血小板激活因子拮抗剂WEB2086对小鼠腹腔巨噬细胞释放肿瘤坏死因子的影响(英文)

小鼠ip3%TG 4天后取其腹腔巨噬细胞,血小板激活因子拮抗剂WEB 2086对LPS诱导的巨噬细胞释放肿瘤坏死因子(TNF)有显著的抑制作用。时效研究表明,TNF的产生和WEB 2086的抑制作用从LPS刺激后4 h开始,持续到22 h,在16 h时达到高峰。本文还首次采用一种用放线菌素D和NaF处理的L-929细胞测定TNF的新方法,此法与另外3种生物测定方法进行比较的结果显示此法具有更敏感、方便的特点。     

12味止血中药对脂多糖诱导小鼠巨噬细胞产生一氧化碳的抑制作用

目的研究12味止血中药对脂多糖(LPS)诱导小鼠巨噬细胞产生一氧化氮(NO)的抑制作用,并探讨其止血机制.方法以1μg·mL-1 LPS刺激小鼠巨噬细胞RAW 264.7,22h后以Griess法测定NO的终产物亚硝酸盐的含量,观察LPS对NO生成的影响.结果12味止血药均可抑制LPS诱导小鼠巨噬细胞RAW 264.7中NO的生成.三七、地榆、仙鹤草、槐花、艾叶、蒲黄、侧柏叶及白茅根的抑制作用显著(P＜0.05).结论本实验为12味止血中药对脂多糖诱导小鼠巨噬细胞产生NO的抑制作用提供了一定的理论依据.     

雷公藤氯内酯醇对小鼠肺泡巨噬细胞体外炎性反应的影响(英文)

目的:探讨雷公藤单体雷公藤氯内酯醇(tripcholorolide,T4)对肺泡巨噬细胞(AM)炎症反应的影响及机制.方法:小鼠AM受脂多糖(LPS)10mg/L刺激的同时,加入T4 500 μg/L或地塞米松 100μmol/L;ELISA法测定上清液中TNFα、IL-1β、IL-6及IL-10浓度:RT-PCR检测上述因子及iNOS基因mRNA的表达.结果:AM受10 mg/L LPS刺激24小时后,上清液中TNFα、IL-1β、IL-6、IL-10及NO释放均明显增加.T4 500 μg/L及地塞米松100μmol/L对上述介质均有不同程度的抑制作用.LPS刺激5小时后,AM中TNFα、IL-6、IL-10和iNOS的mRNA表达均明显增加.T4和地塞米松对上述介质的mRNA表达均有明显抑制作用.另外,T4对TNFα、IL-6、IL-10 mRNA的稳定性无明显影响.结论:T4具有抑制AM中促炎介质和抗炎介质表达的作用.     

12味止血中药对脂多糖诱导小鼠巨噬细胞产生一氧化氮的抑制作用

目的:研究12味止血中药对脂多糖(LPS)诱导小鼠巨噬细胞产生一氧化氮(NO)的抑制作用,并探讨其止血机制。方法:以1μg.mL-1LPS刺激小鼠巨噬细胞RAW264.7,22h后以Griess法测定NO的终产物亚硝酸盐的含量,观察LPS对NO生成的影响。结果:12味止血药均可抑制LPS诱导小鼠巨噬细胞RAW264.7中NO的生成。三七、地榆、仙鹤草、槐花、艾叶、蒲黄、侧柏叶及白茅根的抑制作用显著(P<0.05)。结论:本实验为12味止血中药对脂多糖诱导小鼠巨噬细胞产生NO的抑制作用提供了一定的理论依据。     

可溶性晚期糖基化终末产物受体对脂多糖肺损伤小鼠肺内细胞因子的干预作用

目的探讨应用重组可溶性晚期糖基化终末产物受体(sRAGE)对脂多糖(LPS)介导的急性肺损伤(ALI)小鼠肺内细胞因子水平的影响。方法健康雄性BALB/C小鼠随机分为磷酸盐缓冲液(PBS)组、LPS组和sRAGE组。LPS组和sRAGE组通过气管内滴注LPS(3mg/kg体质量)建立ALI模型,造模后1hsRAGE组腹腔注射100μg重组小鼠sRAGE,各组于24h留取标本,采用Bio-Plex悬浮芯片技术检测支气管肺泡灌洗液(BALF)中8种细胞因子含量,并计数炎症细胞数量和蛋白(TP)含量,对肺组织进行病理学评估。观察sRAGE干预对造模后4h肺组织核因子(NF)-κB P65DNA结合活性的影响。结果 LPS滴注24h后,BALF中8种细胞因子含量均显著升高,白细胞(WBC)总数和中性粒细胞(NEU)数量显著增加,TP含量升高,肺组织出现典型的ALI病理损害。sRAGE干预显著降低了BALF中肿瘤坏死因子(TNF)-α、巨噬细胞炎症蛋白(MIP)-1β和MIP-1α水平,减少了BALF中WBC、NEU数量及TP含量,减轻了LPS引起的肺组织病理改变,并对造模后4hLPS介导的肺内NF-κB活化有抑制作用。结论应用重组sRAGE阻止RAGE信号能调控LPS介导的肺内细胞因子的表达,这构成了sRAGE抑制肺内炎症的重要机制之一。     

多粘菌素B对大鼠内毒素血症血浆中内毒素和肿瘤坏死因子-α水平的影响

目的 :观察多粘菌素B(PMX B)对大鼠内毒素血症血浆中内毒素 (LPS)和肿瘤坏死因子 α(TNF α)水平的影响。方法 :实验分为 :LPS PMX B组 ;LPS 生理盐水 (NS)组 ;LPS模型组。给大鼠一次LPS 0 5mg·kg-1,iv ,前 2组分别于注入LPS后 0 5h给予PMX B 0 2mg·kg-1和 1mLNS ,iv ,LPS模型组大鼠于注入LPS后不作其他处理。在给LPS后 0 5 ,1,2 ,4h抽血 ,用鲎试剂检测血浆中LPS ,ELISA法测定TNF α水平。结果 :LPS PMX B组 ,血浆LPS ,TNF α水平明显下降 ,平均动脉压 (MAP)于 2 ,4h明显高于LPS NS组和LPS模型组。结论 :PML B治疗能明显降低血浆中LPS ,TNF α水平。     

NAC和地塞米松对脂多糖诱导大鼠肺泡巨噬细胞p38蛋白激酶的影响

目的　观察脂多糖 (LPS)诱导肺泡巨噬细胞 (AM )p38蛋白激酶活化及抗炎药物地塞米松 (DEX)和N 乙酰半胱氨酸 (NAC)对其影响。方法　分离培养大鼠肺泡巨噬细胞 ,设正常对照组、LPS刺激组、DEX或NAC干预组 ,共 4组。分别采用Western印迹和放射免疫分析法检测AM核提取物 p38蛋白激酶和细胞培养上清TNF α、IL 8含量。 结果　LPS刺激组核蛋白提取物p38蛋白激酶和细胞培养上清TNF α、IL 8含量较正常对照组升高 (P <0 0 1 )。DEX组和NAC组虽较正常对照组高 ,但均低于LPS刺激组 (P <0 0 1 )。核提取物 p38蛋白激酶和细胞培养上清TNF α、IL 8含量之间分别呈正相关 (r =0 754、0 62 5 ,P <0 0 1 )。结论　LPS诱导肺泡巨噬细胞 p38蛋白激酶活化 ,进而导致TNF α、IL 8表达增多 ;DEX和NAC可能通过抑制 p38活化而减少炎性介质TNF α、IL 8的释放     

四环素抑制活化的内皮细胞表达粘附分子

目的　研究四环素对肿瘤坏死因子 α(TNF α)或细菌内毒素 (LPS)活化的内皮细胞表达粘附分子E 选择素和细胞间粘附分子 1(ICAM 1)的影响 ,为探讨四环素类药物的抗炎作用提供新的实验依据。方法　用TNF α(0 5～ 5 0 μg·L-1)或LPS(0 5～ 5 0mg·L-1)孵育培养的人脐静脉内皮细胞使其活化 ;用四环素 (10 -6～ 10 -4 mol·L-1)预孵育内皮细胞 1h再与TNF α(1μg·L-1)或LPS(5mg·L-1)共同孵育4h或 18h ;应用细胞 酶联免疫吸附分析方法检测E 选择素和ICAM 1的表达。结果　TNF α或LPS孵育内皮细胞 4h明显诱导E 选择素的表达 ,TNF α或LPS孵育内皮细胞 18h明显增加ICAM 1的构成表达 (P <0 0 1) ;用四环素处理后则明显抑制TNF α或LPS诱导的E 选择素和ICAM 1的表达 (P <0 0 1) ,并呈现剂量依赖性。结论　四环素可抑制TNF α或LPS活化的人脐静脉内皮细胞表达粘附分子E 选择素和细胞间粘附分子 1。     

银杏内酯B对脂多糖刺激的小鼠腹腔巨噬细胞TNFα生成及大鼠胸腔多形核白细胞NF-кB活化的影响

目的研究银杏内酯B对脂多糖刺激的小鼠腹腔巨噬细胞TNFα生成及大鼠胸腔多形核白细胞NF-κB活化的影响.方法用L929细胞结晶紫染色法检测TNFα的含量,用电泳迁移率改变检测法检测NF-κB的结合活性.结果1和10 μmol·L-1银杏内酯R能够显著抑制LPS刺激的小鼠腹腔巨噬细胞TNFα的生成,其IC50为0.26μmol·L-1:1 mg·L-1LPS和1 nmol·L-1PAF均可活化大鼠胸腔多形核白细胞NF-κB;银杏内酯B能够抑制LPS或PAF刺激的NF-κR活化.结论银杏内酯B能够抑制LPS刺激的小鼠腹腔巨噬细胞TNFα生成及大鼠胸腔多形核白细胞NF-κB的活化.PAF参与LPS激活NF-κB的过程.     

卡巴胆碱对内毒素刺激大鼠腹腔巨噬细胞炎症细胞因子表达的影响

目的:研究拟胆碱药卡巴胆碱在体外对内毒素(LPS)刺激大鼠腹腔巨噬细胞表达炎症细胞因子的作用.方法:采集雄性Wistar大鼠腹腔巨噬细胞,经卡巴胆碱、烟碱或毒蕈碱预处理后,再给予LPS刺激,4 h后取细胞培养上清液,ELISA法检测TNF-α、IL-6和IL-10水平.结果:LPS刺激后TNF-α、IL-6和IL-10表达均显著增加.卡巴胆碱与烟碱能明显抑制TNF-α、IL-6表达的增加(P<0.01),卡巴胆碱的抑制作用有明显的量效关系,毒蕈碱的抑制作用则明显低于卡巴胆碱和烟碱(P<0.01).卡巴胆碱和烟碱对LPS刺激后的IL-10水平升高均无明显影响.结论:卡巴胆碱在体外LPS刺激腹腔巨噬细胞炎症模型中能抑制促炎细胞因子表达的增加而对抗炎细胞因子表达无明显作用,其作用与烟碱类似.     

Role for intracellular platelet-activating factor in the circulatory failure in a model of gram-positive shock.

1. This study investigates the effects of two structurally different antagonists of platelet-activating factor (PAF), BN52021 and WEB2086, on the circulatory and renal failure elicited by lipoteichoic acid (LTA) from Staphylococcus aureus (an organism without endotoxin) in anaesthetized rats. 2. Administration of LTA (10 mg kg-1, i.v.) caused hypotension and vascular hyporeactivity to noradrenaline (1 microgram kg-1, i.v.) WEB2086 (5 mg kg-1, i.v., 20 min before and 150 min after LTA) inhibited the delayed fall in mean arterial blood pressure (at 300 min: 99 +/- 6 mmHg vs. 75 +/- 6 mmHg, P < 0.01) and prevented the decrease in pressor response to noradrenaline (at 300 min: 36 +/- 5 mmHg min vs. 17 +/- 5 mmHg min, P < 0.01). Surprisingly, BN52021 (20 mg kg-1, i.v., 20 min before and 150 min after LTA) neither prevented the hypotension (74 +/- 6 mmHg) nor the vascular hyporeactivity (21 +/- 5 mmHg min). However, BN52021 inhibited the hypotension to injections of PAF as well as the circulatory failure elicited by lipopolysaccharides (10 mg kg-1, i.v.). 3. LTA caused an increase in plasma concentration of creatinine from 39 +/- 5 microM (sham-operated) to 70 +/- 8 microM and urea from 4.7 +/- 0.1 to 13.1 +/- 1.6 mM. The renal failure elicited by LTA was significantly inhibited by WEB2086 (creatinine: 45 +/- 4 microM and urea: 5.7 +/- 0.7 mM), but not by BN52021. 4. The induction of nitric oxide synthase activity in lungs by LTA was attenuated by WEB2086 from 98 +/- 17 to 40 +/- 15 pmol L-citrulline 30 min-1 mg-1 protein (P < 0.01), but not by BN52021 (148 +/- 21 pmol L-citrulline 30 min-1 mg-1 protein). Similarly, WEB2086, but not BN52021, inhibited the increase in plasma nitrite concentration associated with the delayed circulatory failure caused by LTA. The release of tumour necrosis factor-alpha (TNF-alpha) after injection of LTA was not attenuated by WEB2086. 5. The induction of nitrite release by cultured macrophages activated with LTA (10 micrograms ml-1 for 24 h) was inhibited by 74 +/- 4% by WEB2086 (3 x 10(-4) M), but not by BN52021, indicating that only WEB2086 acts on intracellular PAF receptors. 6. Thus, the intracellular release of PAF contributes to the circulatory and renal failure and induction of nitric oxide synthase elicited by LTA in anaesthetized rats. The difference between the two structurally different PAF antagonists in our septic shock models using either LTA or lipopolysaccharide (LPS), shows the importance of models for Gram-positive sepsis in the elucidation of the pathophysiology of septic shock and for the evaluation of potential drugs.     

Inhibition of platelet-activating factor fails to limit ischemia and reperfusion-induced myocardial damage.

To determine the role of platelet-activating factor (1-O-hexa-decyl-2-acetyl-sn-glyceryl-phosphoryl-choline, PAF) in myocardial ischemic and reperfusion-induced injury, the effects of a PAF receptor antagonist (WEB 2086) were studied in an anesthetized canine model of ischemia (90 min) and reperfusion (6 h). Thirty minutes after onset of ischemia, WEB 2086 was administered as a bolus (20 mg/kg intravenously, i.v.) followed by a continuous 6-h infusion (10 mg/kg/h i.v.). Controls received vehicle alone (0.9% saline). Platelet aggregation was studied at baseline and at 1, 2, 4, and 6 h of drug administration and at the end of the reperfusion period. WEB 2086 treatment did not significantly affect platelet aggregation stimulated by ADP or arachidonic acid (AA). After 1 h of drug infusion, the ex vivo aggregatory response to exogenous (200 nM) PAF was ablated in WEB 2086-treated animals. WEB 2086 administration did not affect heart rate (HR) or mean arterial blood pressure (MAP) during the occlusion or reperfusion phases. During reperfusion of the ischemic tissue, left circumflex coronary artery (LCX) blood flow of WEB 2086-treated animals increased (p < 0.05) above control value. The area of the left ventricle at risk of infarct was not different between control and WEB 2086-treated groups. Infarct size was not significantly reduced in WEB 2086-treated animals. The results of our investigation using a 90-min ischemic period followed by 6-h reperfusion show that pharmacologic antagonism of PAF by WEB 2086 does not protect the heart against ischemia and reperfusion-induced injury.     

Partial agonist effect of the platelet-activating factor receptor antagonists, WEB 2086 and WEB 2170, in the rat perfused heart.

1. WEB 2086 and WEB 2170 are potent platelet-activating factor (PAF) receptor antagonists and have been used widely as pharmacological tools to investigate the actions of PAF in a variety of biological systems. 2. Low concentrations of WEB 2086 and WEB 2170 blocked the vasoconstrictor action of PAF in the rat perfused heart. In this study, we observed that moderate concentrations of WEB 2086 and WEB 2170 increased the perfusion pressure in rat isolated hearts under constant flow perfusion. The vasoconstrictor actions of WEB 2086 and WEB 2170 were not observed with a structurally different PAF receptor antagonist, FR-900452. 3. To determine whether this vasoconstrictor action of WEB 2086 involved non-specific effects or was via the activation of PAF receptors, hearts were pretreated with 1000 pmol PAF or 50 microM FR-900452. These pretreatments attenuated the vasoconstrictor action of 1 microM WEB 2086, suggesting that the action of WEB 2086 may be mediated via PAF receptors. Pretreatment with the leukotriene receptor antagonist (L-649,923, 5 microM) and the leukotriene synthesis inhibitor (MK-886, 10 microM) that are known to block the vasoconstrictor action of PAF receptor activation also attenuated the vasoconstrictor action of WEB 2086. Pretreatment with PAF or MK-886 attenuated the vasoconstrictor action of 0.5 microM WEB 2170. 4. When PAF receptors were activated by PAF in the perfused heart, significant amounts of leukotriene C4 and leukotriene C4/D4/E4 were detected in the coronary effluent. However, no significant amount of these leukotrienes was detected in the coronary effluent when hearts were perfused with 1 microM WEB 2086 or 0.5 microM WEB 2170.(ABSTRACT TRUNCATED AT 250 WORDS)     

蝙蝠葛碱和粉防己碱对[3H]WEB 2086与体外牛脑前动脉平滑肌细胞结合的影响(英文)

用标记的血小板活化因子拮抗剂[³H]WEB 2086,在培养的牛脑前动脉平滑肌细胞上鉴定了血小板活化因子受体。结果表明在25℃时该细胞上存在两种与配基具有不同亲和力的受体结合位点,其中K_d-1=22.8±5.0 nmol·L^-1,K_d-2=186+20.5 nmol·L^-1;B_max-1=2.1±0.3 pmol/10⁴细胞,B_max-2=12.1±1-5 pmol/10⁶细胞。蝙蝠葛碱和粉防己碱均能抑制[³H]WEB2086与上述细胞的结合。     

Platelet activating factor as a proinflammatory mediator in acetic-induced colitis in the rat.

Platelet activating factor (PAF) induces neutrophilia and produces a variety of gastrointestinal lesions. The role of PAF as a proinflammatory mediator was examined by measuring the production of PAF and the efficacy of the PAF antagonists WEB 2086 and Ro 24-0238 in acetic acid (HOAc)-induced colitis. PAF levels within the colonic mucosa, as measured by radioimmunoassay, increased from 259 +/- 119 ng/mg in control tissue, to 616 +/- 266 ng/mg in HOAc inflamed tissue. The accumulation of neutrophils within the mucosa was decreased by 53 +/- 10% by pretreatment with 3 mg/kg WEB 2086, and by 43 +/- 11% by 3 mg/kg Ro 24-0238. PAF-induced neutrophilia had no effect on the severity of HOAc-induced colitis, therefore, PAF my be involved in sustaining the chronic inflammation of colitis.     

Protective effect of WEB 2086, a novel antagonist of platelet activating factor, in endotoxin shock

WEB 2086, a novel specific platelet activating factor (PAF) antagonist derived from triazolodiazepines, inhibited in a dose-related manner the hypotensive and lethal effect of PAF as well as of E. coli endotoxin in the rat. The hypotension induced by endotoxin (15 mg/kg i.v.) or PAF (30 ng/(kg X min) i.v.) in anaesthetized rats was prevented by oral (1-10 mg/kg) and inhibited or reversed by i.v. (0.1-5.0 mg/kg or 0.1-1.0 mg/kg) doses of WEB 2086. Similar oral and i.v. doses of WEB 2086 protected conscious rats from PAF (15 micrograms/kg i.v.)- and endotoxin (7.5 mg/kg i.v.)-induced death. The results obtained with WEB 2086 confirm that PAF has an important role in the pathophysiology of endotoxin shock. This compound may have a therapeutic effect in human septic shock.     

Lipopolysaccharide activates nuclear factor kappaB in rat intestine: role of endogenous platelet-activating factor and tumour necrosis factor

1. We examined the effect of lipopolysaccharide (LPS), a cell wall constituent of Gram negative bacteria, on nuclear factor kappaB (NF-kappaB) activation in the intestine and the roles of endogenous platelet-activating factor (PAF), tumour necrosis factor-alpha (TNF) and neutrophils. We also compared the time course of NF-kappaB activation in response to PAF and LPS. 2. Ileal nuclear extracts from LPS (8 mg kg(-1), IV)-injected rats were assayed for NF-kappaB-DNA-binding activity and identification of the subunits. Some rats were pretreated with WEB2170 (a PAF receptor antagonist), anti-TNF antibody, or anti-neutrophil antiserum. NF-kappaB p65 was localized by immunohistochemistry. An additional group was challenged with PAF (2 microg kg(-1), IV) for comparison. 3. LPS activates intestinal NF-kappaB, both as p50-p50 and p50-p65 dimers within 15 min, and the effect peaks at 2 h. The effect is slower and more sustained than that of PAF, which peaks at 30 min. Activated NF-kappaB was immunolocalized within epithelial and lamina propria cells. LPS effect was reduced by 41, 37 and 44%, respectively, in animals pretreated with WEB2170, anti-TNF antibody, or anti-neutrophil antiserum (P<0.05). 4. LPS activates intestinal NF-kappaB in vivo and neutrophil activation is involved in its action. The LPS effect is mediated by both endogenous PAF and TNF.     

Long-lasting inhibitory activity of the hetrazepinic BN 50730 on exudation and cellular alterations evoked by PAF and LPS.

1. Inhibitory effects of the hetrazepinic derivative BN 50730 on the rat pleural inflammatory response, triggered by PAF or lipopolysaccharides (LPS), were examined. The type of pharmacological blockade exerted by this antagonist in in vitro assays of eosinophil chemotaxis and platelet aggregation were also investigated. 2. Intrathoracic injection of PAF (1 microgram per cavity) caused a 4 fold increase in the extravasated protein within 15 min and led to a marked eosinophil accumulation 24 h post-challenge. BN 50730 (0.5-10 micrograms per cavity) inhibited exudation by PAF dose-dependently without modifying the response induced by histamine, bradykinin or 5-hydroxytryptamine (5-HT). 3. The kinetics of the inhibitory effect on exudation revealed that the actions of WEB 2086 and BN 52021 (10 micrograms per cavity) were over within 2 and 4 h respectively, whereas BN 50730 (10 micrograms per cavity) retained 80% of its inhibitory activity for 4 days. 4. Oral treatment with BN 50730 (10-20 mg kg-1, 1 h beforehand) suppressed the leucocyte accumulation and late eosinophilia observed 6 and 24 h after PAF respectively, but did not modify the eosinophilia induced by leukotriene B4 (LTB4) or bradykinin. BN 50730 also failed to reduce the eosinophil accumulation induced by LPS but drastically inhibited the neutrophil influx. 5. The pre-incubation of rat peritoneal eosinophils for 10 min with BN 50730 (30 nM-1 microM) dose-dependently inhibited the chemotaxis induced by PAF (0.1 microM) in vitro. The IC50 values for BN 52021, WEB 2086 and BN 50730 in this system were 5, 5 and 0.05 microM respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

苦参碱对脂多糖/痤疮丙酸杆菌诱导的小鼠肝炎及产生肿瘤坏死因子的影响

用小鼠致死性肝炎模型和TNF体外诱生的方法,研究苦参碱(Mat)对脂多糖(lipopolysaccharides,LPS)诱导的经痤疮丙酸杆菌(propionibacterittm acnes,PA)预刺激的小鼠产生肿瘤坏死因子(TNF)以及致死性肝炎的影响。结果表明:Mat(10,50mg·kg^-1,ip,bid×3d)可降低血清TNF和ALT水平及小鼠对LPS致死毒性的敏感性,并可在体外抑制LPS诱导的经PA预刺激的小鼠腹腔巨噬细胞释放TNF。提示Mat的保肝作用与其抑制TNF释放有关。