Effects of platelet activating factor and ginkgolid B on the cAMP levels in washed rabbit platelets

研究了血小板激活因子（ＰＡＦ）和ＰＡＦ拮抗剂银杏内酯Ｂ对洗涤兔血小板中ｃＡＭＰ含量的作用．结果表明ＰＡＦ（０．１－１．０μｍｏｌ·Ｌ－１）对血小板的基础ｃＡＭＰ水平无影响，但对前列腺素Ｅ１（ＰＧＥ１）２μｍｏｌ·Ｌ－１及４，５－二氢－６－［４－（１Ｈ－咪唑－１－）苯基］－５－甲基－３－（２Ｈ）－哒嗪酮（ＣＩ－９３０）２０μｍｏｌ·Ｌ－１引起的ｃＡＭＰ升高有显著的抑制作用．银杏内酯Ｂ能完全拮抗ＰＡＦ抑制ＰＧＥ１和ＣＩ－９３０升高ｃＡＭＰ的作用，ＩＣ５０分别为４．７和１２．５μｍｏｌ·Ｌ－１．合用磷酸肌酸／磷酸肌酸激酶和阿司匹林对ＰＡＦ和银杏内酯Ｂ的作用均无影响．提示ＰＡＦ对磷酸二酯酶的激活作用及腺苷酸环化酶的抑制作用是ＰＡＦ的直接作用，与其同ＰＡＦ受体结合有关．     

血小板激活因子及其拮抗剂银杏内酯B对大鼠心肌缺血再灌注损伤的影响(英文)

目的　探讨外源性血小板激活因子 (PAF)激活的多核形中性粒细胞 (PMN)是否具有低浓度氧自由基模拟缺血预适应 (IP)对缺血再灌注 (IR)所致心肌损伤的保护作用。方法　结扎冠脉左前降支 30min ,再灌注 3h制备大鼠心脏IR模型。以缺血前给予 2次 5min缺血 ,10min再灌注作为IP。实验分为6组 ,IR组、IR +PAF组和IR +银杏内酯B(GB、PAF拮抗剂 )组 ,分别于缺血前 2 5和 10miniv生理盐水、PAF(3μg·kg- 1)和GB (5mg·kg- 1) ;IP +IR组、IP +IR +PAF组和IP +IR +GB组分别于IP 2次 10min再灌注开始时iv相应药物。观察分析心功能、梗死面积、PMN计数、心肌髓过氧化物酶活性、TUNEL阳性细胞计数等指标。结果　给予PAF明显加重IR引起的心脏损伤、PMN浸润及凋亡的程度 ;GB对IR引起的心功能下降没有明显影响 ,但明显减少梗死面积、PMN浸润及凋亡细胞 ;PAF明显削弱IP的保护作用 ;GB对IP作用未见明显影响。结论　IR后PMN浸润可能是引起心肌细胞凋亡的一个重要原因 ;PAF激活PMN可能不具有模拟IP对IR所致心肌损伤的保护作用 ,反而取消IP的保护作用。     

HPLC-ELSD法测定银杏内酯B滴丸中银杏内酯B的含量

目的：建立银杏内酯B滴丸中银杏内酯B的含量测定方法。方法：采用高效液相色谱法。色谱柱为E ZORBAX Eclipse Plus C18（5μm,4.6mm×150mm,Agilent）,流动相为正丙醇-四氢呋喃-水（1∶33∶66）,流速为1.0ml/min,柱温为30℃,采用蒸发光散射检测器（ELSD）检测,外标法测定。结果：银杏内酯B滴丸中银杏内酯B的进样量在1.071～21.42μg（r=0.9996）范围内与各自峰面积对数值呈良好的线性关系;平均加样回收率为98.049%,RSD为0.872%,样品在10小时内稳定。结论：所建立的方法可行,准确、重复性好,适用于银杏内酯B滴丸中银杏内酯B的质量控制。     

HPLC-ELSD法测定白果中银杏内酯A、银杏内酯B和银杏内酯C

目的建立HPLC-ELSD法同时测定白果药材中银杏内酯A、银杏内酯B和银杏内酯C 3个萜杏内酯成分。方法采用Agilent 5TC C18色谱柱(250 mm×4.6 mm,5μm),以甲醇–四氢呋喃–水(25∶10∶65)为流动相,体积流量1 m L/min,柱温30℃,蒸发光散射检测器检测,对照品进样量为10、20μL,供试品溶液进样量为5~20μL。结果银杏内酯A在0.37~5.92μg、银杏内酯B在2.8~44.8μg、银杏内酯C在0.65~10.4μg时线性关系良好。银杏内酯A、银杏内酯B、银杏内酯C平均回收率分别为92.40%、95.03%、91.29%,RSD值分别为2.75%、2.06%、2.88%。结论本法操作简单,重复性好,为白果药材的质量控制提供了依据。     

银杏内酯药理作用研究进展

血小板激活因子是一种由多种细胞产生又可作用于多种细胞的内源性活性物质，个有 生物学作用。银杏内酯为天然银杏提取物中的主要活性成分，是一类特异有效的ＰＡＦ受体损坏抗剂。药理作用最新研究表明其具有抗过敏，抗炎，抗休克作用，对缺血损伤及器移植的排斥反应亦有保护作用等。广泛的代理作用使其有广阔的临床应用前景。     

高效液相色谱法测定银杏内酯B注射液含量

目的 研究并建立银杏内酯B 注射液的含量测定方法。方法 采用高效液相色谱法,以甲醇-四氢呋喃 水（2.5：1：6.5）为流动相,检测波长为225 nm,测定银杏内酯B 的含量。结果 银杏内酯B在9.98～99.8 μg之间呈良好的线性关系,加样回收率平均值为100.70%,RSD 为0.14%;精密度良好,RSD为0.21%;重复性也很好,RSD为0.21%。结论 该方法稳定可靠,灵敏简便,重复性好,可作为银杏内酯B 注射液的质量控制方法。     

银杏内酯B对血小板活化因子刺激的大鼠中性粒细胞功能的影响银杏内酯B对血小板活化因子刺激的大鼠中性粒细胞功能的影响

目的 研究银杏内酯B对血小板活化因子(PAF)刺激的大鼠中性粒细胞粘附、趋化及脱颗粒功能的影响。方法 从大鼠外周血分离中性粒细胞,用MTT比色法、Boyden小室法及β-葡萄糖苷酸酶释放法分别检测PAF诱导的粒细胞粘附、趋化及脱颗粒反应。结果10 μmol·L^-1 银杏内酯B可显著抑制中性粒细胞的粘附反应;1～1 000 nmol·L^-1 可剂量依赖性抑制10 nmol·L^-1 PAF诱发的粒细胞趋化反应,其IC₅₀为4.84 nmol·L^-1; 0.01～10 μmol·L^-1可抑制1 μmol·L^-1 PAF诱发的粒细胞释放β-葡糖苷酸酶,其IC₅₀为3.56 μmol·L^-1。结论银杏内酯B能够抑制PAF刺激的大鼠中性粒细胞粘附、趋化及脱颗粒反应。     

银杏内酯B对ApoE基因敲除小鼠动脉粥样硬化的影响

目的探讨银杏内酯B(ginkgolide B,GB)对ApoE基因敲除(ApoE-/-)小鼠动脉粥样硬化的影响。方法 12只8周龄C57BL/6J小鼠为正常组,24只8周龄♂ApoE-/-小鼠为阳性模型组以及药物组;药物组每天给予GB 0.6 mg灌胃。8周后处死小鼠,用病理学方法观察小鼠动脉斑块、脂质沉积以及巨噬细胞表达。用ELISA方法测定血浆RAN-TES含量。结果电镜结果显示模型组动脉斑块较大,血管内膜损伤明显,而GB组小鼠动脉内表面损伤明显减轻,斑块较小。HE染色显示了同样的结果,模型组斑块较大,GB组斑块较小。血浆RANTES测定正常组为123.81 ng.L-1,模型组为359.16 ng.L-1,GB组为193.36 ng.L-1,各组之间差异存在统计学意义(P<0.01)。结论 GB能有效地减轻ApoE-/-小鼠的动脉粥样硬化损伤,并降低血浆RANTES含量,其药理学机制可能与抑制血小板功能相关。     

银杏内酯注射液和银杏内酯ABC对血小板活化因子诱导的家兔血小板聚集作用比较

目的 观察银杏内酯注射液、银杏内酯ABC以及银杏叶提取物注射液（EGb761）对家兔血小板聚集功能、超微结构及PF-4和β-TG含量的影响比较。方法 将日本大耳兔分为对照（生理盐水）组、EGb761（1.02 mL/kg）组、银杏内酯ABC （5.13 mg/kg）组、银杏内酯注射液（1.02 mL/kg,以萜内酯计5.1 mg/kg）组、白果内酯（5.13 mg/kg）组,分别iv相应药物1周。给药结束后,家兔心脏取血,观察PAF诱导下血小板聚集率;电镜观察血小板超微结构;试剂盒法观察PAF诱导下血清中血小板因子-4（PF-4）和β-血小板球蛋白（β-TG）含量。结果 与对照组比较,EGb761组、银杏内酯ABC组、银杏内酯注射液组的血小板聚集率均显著降低（P<0.05、0.01）,白果内酯组无显著变化;血小板聚集抑制率：银杏内酯注射液（43.76%） > 银杏内酯ABC （35.3%） > EGb761（26.52%） > 白果内酯（5.48%）。与对照组比较,EGb761、银杏内酯ABC、银杏内酯注射液均能减少聚集型血小板数量,使树突型血小板突起变少变短。与对照组比较,银杏内酯注射液和银杏内酯ABC均使PF-4和β-TG表达显著降低（P<0.01）,EGb761仅显著降低β-TG表达（P<0.05）。结论 银杏内酯注射液通过PAF途径发挥抗血小板聚集作用,其作用强于银杏内酯ABC,可能是白果内酯发挥了协同增效作用。     

高效液相色谱法测定银杏内酯B滴丸的含量

目的建立银杏内酯B滴丸中银杏内酯B的含量测定方法。方法采用HPLC,选择Ec lipseXDB-C18(150mm×4.6mm,5μm)色谱柱,以甲醇-0.1%磷酸溶液(33.3∶66.7)为流动相;流速:1.0mL.m in-1;检测波长:225nm。结果银杏内酯B在0.2650~4.240mg.mL-1范围内线性关系良好(r=1.000);平均加样回收率为99.6%(RSD=1.3%)。结论此方法简便、快速、准确,可作为银杏内酯B滴丸的含量测定方法。     

粉防己碱对兔血小板聚集和血小板活化因子生成的影响(英文)

目的:探讨粉防己碱(Tet)对兔血小板聚集和PAF生成的影响.方法:卡西霉素(Cal)和PAF诱导血小板聚集的聚集率和Tet对血小板聚集的抑制率被测定;给予或未给予Tet处理之血小板用Cal刺激释放PAF的量也被测定.结果:在4—64 μmol·L~(-1)浓度范围,Tet明显抑制Cal和PAF诱导的血小板聚集.IC_(50)值分别为8.6μmol·L~(-1)和14.0μmol·L~(-1).Tet也浓度依赖性的抑制Cal诱导血小板释放PAF,IC_(50)值为21.0μmol·L~(-1).结论:Tet抑制血小板聚集作用与抑制内源性PAF生成有关.     

丹酚酸B镁盐对兔洗涤血小板聚集和5-HT释放反应的影响(英文)

AIM: To study the effects and mechanism of magnesium lithospermate B(MLB) on rabbit platelet aggregation and 5-HT release. METHODS: The platelet aggregation was determined by Born's method. Release of serotonin (5-HT) and formation of thromboxane A2 (TXA2) were measured by fluorophotometry and radioimmunoassay (RIA) respectively. Cytoplasmic free Ca2+ concentration ([Ca2+]i) in platelets was measured by Fura 2-AM fluorescence technique. RESULTS: In washed platelets, thrombin (200 U/L) or arachidonic acid (AA) (30 mumol/L)-induced aggregation was inhibited by MLB 50-800 mg/L in a concentration-dependent manner. In addition, MLB had more inhibitory effects on platelet aggregation in the absence of extracellular calcium with IC50 of 102 mg/L than in the presence of CaCl2 1 mmol/L with IC50 of 194 mg/L. MLB concentration-dependently decreased the thrombin-activated release of 5-HT, whereas it did not affect the formation of TXA2 in platelets. Furthermore, MLB not only inhibited the rise of [Ca2+]i in thrombin stimulated platelets, but decreased the [Ca2+]i in resting platelets. CONCLUSION: MLB inhibited the aggregation and 5-HT release in rabbit platelets and it is probably by attenuating intracellular calcium concentration.     

Correlation of platelet activating factor and age-related macular degeneration

Objective: To investigate the role of Platelet Activating Factor (PAF) in the pathogenesis and development of Age-Related Macular Degeneration (ARMD).

Research design and methods: Fifty six patients with ARMD (24 patients with dry ARMD and 32 patients with wet ARMD) and 25 age-matched control participants underwent ophthalmological examination, including visual acuity measurement and evaluation of the retina. The participants were classified into three groups according to their retinal status, based on indirect fundoscopy, Optical Coherence Tomography and fluorescein angiography findings. In order to evaluate the concentrations of PAF in serum, blood samples were collected from all participants and were analyzed with ELISA technique.

Results: The concentrations of PAF differed significantly according to macular lesions and were found to be lower in patients with ARMD than control participants.

Conclusions: PAF levels are decreased along with the severity of ARMD. Understanding the role of PAF in pathogenesis of ARMD could be the impetus for the development of new therapies field of treatment of ARMD or even other retinal diseases.     

全身广谱治疗仪对大鼠免疫器官cAMP和CA含量的影响

为探讨全身广谱治疗仪照射对大鼠免疫器官ｃＡＭＰ和ＣＡ的影响。采用全身广谱治疗仪照射７天后,用ＳＲＢＣＶ免疫Ｗｉｓｔａｒ大鼠。在免疫后４天和７天脾脏,胸腺和下丘脑ｃＡＭＰ含量均降低。而在免疫后４天脾脏去甲肾上腺素、肾上腺素均增高,提示：全身广谱治疗仪照射大鼠,使免疫器官儿茶酚胺释放增加,ｃＡＭＰ代谢降低,可能导致淋巴细胞增殖和免疫功能增强。     

蓝萼甲素对兔血小板生成血小板活化因子及花生四烯酸代谢的影响

蓝萼甲素在浓度0.1～100μmol·L~(-1)时可抑制A23187刺激的免血小板生成血小板活化因子,其IC_(50)为0.47μmol·L~(-1),当其浓度为10,100μmol·L~(-1)时能抑制花生四烯酸诱导的兔血小板血栓素A_2生成同时升高前列腺素E_2,推测蓝萼甲素通过抑制血小板活化因子生物合成及选择性抑制血栓素A_2生成而抑制血小板激活。     

Regulatory role of protein tyrosine phosphorylation in platelet activating factor induced signal transduction in platelets 1

目的：研究蛋白酪氨酸磷酸化（ＰＴＰ）在血小板激活因子（ＰＡＦ）诱导血小板信号传导中的作用．方法：用水洗兔血小板考查Ｇｅｎ抑制聚集及５羟色胺释放,Ｆｕｒａ２和ＢＣＥＣＦ负载测胞内钙及ｐＨ,特异性抗酪氨酸单抗及免疫印迹法检测ＰＴＰ．结果：Ｇｅｎ１００和２００μｍｏｌ·Ｌ－１分别抑制ＰＡＦ诱导的５羟色胺释放为２３７％±２０％及４１％±８％,对胞内钙增加和Ｎａ＋／Ｈ＋交换也有抑制作用．ＰＡＦ增加Ｍｒ为７００００,６００００,５００００,４２０００／４００００,３４０００的ＰＴＰ．Ｇｅｎ２００,４００μｍｏｌ·Ｌ－１明显抑制该效应．用Ｓｔａ２０ｎｍｏｌ·Ｌ－１,ＢＡＰＴＡ２００μｍｏｌ·Ｌ－１,依他酸２ｍｍｏｌ·Ｌ－１,分别阻断ＰＫＣ及胞内钙增加和内流,也减少ＰＴＰ形成．结论：ＰＴＰ参与ＰＡＦ诱导血小板信号传导途径,ＰＫＣ活化和胞内钙动员对ＰＴＰ有调节作用．     

The effect of platelet activating factor on nasal hypersensitivity

Summary Platelet activating factor (PAF) is known to have a wide range of biological activities. In the lower airways PAF has been suggested as the biochemical mediator partly responsible for the bronchial hyperreactivity which is a feature of asthma. In order to study whether PAF has a similar effect in the upper airways, we carried out a double blind, placebo-controlled cross-over study in twelve patients with strictly seasonal allergic rhinitis. The study was performed in pollen-free winter months.26 µg PAF or placebo was sprayed into each nasal cavity 8 h and 1 h before a nasal allergen challenge. The nasal response to PAF and the allergen challenge that followed was monitored by repeated measurements of nasal expiratory peak flow rate and symptom scores.PAF induced only minor changes in nasal patency and nasal symptoms as compared to placebo. However, pretreatment with PAF induced an increase in responsiveness of the nasal vasculature to the allergen challenge that followed. This was registered as a small, but statistically significant increase in the symptom scores for nasal blockage, from 1.7 (0.3; SEM) after placebo pretreatment to 2.4 (0.36; SEM) after PAF (p<0.05). A similar trend was also noted for the measurements of nasal peak flow. The other response parameters, sneezes and secretion, remained identical.These results suggest that PAF may play a role in human nasal hyperreactivity, but it appears that PAF is not a major mediator involved in the induction of this phenomenon.     

Triazolodiazepines are potent antagonists of platelet activating factor (PAF) in vitro and in vivo

Summary The triazolodiazepines brotizolam, triazolam and alprazolam inhibited PAF-induced human platelet aggregation in vitro (IC₅₀ = 0.54, 7.6 and 13.7 M, respectively) but showed only a weak or no effect against other aggregating agents (ADP, adrenaline, collagen, serotonin, arachidonic acid). In comparison, flunitrazepam and diazepam, two diazepines without the triazole ring, showed IC₅₀-values of 42 and 260 M, respectively. Flunitrazepam does not possess the specificity shown by the other compounds. Brotizolam and triazolam also inhibited PAF-induced human neutrophil aggregation in vitro, with IC₅₀-values 0.21 and 6.6 M, respectively.In anaesthetized guinea pigs, brotizolam (2.5 to 10 mg/kg p.o. or 0.1 to 0.5 mg/kg i.v.) or triazolam (20 to 100 mg/kg p.o.) inhibited dose-dependently the intrathoracic accumulation and aggregation of ¹¹¹Indium labelled platelets induced by an i. v. infusion of PAF (30 ng/kg × min).Brotizolam at doses of 1 to 10 mg/kg p. o. and 0.1 to 0.5 mg/kg i. v. inhibited dose-dependently the reduction in tidal volume (bronchoconstriction), the systemic hypotension and the lethal effect due to i. v. PAF in guinea pigs. Triazolam inhibited these effects of PAF at doses of 50 to 200 mg/kg p.o.PAF-induced systemic hypotension in rats can be reversed by cumulative i. v. doses (0.05 to 1.0 mg/kg) of brotizolam.In conclusion, these results show that triazolodiazepines, like brotizolam and triazolam, are potent inhibitors of PAF-induced effects in vitro and in vivo. Send offprint requests to J. Casals-Stenzel at the above address     

Platelet-released ADP stabilizes PAF-induced rabbit platelet aggregation by stabilizing intracellular calcium

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