蝙蝠葛碱对血小板聚集及花生四烯酸代谢的影响

蝙蝠葛碱(Dau) 抑制AA及ADP诱导的大鼠血小板聚集,也能抑制AA,ADP及Adr诱导的人血小板聚集。这种抑制作用与Dau剂量呈依赖关系。Dau抑制大鼠洗涤血小板对[1-¹⁴C]AA经环氧酶途径的代谢,TXB₂与HHT的形成均呈剂量依赖性减少。当Dau浓度达到0.1 mmol/L时亦能抑制12-HETE的形成。Dau对AA代谢的上述影响可能是其抑制血小板聚集的机理之一。     

山莨菪碱对大鼠胸水中性白细胞花生四烯酸代谢的影响

本文研究了654-2对大鼠胸水中性白细胞代谢[1-¹⁴C]AA及内源性AA的影响。大鼠白细胞AA经5-LPO代谢途径形成的主要产物为LTB₄及5-HETE,经CO途径的主要产物为HHT及TXB₂。654-2对白细胞代谢[1-¹⁴C]AA无抑制作用,但显著减少白细胞从内源性AA形成的LTB₄,5-HETE,HHT及TXB₂。这种抑制作用与654-2呈剂量依赖关系。本实验结果表明,654-2抑制PG及LT的形成可能是影响了AA从胞膜的释放,而并非直接抑制CO及5-LPO。     

20-羟-二十烷四烯酸在糖尿病心血管并发症中的作用

糖尿病是以高血糖为特征的代谢紊乱综合征,其心血管并发症是导致糖尿病患者致残、致死的主要原因.20-羟-二十烷四烯酸(20-hydroxyeicosatetraenoic acid,20-HETE)是近年来发现的花生四烯酸(Arachidonic acid,AA)第三条代谢通路,细胞色素P-450(Cytochrome P-450,CYP450)途径的活性代谢产物.现有研究表明其在调节肾功能和血管平滑肌紧张度中起到重要作用,但对20-HETE与糖尿病的关系研究甚少.本文对近年来国际上20-HETE与糖尿病心血管并发症的研究报道进行综述.     

15-羟廿碳四烯酸对缺氧性大鼠颈内动脉环的收缩作用及机制

目的研究15-羟廿碳四烯酸(15-HETE)对正常及缺氧状态的离体大鼠颈内动脉环的作用,探讨15-HETE在缺血/缺氧性脑损伤的病理生理过程中的作用。方法16只W istar大鼠随机分为两组即对照组:其氧浓度(F iO2)为21%和缺氧组:置于F iO2为10%的缺氧箱中(n=8)。9 d后将大鼠处死,游离颈内动脉(ICA)制备血管环,KH液中分别加入不同浓度15-HETE以及2 mmol.L-14-AP、10-2mol.L-1TEA、10-6mol.L-1GLYB后平衡15 min后,再加入15-HETE,观察不同浓度15-HETE、不同K+通道阻断剂对颈内动脉环收缩反应程度的变化。结果15-HETE以浓度依赖方式(10-8mol.L-1~10-6mol.L-1)使游离颈内动脉环张力增加,对缺氧组大鼠颈内动脉环张力作用更为明显,与正常对照组相比差异有显著性(15-HETE浓度为10-8mol.L-1、10-7mol.L-1时,P<0.05,10-6mol.L-1时,P<0.01,n=8);2 mmol.L-14-AP使颈内动脉环血管张力增高(P<0.05,n=8),但预先应用2 mmol.L-14-AP后浴液中加入10-6mol.L-115-HETE,血管张力无进一步增加的趋势;10-2mol.L-1TEA、10-6mol.L-1GLYB,对血管环张力差异无显著性(P>0.05,n=8);但进一步应用15-HETE使血管环张力增加,变化幅度接近单纯应用15-HETE组。结论15-HETE收缩ICA,缺氧情况下尤为明显;Kv通道在15-HETE所致的ICA收缩中起作用。     

花生四烯酸及其3种代谢产物对兔肺动脉环作用的比较

目的通过比较花生四烯酸(AA)及其3种代谢产物:15-羟基二十碳四烯酸(15-HETE)、8(S),15(S)-二羟基二十碳四烯酸〔8(S),15(S)-D iHETE〕、15-酮基二十碳四烯酸(15-KETE)对缺氧组与正常组幼兔的肺动脉环张力的影响,以探讨AA及其3种代谢产物在缺氧性肺动脉高压形成中的作用。方法12只新生幼兔随机分为两组(n=6),一组置于正常的培养箱中,其吸入氧(F iO2)浓度为0.21,作为正常组;一组置于缺氧的培养箱中,吸入氧(F iO2)浓度为0.10,作为缺氧组。9 d后将两组幼兔处死,采用组织浴槽血管环法观察AA、8(S),15(S)-D iHETE、15-HETE、15-KETE对正常和缺氧幼兔肺动脉收缩的影响。结果①AA、8(S),15(S)-D iHETE、15-HETE、15-KETE以浓度依赖方式收缩正常组幼兔肺动脉环,其中15-KETE和15(S)-D iHETE收缩血管作用明显,而AA、15-HETE收缩血管作用不明显;②AA、15-KETE、15-HETE、8(S),15(S)-D iHETE使缺氧幼兔肺动脉环张力增加,AA、15-HETE使缺氧组幼兔肺动脉环张力较正常组增高;但缺氧肺动脉环对四种物质的反应力差异无显著性;③正常组幼兔肺动脉环对15-KETE的反应明显高于缺氧组。结论15-HETE的代谢产物8(S),15(S)-D i-HETE在缺氧性肺动脉高压形成中起一定的作用,而15-KETE可能不参与慢性缺氧导致的幼兔肺动脉的收缩过程。     

柴胡皂苷体外干预大鼠胸腔炎性白细胞5-LO活性的实验研究

目的:观察柴胡皂苷体外对大鼠胸腔炎性白细胞脂氧酶(5-LO)活性的影响,探讨其干预花生四烯酸代谢的抗炎机制.方法:以角叉菜胶所致急性胸膜炎模型大鼠的胸腔炎性白细胞为基本反应体系,分别加入CaCl2,MgCl2,花生四烯酸(AA),A23187以及柴胡皂苷(0.04,0.08,0.16,0.24 mg·mL-1)或齐留通(0.625 μg·mL-1),提取完整白细胞的5-LO代谢产物白三烯B4(LTB4)和5-羟基二十碳四烯酸(5-HETE),运用反相高效液相色谱(RP-HPLC)紫外检测分离测定LTB4和5-HETE的合成水平,来反映5-LO的活性.结果:柴胡皂苷在0.08～0.24 mg·mL-1浓度范围内(经MTT法测定为安全剂量范围),对大鼠胸腔炎性白细胞LTB4与5-HETE 的生物合成呈现抑制作用,其中0.16、0.24 mg·mL-1剂量作用明显(p<0.01).结论:柴胡皂苷(0.04～0.24 mg·mL-1浓度)体外可抑制大鼠胸腔白细胞花生四烯酸代谢酶5-LO的活性,降低LTB4与5-HETE合成水平,提示其抗炎作用可能与抑制5-LO 活性,减少致炎代谢产物的生成有关.     

烙铁头蛇毒血小板聚集素对血小板聚集和抑制剂关系的研究

研究山莨菪碱(654-2)、阿斯匹林(ASA)、前列环素(PGI_2)样物质对二磷酸腺苷(ADP)、花生四烯酸(AA)以及烙铁头蛇毒血小板聚集素(TMVA)诱导的阻抗法全血血小板聚集(简称全血聚集)的影响。结果表明:ADP.AA.TMVA诱导的全血聚集随剂量增加而增强.阻抗技术对于估价生理条件下的血小板功能较为敏感。654-2促进ADP诱导的全血聚集.而ASA和PGI_2样物质对ADP和AA诱导的聚集均有抑制作用.但不能防止TMVA诱导的聚集。提示TMVA可能通过第3途径(类似于血小板活化因子.PAF)诱导血小板聚集.经ADP和AA作用过的血小板对TMVA的活化仍有反应,其机理值得研究。     

花生四烯酸 12-脂氧化酶– 12-氢基二十碳四烯酸– GPR31轴在肝脏再灌注肝缺血损伤中的作用

目的 探究花生四烯酸12-脂氧化酶(ALOX12)–12-氢基二十碳四烯酸(HETE)–GPR31轴在肝脏再灌注肝缺血损伤中的作用机制.方法 采用8周龄雄性B6.Cg-Tg(MX1-cre)Cgn/J小鼠为研究对象,采用随机数字表法分为三组:空白对照组(n=12)、实验对照组(n=12)和实验组(n=12).实验组小鼠进行基于胚胎干细胞的基因打靶技术制备基因敲除手术;进行肝血流阻断.肝血流阻断45 min后发送移走血管夹,以恢复血液供应.实验对照组进行肝血流阻断.空白对照组同样进行开腹但并不进行肝血流阻断.采用蛋白质印迹法(Western blotting)检测ALOX12–12-HETE–GPR31轴中基因表达水平.采用酶联免疫吸附测定(ELISA)法分别检测肝脏血清中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)三项炎性因子水平和12-HETE含量.采用原位细胞凋亡检测试剂盒检测细胞凋亡情况.采用日立7180全自动生化分析仪检测小鼠肝脏血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、AST/ALT.结果 肝脏再灌注肝缺血损伤小鼠的ALOX12–12-HETE-GPR31轴中基因转录水平均高于空白对照组(P<0.05),且随着再灌注时间的延长而逐渐增大(P<0.05).空白对照组和实验组小鼠,在肝血流阻断并再灌注后,随着再灌注时间延长ALOX12–12-HETE–GPR31轴中基因转录水平间差异无统计学意义(P>0.05).实验对照组小鼠肝脏中IL-1β[(20.53±1.32)ng/L比(10.61±0.83)ng/L]、IL-6[(322.1±11.41)ng/L比(107.34±9.02)ng/L]、TNF-α水平[(31.78±2.42)ng/L比(11.41±0.98)ng/L]、12-HETE含量、肝脏组织细胞凋亡率、ALT[(47.94±1.48)U/L比(24.85±1.50)U/L]、AST[(54.45±3.17)U/L比(30.69±2.08)U/L]和AST/ALT[(1.23±0.04)比(0.69±0.03)]均高于空白对照组(P<0.05).实验组小鼠各项指标低于实验对照组(P<0.05),且与空白对照组差异无统计学意义(P>0.05).结论 ALOX12表达量的上调会促进12-HETE的积累,特异性敲除ALOX12基因,阻断12-HETE的积累能够有效抑制肝脏再灌注肝缺血损伤.     

在大鼠中15-LO/15-HETE参与缺氧对K_V1.5表达的抑制作用

目的采用分子生物学技术从组织和细胞水平上观察阻断15-LO/15-HETE后,缺氧对KV1.5表达的影响。方法通过酶法分离、培养Wistar大鼠肺动脉血管平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)和大鼠肺动脉,应用Western blot和RT-PCR方法分别从蛋白质水平和mRNA水平上观察在15-LO阻断剂CDC和NDGA作用下,缺氧对KV1.5表达的影响。结果从组织和细胞水平上,用CDC和NDGA阻断15-LO即阻断了内源性15-HETE的产生,KV1.5的表达量在蛋白质水平和mRNA水平与未阻断组比较都增加。在阻断了内源性15-HETE的产生以后,加入外源性15-HETE,KV1.5的表达量减低。说明不仅内源性15-HETE参与诱导缺氧对KV1.5表达的影响,外源性15-HETE也同样能影响缺氧条件下KV1.5的表达量。但在阻断了内源性15-HETE的产生以后,加入外源性15-HETE,KV1.5的表达量减低。说明不仅内源性15-HETE参与诱导缺氧对KV1.5表达的影响,外源性15-HETE也同样能影响缺氧条件下KV1.5的表达量。结论上述结果表明,从大鼠肺动脉组织和细胞水平上,内源性15-HETE介导了缺氧对KV1.5表达的抑制作用。     

新灯盏素对血细胞与内皮细胞花生四烯酸代谢影响的差异

新灯盏素是从灯盏花中提取的4－羟基－7－0－葡萄糖醛酸甙的可溶性钠盐与钙盐，该药抑制血小板TXB2生成而不影响羟廿碳四烯酸的产量；抑制内皮细胞6－酮PGF1a的生成；对白细胞TXB2生成无影响，但明显加 强钙离子载体刺激LTB4生成的作用，结果表明新灯盏素对血细胞与内皮细胞的花生四烯酸代谢有不同的影响。     

5-Hydroxytryptamine and the metabolism of arachidonic acid by the lipoxygenase and cyclooxygenase of washed human platelets

During secondary aggregation, platelets release 5-hydroxytryptamine (5-HT) from their dense granule stores concurrent with arachidonic acid (AA) metabolism. To examine the hypothesis that released 5-HT has a modulatory effect on the metabolism of AA by platelets, we incubated nonaggregating washed human platelets with 5-HT in the presence of [3H]AA. Stimulation with 10(-4) M 5-HT, followed by incubation with 3 microM AA and 1 microCi [3H]AA for 5 min, resulted in a decrease in the formation of thromboxane B2 (TxB2) and 12-hydroxyheptadecatrienoic acid (HHT, P less than 0.05). The same treatment conditions and stimulation with 10(-7) to 10(-4) M 5-HT resulted in an elevation of 12-hydroxyeicosatetraenoic acid (12-HETE) formation (P less than 0.05). Treatment with the monoamine uptake inhibitor imipramine (20 microM) further increased the stimulation of 12-HETE formation observed in the presence of 10(-4) M 5-HT, suggesting that 5-HT may act at the platelet surface. A 5-HT1A receptor agonist, 8-hydroxy-dipropylaminotetralin (DPAT, 10(-6) to 10(-4) M) stimulated the formation of platelet cyclooxygenase (CO) products, whereas (+/-)1-(2,5-dimethoxy-4-iodo phenyl)-amino propane hydrochloride (DOI, 10(-6) to 10(-4) M), a 5-HT2 receptor agonist, had no significant effect on CO product formation. In addition, the 5-HT2 receptor antagonist ketanserin (10(-7) M) did not block the changes in CO or lipoxygenase metabolism induced by 5-HT. Since both DOI and DPAT stimulated 12-HETE formation whereas ketanserin was unable to reverse the 5-HT-enhanced 12-HETE formation, it seems unlikely that the stimulation of a 5-HT2 receptor is responsible for this action of 5-HT on platelets. We conclude that 5-HT depresses CO product formation while increasing 12-HETE formation through interaction with a platelet serotonergic binding site other than the 5-HT2 receptor.     

咪苯嗪酮对TXA2和HHT生成的选择性抑制作用

咪苯嗪酮(CI-914)能抑制大鼠血小板环氧酶和TXA₂合成酶产物HHT的生成,而对脂氧酶产物12-HETE的生成仅高浓度药物才有弱的抑制作用,提示CI-914主要影响花生四烯酸(AA)环氧酶途径,而对脂氧酶途径影响较少。在大鼠血小板和中性白细胞CI-914能抑制TXA₂的生成,同时CI-914还可使白细胞6-keto-PGF_1a和血小板PGE₂的产生量显著增加,提示CI-914在这两种细胞引起了AA的转向合成。上述结果基本证实,CI-914在大鼠中性白细胞和血小板对TXA₂合成酶具有选择性抑制作用。     

Effects of Nonanal,trans-2-Nonenal and 4-Hydroxy-2,3-trans-nonenal on Cyclooxygenase and 12-Lipoxygenase Metabolism of Arachidonic Acid in Rabbit Platelets

The effects of nonanal, trans-2-nonenal and 4-hydroxy-2,3-trans-nonenal on the formation of thromboxane B₂ (TXB₂), 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid in washed rabbit platelets were examined. Nonanal and trans-2-nonenal at concentrations ranging from 0.25 to 2 μm inhibited TXB₂, HHT and 12-HETE formation, reducing the amounts of these three arachidonic acid metabolites by 50% at nonanal and trans-2-nonenal concentrations of approximately 0.25 μm. The inhibition of TXB₂, HHT and 12-HETE formation induced by 4-hydroxy-2,3-trans-nonenal (50% inhibition by 4-hydroxy-2,3-trans-nonenal at a concentration of approximately 100 μm) was 400 times weaker than that induced by nonanal and trans-2-nonenal. These results suggest that nonanal and trans-2-nonenal can be modulators of platelet arachidonic acid metabolism by affecting the activity of cyclooxygenase and 12-lipoxygenase.     

Effects of endocrine disruptors on arachidonic acid metabolism in rabbit platelets

To explore the possible actions of endocrine disruptors on the autacoid synthesis in the body, we investigated the effects of nonylphenol (NP), bisphenol A (BPA), di-n-butyl phthalate (DBP), benzyl-n-butyl phthalate (BBP), and di-2-ethylhexyl phthalate (DEHP) on the formation of 12-lipoxygenase metabolite, 12-HETE, and cyclooxygenase metabolites, TXB(2) and 12-HHT, from exogenous arachidonic acid (AA) in rabbit platelets. NP (10-50 microM) showed strong inhibition on the formation of cyclooxygenase metabolites (TXB(2), 34-95% inhibition; 12-HHT, 13-78% inhibition) and weaker inhibition on the formation of 12-HETE (0-49% inhibition). BPA, DBP, BBP, DEHP, and 17beta-estradiol (endogenous estrogen) failed to show any effect on the formation of cyclooxygenase and 12-lipoxygenase metabolites at concentrations up to 100 microM. These results suggest that NP inhibits AA metabolism in platelets and that its effects on the cyclooxygenase pathway predominate over those exerted via the 12-lipoxygenase pathway.     

Effect of adenine nucleotides on cyclooxygenase and lipoxygenase enzyme products of arachidonic acid in human platelets

Nucleotides are known to enhance cyclooxygenase product formation in several tissues and, in addition, are believed to function as cofactors for mammalian 5-lipoxygenases. Since nucleotides are released by stimulated platelets and by damaged tissue, we examined the hypothesis that nucleotides can affect the metabolism of arachidonic acid (AA) in washed human platelets. The various nucleotides were given 15 sec prior to the addition of 3 microM arachidonic acid and 1 muCi [3H]AA. We found that the phosphorylated adenine derivatives (ATP, ADP, and AMP) increased the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) by 2-fold without altering the formation of cyclooxygenase products. Adenosine was without effect on 12-HETE formation. ATP also stimulated 12-HETE formation in lysed platelets. This suggests that the 12-lipoxygenase enzyme of platelets can be regulated by adenine nucleotides. We next determined the portion of the nucleotide molecule responsible for the enhanced 12-lipoxygenase activity of platelets. Alteration of the nucleotide base led to a decrease in stimulation, with GTP less active than ATP, and UTP even less active than GTP. Studies with adenine nucleotides showed that the length of the phosphate chain was not important. We also found that the stable methylene isosters of ATP (alpha, beta-methylene ATP and beta, gamma-methylene ATP) increased 12-HETE formation, suggesting that the conformation and hydrolysis of the phosphate chain are not responsible for the stimulatory activity. Cyclic 3',5'AMP and 3'AMP were inactive, implying the necessity for a free phosphate at the 5' position for nucleotide stimulation of 12-HETE synthesis. In conclusion, platelet 12-lipoxygenase was stimulated by ATP, as is true for several mammalian 5-lipoxygenases. However, cyclooxygenase product formation by platelets was not altered by nucleotide addition. These studies suggest that following in vivo injury or platelet aggregation, when local concentrations of nucleotides are high, platelet lipoxygenase activity may be stimulated.     

Arachidonic acid metabolism of cultured peritoneal rat macrophages and its manipulation by nonsteroidal antiinflammatory agents

[3H]Arachidonic acid (AA) metabolism by cultured rat peritoneal macrophages (M phi) was examined. Ninety percent of incorporated [3H]AA localized in phospholipids (phosphatidylcholine, 30%; phosphatidylinositol, 23.9%; phosphatidylethanolamine, 23.7%) whereas 8.3% and 1.5% was found in the free fatty acid and neutral lipid fractions. 12-O-tetradecanoate phorbol-13-acetate (TPA) induced the reduction of label from phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine (42%, 46% and 47%, respectively) and an accumulation of label into free fatty acids (34%) or neutral lipid (61%) fractions. Simultaneously, 12% of the incorporated label was released into the medium as [3H]6-keto prostaglandin (PG)F1 alpha, [3H]thromboxane B2 (TxB2), [3H]PGE2, [3H]hydroxyheptadecanoic acid (HHT), [3H]15-hydroxyeicosatetraenoic acid (HETE), [3H]12-HETE and [3H]AA. Exposure to 0.3-3 microM indomethacin reduced TPA-induced label release into the medium which was distinguished by dose-dependent reductions in all [3H]prostanoids as well as [3H]15- and [3H]12-HETE and a reciprocal increase in [3H]AA. Nordihydroguaiaretic acid (NDGA) altered AA metabolism at concentrations which approached its toxic dose (greater than 20 microM). Cells exposed to 10 microM NDGA reduced TPA-induced label release into the medium which was characterized by reductions in [3H]-TxB2, [3H]PGE2 and [3H]HHT, no change in [3H]15-HETE, [3H]12-HETE or [3H]AA and the appearance of [3H]PGF2 alpha. Cellular label redistributions of lipid fractions in cells exposed to NDGA or indomethacin were significantly less than that of control cultures indicating inhibition of acylhydrolase activity. Indomethacin or NDGA, therefore modify AA metabolism of cultured rat M phi by influencing more than one target enzyme.     

Spectrophotometric monitoring of lipoxygenase and cyclooxygenase pathway activity using ionophore stimulated whole blood

We have employed clotting human blood stimulated with ionophore to develop a system for measuring cyclooxygenase, 5-lipoxygenase, and 12-lipoxygenase pathway products released into the serum fraction. In a single chromatographic run, 5-HETE, 12-HETE, 12-OH-heptadecatrienoic acid (HHT), LTB4, 20-OH-LTB4, and 20-COOH-LTB4 are quantitated by UV monitoring after separation by HPLC. The kinetics of product formation/release of all fatty acid products into the serum show an apparent lag of approximately 2 min, after which time the amounts of HHT, 5-HETE, and LTB4, respectively, plateau at 10 min while 12-HETE increases over a 60 min period. The system is responsive to standard cyclooxygenase and lipoxygenase inhibitors, and is of value of evaluating prospective blockers of AA metabolism in a whole blood setting.