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。     

山莨菪碱对洗涤大鼠血小板代谢花生四烯酸的影响

作者建立了一个用大鼠洗涤血小板研究药物对外源性及内源性AA代谢影响的方法。采用HPLC测定血小板AA代谢物HHT及12-HETE,观察了底物(AA)浓度、孵育时间、A23187加量等对血小板代谢AA的影响。并用此法研究了654-2对洗涤血小板AA代谢的影响。654-2显著减少血小板从内源性AA形成HHT及12-HETE,且作用随剂量增加而增强,但不影响血小板对外源性AA的代谢。上述结果表明,654-2是通过抑制AA释放而减少AA代谢物的形成。     

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。     

紫草素抗炎及对白三烯B4生物合成的抑制作用

皮下注射紫草素(shikonin)10mg.kg^-1对小鼠巴豆油耳炎症和大鼠酵母性足肿有明显抑制作用;在白细胞体外温孵系统中,紫草素在10^-4～10^-7mol·L^-1浓度范围内可浓度依赖性地抑制LTB₄和5-HETE生物合成,其50%抑制浓度(IC₅₀)分别为6.2×10^-7mol.L^-1和2.6×1O^-7mol·L^-1。此外,还报道紫草素几个天然衍生物对LTB₄生物合成的影响。     

银杏内酯B对大鼠中性白细胞花生四烯酸代谢酶和细胞内钙水平的影响

目的　观察银杏内酯B对大鼠中性白细胞花生四烯酸代谢酶及细胞内钙水平的影响。方法　反相高效液相色谱及Fura-2/AM荧光指示剂法。结果　银杏内酯B在0.1-10μmol·L^-1范围内,使花生四烯酸释放量降低10.9%-22.2%;0.1-50μmol·L^-1时,LTB₄和5-HETE生成量分别降低29.4%-88.6%和26.2%-89.3%;在0.1-100μmol·L^-1使PAF和fMLP刺激引起的细胞内游离钙浓度分别降低13.9%-51.4%和2.2%-36.6%。结论　银杏内酯B能抑制体外大鼠中性白细胞花生四烯酸代谢酶的活性及细胞内游离钙浓度的升高。     

阿魏酸钠对花生四烯酸代谢的影响

利用放射薄层方法测定兔血小板花生四烯酸代谢产物TXB₂,PGE₂和PGF_2α。用放射免疫法测定兔血小板TXB₂及主动脉6-keto-PGF_1α。阿魏酸钠(SF,0.1～3.2 mmol/L),抑制¹⁴C-花生四烯酸转化为TXB₂,呈剂量效应关系,IC₅₀为0.762 mmol/L。SF在较高浓度(0.8～3.2mmol/L)时亦抑制PGE₂,PGF_2α的生成。用放免法观察到,SF对血小板TXB₂和动脉壁6-keto-PGF_1α的生成均有抑制作用,对TXB₂的作用较强。结果提示,SF可抑制兔血小板和动脉壁环氧酶活性。     

三乙酰莽草酸对血小板聚集的抑制作用

目的：研究三乙酰莽草酸（TSA）对血小板聚集功能的抑制作用及其作用机理。方法：用比浊法测定血小板聚集功能,分光光度法测定MDA的含量,放免法测定TXB₂,6-酮-PGF_1α,cAMP和cGMP的含量。结果：TSA 12.5,25,50,100和200 mg.kg^-1 ig明显抑制ADP和胶原诱导的大鼠血小板聚集;TSA 12.5,50和200　mg.kg^-1 ig显著增加大鼠血小板内cAMP水平,但不影响cGMP水平。TSA 200 mg.kg^-1对AA诱导的血小板中MDA的生成,ADP诱导的血小板中TXB2和腹主动脉壁6-酮-PGF_1α的生成有轻度抑制作用。结论：TSA抑制血小板聚集作用部分与血小板内cAMP水平升高有关。     

拟人参皂苷F11在大鼠体内的药物代谢研究

目的探讨拟人参皂苷F11在大鼠体内的药物代谢产物及其过程.方法ip拟人参皂苷F₁₁后,应用TLC分析排泄物中的代谢产物,并利用制备薄层分离制备代谢产物,通过波谱解析(MS,¹HNMR,¹³CNMR,¹H-¹HCOSY)确定其结构.结果从粪便中分离鉴定了3种代谢产物,分别为拟人参皂苷R_T5,ocotillol和1个新的代谢产物F-3-1,并确定其结构为6-O-α-L-吡喃鼠李糖基(1-2)-β-D-吡喃葡糖基-(20S,23S,24R)-达玛-20(24)-环氧-3β,6α,12β,23,25-五醇(6-O-α-L-rhamnopyranosyl-(1-2)-β-D-glucopyranosyl-(20S,23S,24R)-dammar-20(24)-epoxy-3-β,6α,12β,23,25-pentanol).但在尿液和胆汁中并未发现任何代谢产物.结论拟人参皂苷F₁₁不被肝脏代谢,但胆汁排泄物可在肠道被代谢为水解和氧化产物.     

丁基苯酞对原代培养的大鼠皮层神经细胞外液6-keto-PGF1α和TXB2及其比值的影响

目的旨在观察丁基苯酞(NBP)对神经细胞培养液中6-酮-PGF_1α和TXB₂含量及其比值的影响。用放射免疫方法,结果发现神经细胞在低糖低氧5h或低糖低氧5h/恢复糖氧3h条件下,d-,l-和dl-NBP(0.1～100μmol·L^-1)能够剂量依赖性升高细胞外液中的6-酮-PGF_1α含量,降低TXB₂水平,从而使6-酮-PGF_1α与TXB₂比值升高。而阿司匹林仅在小剂量(0.1,1μmol·L^-1)时能升高6-酮 PGF^1α与TXB^{2比值,大剂量(10,100μmol·L-1)时无影响。提示:NBP对6-酮-PGF_1α/TXB₂比值的升高可能与其增加局部脑血流和改善缺血性脑损伤有关。}     

白三烯类化合物(Leukotrienes)对小鼠腹腔巨噬细胞生成白细胞介素6的影响

探讨了白三烯B₄(LTB₄)、白三烯C₄(LTC₄)及白三烯D₄(LTD₄)对小鼠腹腔巨噬细胞分泌白细胞介素6(interleukin6,IL-6)的影响。用IL-6依赖细胞株B9的MTT方法测定样品中IL-6的含量。结果显示,LTB₄能剂量依赖性地增加巨噬细胞培养上清液中IL-6的含量。LTC₄及LTD₄促进巨噬细胞培养上清液中IL-6含量的最适浓度分别为:6.9×10^-8和8.05×10^-8mol·L^-1。结果提示:LTB₄与LTC₄及LTD₄在某些生物学功能方面有一致性。与LTC₄及LTD₄相比,LTB₄促进巨噬细胞培养上清液中IL-6的含量的最适浓度则要大1～2个数量级,提示在炎症反应中,肽白三烯与IL-6的相关性较强。     

Effects of dauricine on the metabolism of arachidonic acid in rat pleural neutrophils

The effects of dauricine (Dau), an isoquinoline alkaloid and anti-arrhythmic agent used in China recently, on the biosynthesis of metabolites of arachidonic acid in rat pleural neutrophils, comparing with dazoxiben, indomethacin and BW-755 c, were studied. The major products of metabolism by 5-lipoxygenase (5-LPO), measured by HPLC, were LTB4 and 5-HETE, whereas the major cyclooxygenase products measured by HPLC and RIA, were HHT and TXB2. The formation of all products by neutrophils was significantly depressed by Dau in a dose-dependent manner. The concentration of Dau required to obtain 50% inhibition (IC50) of formation of HHT, TXB2, LTB4 and 5-HETE was 25.3, 59.3, 31.8 and 59.5 mumol/L, respectively. These results indicate that the two major metabolic pathways of arachidonic acid in rat pleural neutrophils are inhibited by Dau.     

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.     

山莨菪碱对乙酰胆碱和去甲肾上腺素引起的兔虹膜释放前列腺素的影响

本文研究了654-2对Ach及NE引起的兔虹膜释放PGE₂及6-keto-PGF_1α作用的影响。Ach及NE使虹膜释放PGs增加,654-2对Ach释放PGs的作用呈剂量依赖性抑制,当654-2浓度为3×10^-5mol/L时,Ach(5x10^-5mol/L)引起的PGE₂及6-keto-PGF_1α的释放量分别降低31%及30%。654-2浓度高于6x10^-5mol/L时显著抑制NE(5x10^-5mol/L)释放虹膜PGs的作用,当654-2浓度为3x10^-4mol/L时,使NE增加虹膜释放PGE:及6-keto-PGF_1α的量分别从62%及34%降至7.5%及4%(p<0.01)。654-2抑制PGs释放对其抗感染性休克等作用可能是有利的。     

Inhibition of the arachidonic acid cascade by norathyriol via blockade of cyclooxygenase and lipoxygenase activity in neutrophils

Recent studies have suggested that dual inhibitors of cyclooxygenase (COX) and lipoxygenase (LO) may be more beneficial in the treatment of inflammatory diseases in which platelet-leukocyte interaction dominates the underlying inflammatory process, than inhibitors of COX or LO alone. In this study, we examined oxygenated xanthones, shown previously to inhibit platelet and neutrophil activation, with respect to the potency of COX inhibition. 1,3,6,7-Tetrahydroxyxanthone (norathyriol) was the most potent. Norathyriol suppressed thromboxane B₂ (TXB₂) and leukotriene B₄ (LTB₄) formation in calcium ionophore (A23187)- and formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rat neutrophils. Norathyriol was 3–4 times more active against LTB₄ formation than against TXB₂ formation (IC₅₀ about 2.8 vs. 10 µM, respectively). Norathyriol also inhibited prostaglandin D₂ (PGD₂) formation in A23187-stimulated rat mast cells (IC₅₀ 3.0±1.2 µM) and in arachidonic acid (AA)-activated mast cell lysate. Norathyriol was a more effective inhibitor of 5-LO activity than of COX, as shown also by analyses of enzyme activities in a cell-free system, of COX and 5-LO metabolic capacity in neutrophils and of ex vivo TXB₂ and LTB₄ formation in A23187-stimulated neutrophils. Moreover, norathyriol inhibited COX-2 and 12-LO with IC₅₀ values (19.6±1.5 and 1.2±0.1 µM, respectively) similar to those required for the inhibition of COX-1 and 5-LO (16.2±1.5 and 1.8±0.4 µM, respectively). Inhibition of 15-LO by norathyriol was slightly less active. Norathyriol had no effect on A23187-induced AA release from neutrophils and did not affect phospholipase A₂ (PLA₂) activity in a cell-free system. These results indicate that norathyriol inhibits the formation of PGs and LTs in neutrophils probably through direct blockade of COX and 5-LO activities. Norathyriol, a single molecule with multiple targets, might provide a potential therapeutic benefit in the treatment of inflammatory diseases.     

Patent Evaluation: Hydroxylamine derivatives as lipoxygenase inhibitors

Summary

Novelty: Novel substituted heteroaryl hydroxylamine derivatives are claimed to be lipoxygenase (LPO) inhibitors. They are said to be particularly effective against 5-LPO. They are potentially useful for the treatment of inflammatory and allergic disorders, asthma, dermatitis, ischaemia and endotoxin shock.

Biology: 5-LPO inhibition was determined by measuring the inhibition of the synthesis of 5-HETE and LTB₄ in guinea-pig polymorphonuclear leukocytes. Methods to measure antiinflammatory and anti-asthmatic activity are also described. However, no specific biological data are presented.

Chemistry: A total of eighty-six compounds are disclosed. Syntheses are given in thirteen examples. Nine compounds are specifically claimed including 2-[3-(4-fluorophenyl)propyl]-6-[2-(N-aminocarbonyl-N-hydroxyamino)ethoxy]-1-oxo-1,2,3,4-tetrahydroisoquinoline.     

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.     

Isoliquiritigenin induces growth inhibition and apoptosis through downregulating arachidonic acid metabolic network and the deactivation of PI3K/Akt in human breast cancer

Arachidonic acid (AA)-derived eicosanoids and its downstream pathways have been demonstrated to play crucial roles in growth control of breast cancer. Here, we demonstrate that isoliquiritigenin, a flavonoid phytoestrogen from licorice, induces growth inhibition and apoptosis through downregulating multiple key enzymes in AA metabolic network and the deactivation of PI3K/Akt in human breast cancer. Isoliquiritigenin diminished cell viability, 5-bromo-2′-deoxyuridine (BrdU) incorporation, and clonogenic ability in both MCF-7 and MDA-MB-231cells, and induced apoptosis as evidenced by an analysis of cytoplasmic histone-associated DNA fragmentation, flow cytometry and hoechst staining. Furthermore, isoliquiritigenin inhibited mRNA expression of multiple forms of AA-metabolizing enzymes, including phospholipase A2 (PLA2), cyclooxygenases (COX)-2 and cytochrome P450 (CYP) 4A, and decreased secretion of their products, including prostaglandin E₂ (PGE₂) and 20-hydroxyeicosatetraenoic acid (20-HETE), without affecting COX-1, 5-lipoxygenase (5-LOX), 5-lipoxygenase activating protein (FLAP), and leukotriene B₄ (LTB₄). In addition, it downregulated the levels of phospho-PI3K, phospho-PDK (Ser²⁴¹), phospho-Akt (Thr³⁰⁸), phospho-Bad (Ser¹³⁶), and Bcl-x_L expression, thereby activating caspase cascades and eventually cleaving poly(ADP-ribose) polymerase (PARP). Conversely, the addition of exogenous eicosanoids, including PGE₂, LTB₄ and a 20-HETE analog (WIT003), and caspase inhibitors, or overexpression of constitutively active Akt reversed isoliquiritigenin-induced apoptosis. Notably, isoliquiritigenin induced growth inhibition and apoptosis of MDA-MB-231 human breast cancer xenografts in nude mice, together with decreased intratumoral levels of eicosanoids and phospho-Akt (Thr³⁰⁸). Collectively, these data suggest that isoliquiritigenin induces growth inhibition and apoptosis through downregulating AA metabolic network and the deactivation of PI3K/Akt in human breast cancer.     

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.