directly reducing 5-LO activity and LT synthesis that may be important in limiting inflammation and maintaining homeostasis.     

Suppression of 5-lipoxygenase activity by anionic cholesterol derivatives

5-Lipoxygenase (5-LO) is a key enzyme involved into biosynthesis of leukotrienes (LTs), mediating the host defense system, and acting simultaneously as inflammatory agents. In this work the effect of anionic cholesterol derivatives on 5-LO activity has been investigated. Cholesterol sulfate activates human polymorphonuclear leukocytes (PMNL) and stimulates their adhesion to endothelium and collagen. Cholesterol sulfate and cholesterol phosphate suppressed leukotriene production in PMNL and in rat basophil leukemia (RBL-1) cell line as well as in homogenates of these cells. Kinetic characteristics of the effect of anionic cholesterol derivatives on leukotriene synthesis have been obtained. In all experiments cholesterol phosphate (charge-2) was shown to be more potent inhibitor than cholesterol sulfate (charge-1). We believe that this fact highlights the importance of negatively charged ester groups for suppression of 5-LO activity.