12/15-脂氧合酶对骨质疏松的影响

12/15-脂氧合酶是催化不饱和脂肪酸代谢的关键酶之一。研究表明,12/15-脂氧合酶及其代谢产物可通过多种途径阻碍干细胞成骨分化,促进破骨细胞生成与骨吸收,但可防止炎性反应相关的骨丢失。12/15-脂氧合酶对绝经前后女性的骨密度(BMD)产生不同的影响。本文就12/15-脂氧合酶对骨代谢调控的研究进展进行综述。     

12/15-脂氧合酶通过15-HETE/PPARγ途径减轻大鼠脑缺血再灌注损伤

目的通过观察脑缺血再灌注后脑内12/15-脂氧合酶代谢产物15-羟基二十碳四烯酸(HETE)含量和过氧化物酶体增殖物激活受体(PPAR)γ表达的变化,探究12/15脂氧合酶与脑缺血再灌注损伤的相关性。方法将SD大鼠90只随机分为15-HETE治疗组(干预组,30只)、假手术组(30只)和缺血再灌注模型组(模型组,30只)。应用线栓法制备缺血再灌注损伤模型,干预组在手术前30 min脑内注射15μl 15-HETE,模型组脑内注射等量的磷酸盐缓冲液(PBS)。应用Western印迹实验考察脑组织中12/15-脂氧合酶和PPARγ在蛋白水平的变化。应用Real Time-PCR考察脑组织中12/15-脂氧合酶和PPARγ在基因水平的变化。应用酶联免疫吸附试验(ELISA)试剂盒考察脑内15-HETE含量变化。结果模型组12/15-脂氧合酶和PPARγ在基因和蛋白水平表达均明显增加。与模型组相比,给予15-HETE治疗后PPARγ基因和蛋白水平明显增加。结论外源性给予15-HETE能够有效激活PPARγ,降低脑梗死面积,为脑缺血再灌注损伤治疗提供根据。     

12-脂氧合酶在糖尿病及其并发症中的作用

花生四烯酸是人体内重要的不饱和脂肪酸,花生四烯酸及其活性代谢产物在调节细胞的生长、增殖、生存和凋亡方面发挥了重要作用。其主要通过环氧合酶、脂氧合酶和细胞色素P450途径进行代谢,在哺乳动物体内,脂氧合酶又可以分为5-脂氧合酶、12-脂氧合酶和15-脂氧合酶,目前发现它们可能参与了糖尿病及其慢性并发症的发生和发展。     

基于5-脂氧合酶代谢通路的中药复方干预动脉粥样硬化炎症的新思路

动脉粥样硬化(AS)是一种慢性炎症性病理过程，具有慢性炎症的“共同点”,即炎症消散的缺失和炎症介质的持续释放。抗炎一直是防治AS的有效途径，而促炎症消散则是近年来研究的热点，但很少有兼顾二者研究的报道，主要原因还是炎症介质和促炎症消散介质一般都由不同的信号通路所介导。5-脂氧合酶代谢产物白三烯B₄(LTB₄)和脂氧素(LX)在AS炎症中具有截然相反的作用特点。现系统阐述LTB₄和LX在AS炎症中的表达及作用机制，再结合中药复方的多靶点多途径的整合调节作用，从抗炎和促炎症消散两方面干预AS炎症更具有优势，为中药复方干预AS炎症提供一种可能的理论依据和研究新思路。     

5-脂氧合酶促癌机制研究进展

5-脂氧合酶蛋白是花生四烯酸代谢途径中的一种关键酶,在恶性肿瘤的发生、发展过程中起了重要作用．抑制5-脂氧合酶及其产物的表达有可能预防和逆转恶性肿瘤的发生,本文结合国内外文献,就5-脂氧合酶分子生物学特征及促癌机制的研究进展作一综述．     

动脉粥样硬化与12/15-LOX基因启动子区甲基化状态的关系

目的 探讨动脉粥样硬化（AS）与12/15-脂氧合酶（12/15-LOX）基因启动子区甲基化状态的关系。方法 将40只SD大鼠随机分为两组,即模型组和对照组,每组20只。模型组:给予高脂＋维生素D3粉剂饮食;对照组正常饮食。3个月后,处死大鼠,取其主动脉弓至腹主动脉的血管组织,用改进的半定量甲基化特异性PCR法检测血管组织中12/15-LOX基因启动子区甲基化状态。结果 ①经苏木精-伊红染色,显微镜下观察可见模型组大鼠的主动脉有明显的粥样斑块形成,对照组未见明显变化。②按照吸光度(A)比值把全部血管组织分为低甲基化状态和高甲基化状态,模型组大鼠的血管组织的低甲基化率显著高于对照组（P<0.01）。结论 血管斑块处12/15-LOX基因启动子区的甲基化状态与AS的形成相关,在AS发病过程中可能发挥着重要的作用。     

5-脂氧合酶与食管癌

食管癌是严重威胁我国人们生命健康的恶性肿瘤,深入了解食管浸润、转移的机制,寻求更新、更好的治疗靶点具有重要意义.越来越多的研究显示,以不饱和脂肪酸氧化代谢为标靶探求抗癌的干预措施在临床前期的研究中具有广阔的前景.肿瘤发生学的研究表明,不饱和脂肪酸必须经历氧化代谢来促进肿瘤生成.5-脂氧合酶（5-LOX）和环氧合酶-2（COX-2）分别是花生四烯酸脂氧合酶、环氧合酶两条代谢途径中重要的限速酶.COX-2参与调节肿瘤细胞的增生与凋亡、肿瘤微血管的生成、癌细胞的侵袭与转移以及宿主免疫系统的抑制等多个病理生理过程.食管癌组织中有COX-2的高表达,并与预后密切相关[1,2].人们已经在探索COX-2的抑制剂在肿瘤防治中的作用.随着研究的深入,参与花生四烯酸代谢的另一关键酶类-脂氧合酶受到了越来越多的关注,有学者发现NSAIDs可通过花生四烯酸代谢的另一主要途径-脂氧合酶途径而触发凋亡[3].不断增多的数据表明,脂氧合酶代谢途径有望成为一个新的抗肿瘤靶点.     

5-脂氧合酶与消化系肿瘤的关系

5-脂氧合酶是催化花生四烯酸代谢产生5-OH-6,8,11,14-廿碳四烯酸和白三烯等类花生酸产物的重要酶类。近年来研究发现5-脂氧合酶与人类多种肿瘤的发生发展有关。本文主要综述5-脂氧合酶及其抑制剂与消化系肿瘤的关系。     

5脂氧合酶激活蛋白基因单核苷酸多态性与冠心病的关联研究

目的探讨5-脂氧合酶激活蛋白基因2354T/A和16699G/A多态性与冠心病的关系。方法采用聚合酶链反应—限制片长多态性技术,对282例经冠状动脉造影证实的冠心病患者和79例对照者进行检测,分析5-脂氧合酶激活蛋白基因2354T/A和16699G/A多态性的基因型和等位基因频率分布情况,检验多态位点与冠心病的关联。结果 5-脂氧合酶激活蛋白基因2354T/A和16699A/G多态性在病例组和对照组均以TT、GG基因型为主。A等位基因为少见型。连锁不平衡分析显示2354T/A,16699G/A间不存在连锁不平衡(D’=0.798,r2=0.037)。单因素分析显示以上2个多态未显示与冠心病的相关性,应用Logistic回归模型将混杂因素矫正后进行分析单个多态位点与疾病的关系时,以上2个多态同样未显示与冠心病的相关性。将以上多态全部纳入构建单体型,有两种单体型频率较高分别为:AG28.6%、TG68.9%,单体型分析显示,两组间比较差异无统计学意义(P>0.05)。结论 5-脂氧合酶激活蛋白基因2354T/A和16699G/A多态性与中国天津汉族人群冠心病易感性无明显关联。     

12/15-脂加氧酶与糖尿病微血管并发症

最近研究发现,12/15-脂加氧酶的过表达或激活可能促进了糖尿病肾病和神经病变的进展.该酶可催化花生四烯酸产生12-羟基二十碳四烯酸和15-羟基二十碳四烯酸,从而影响细胞结构、代谢和信号转导.研究发现12/15-脂加氧酶及其代谢产物可直接诱导或间接调节细胞因子导致细胞肥大、增殖和细胞外基质堆积,参与糖尿病肾病的发生.此外,该酶的过表达和激活与外周神经系统的氧化-硝化应激相关,可能参与糖尿病神经病变的发生.因此,12/15-脂加氧酶抑制剂或包含抑制剂的联合治疗可能为糖尿病微血管并发症的治疗提供新的研究方向.     

Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.

The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, we have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.     

Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase.

Arachidonate 15-lipoxygenase (arachidonate:oxygen 15-oxidoreductase, EC 1.13.11.33) is a lipid-peroxidating enzyme that is implicated in oxidizing low density lipoprotein to its atherogenic form. Monocyte/macrophage 15-lipoxygenase is present in human atherosclerotic lesions. To pursue a basis for induction of the enzyme, which is not present in blood monocytes, the ability of relevant cytokines to regulate its expression was investigated. Interleukin 4 (IL-4), among 16 factors tested, specifically induced 15-lipoxygenase mRNA and protein in cultured human monocytes. Interferon gamma and hydrocortisone inhibited this induction. High-performance liquid chromatography analysis of lipid extracts from IL-4-treated monocytes detected 15-lipoxygenase products esterified to the cellular membrane lipids, indicating enzymatic action on endogenous substrates. Stimulation of IL-4-treated monocytes with calcium ionophore or opsonized zymosan A enhanced the formation of 15-lipoxygenase products. These data identify IL-4 and interferon gamma as physiological regulators of lipoxygenase expression and suggest an important link between 15-lipoxygenase function and the immune/inflammatory response in atherosclerosis as well as other diseases.     

Cloning of the cDNA for human 12-lipoxygenase.

A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.     

12/15-lipoxygenase, oxidative modification of LDL and atherogenesis.

Lipoxygenases comprise a family of non-heme iron-containing dioxygenases that stereospecifically insert molecular oxygen into free or esterified polyunsaturated fatty acids. The dual specificity 12/15-lipoxygenases have been implicated in the oxidative modification of low-density lipoproteins and foam cell formation primarily based on in vitro studies. Recent in vivo data obtained with 12/15-lipoxygenase-deficient mice crossbred to apolipoprotein E-deficient mice have established a proatherogenic role for this pathway. In contrast, previous experiments with macrophage expressing 15-lipoxygenase transgenic rabbits have suggested an anti-atherogenic role. Possible explanations are presented that may elucidate these differences.     

Expanding expression of the 5-lipoxygenase pathway within the arterial wall during human atherogenesis

Oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. In addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. Here, we studied both lipoxygenase pathways in human atherosclerosis. The 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients afflicted with various lesion stages of atherosclerosis of the aorta and of coronary and carotid arteries. 5-lipoxygenase localized to macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes, and the number of 5-lipoxygenase expressing cells markedly increased in advanced lesions. By contrast, reticulocyte-type 15-lipoxygenase was expressed at levels that were several orders of magnitude lower than 5-lipoxygenase in both normal and diseased arteries, and its expression could not be related to lesion pathology. Our data support a model of atherogenesis in which 5-lipoxygenase cascade-dependent inflammatory circuits consisting of several leukocyte lineages and arterial wall cells evolve within the blood vessel wall during critical stages of lesion development. They raise the possibility that antileukotriene drugs may be an effective treatment regimen in late-stage disease.     

12/15-lipoxygenase–derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H₂O₂, as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF β-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX–derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H₂O₂ in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation.     

Evidence for 15-HETE synthesis by human umbilical vein endothelial cells

Incubation of cultured human umbilical vein endothelial cells with [1-14C]-arachidonic acid, followed by RP-HPLC analysis, resulted in the appearance of two principal radioactive products besides 6-keto-PGF1 alpha. The first peak was HHT, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by GC/MS and was shown to be the lipoxygenase product 15-HETE. Stimulation of endothelial cells with thrombin enhanced 15-HETE synthesis from arachidonate. Subsequent experiments showed that 5-HETE and 12-HETE were also synthesized by endothelial cells, but no evidence of leukotriene synthesis was found. Incubation of the 15-HETE precursor 15-HPETE with endothelial cells resulted in the formation of four distinct ultraviolet light-absorbing peaks. Ultraviolet and GC/MS analysis showed these peaks to be 8,15-diHETEs that differed only in their hydroxyl configuration and cis-trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14,15-LTA4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggest that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-LTA4.