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

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B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0.26 μmol·L^-1；1 mg·L^-1 LPS和1 nmol·L^-1 PAF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-κB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。     

阿的平预防中暑的研究

本研究旨在观察预先给予阿的平对大鼠中暑模型的保护作用，同时采用体外培养的神经元热损伤模型探讨阿的平的作用机制。清醒大鼠分别灌胃给予阿的平4.5，9.0和18 mg·kg^-1，1 h后进入(41.0±0.5) ℃的水循环热仓，持续观察动物直肠温度直至死亡，直肠温度和动物存活时间分析表明,预先给予阿的平能够延缓热环境中大鼠的体温快速增加，推迟动物死亡。在细胞水平，原代培养的大鼠纹状体神经元在43.0 ℃培养条件1 h造成细胞热损伤模型，阿的平处理组预先在培养上清液中加入20 μmol·L^-1阿的平，1 h后接受热环境处理。采用荧光分光光度法测定细胞膜流动性，细胞裂解后采用［³H］标记花生四烯酸作为底物测定细胞内磷脂酶A₂活性，采用气相色谱-质谱联用的方法测定细胞内活性脂肪酸的水平。结果表明热环境导致纹状体神经元细胞膜流动性下降，细胞内磷脂酶A₂活性升高，细胞内花生四烯酸浓度升高，而阿的平稳定细胞膜、抑制细胞内磷脂酶A₂活性以及减少花生四烯酸及其代谢产物的释放，可能部分解释阿的平延缓中暑和对抗热环境损伤的机制。     

人参皂甙Rb1降低细胞内Ca2+作用的机制

使用荧光探针Fura-2/AM,采用双波长荧光分光光度法观察到,人参皂甙Rb₁(10,50,100μmol·L^-1)能剂量依赖性减少新生鼠脑细胞内钙浓度,并能增加由硫酸亚铁及半胱氨酸所降低的膜流动性,Rb₁(10μmol·L^-1)能使离体大鼠尾动脉去甲肾上腺素量—效曲线右移,最大效应降低;Rb₁(10,100μmol·L^-1)能降低离体鼠基底动脉5-HT所引起的收缩。使用全细胞膜片钳技术发现人参皂甙Rb₁(50,100μmol·L^-1)对钙电流无明显影响;Rb₁在低剂量能增加大鼠突触体Na⁺-K⁺ATPase及Ca²⁺-Mg²⁺ATPase活性。从而揭示Rb₁降低胞内钙含量可能通过增加ATP酶活性而产生。     

蚓激酶的心肌保护作用及机制

目的研究蚓激酶对心肌缺血的保护作用，并进一步探讨其可能机制。方法采用结扎大鼠左冠状动脉前降支制备急性心肌缺血模型，观察蚓激酶对心肌缺血的保护作用；应用全细胞膜片钳和激光扫描共聚焦技术，研究蚓激酶对L-型钙电流(I_Ca-L)和细胞内游离钙离子浓度的影响。结果蚓激酶80，40和20 mg·kg^-1剂量组均可缩小心肌梗死面积。膜片钳研究结果表明，当刺激电压为+10 mV时，10和50 μmol·L^-1蚓激酶使I_Ca-L降低共聚焦结果显示，在静息状态下，10 μmol·L^-1蚓激酶对［Ca²⁺］_i无明显影响；但10 μmol·L^-1蚓激酶对60 mmol·L^-1 KCl诱导的［Ca²⁺］_i升高却有明显抑制作用，并且在整个实验过程中(240 s)并未出现明显的峰值。结论蚓激酶对大鼠心肌缺血具有保护作用，其机制可能与抑制I_Ca-L及下调［Ca²⁺］_i有关。     

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。     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

粉防己碱、颅通定对兔窦房结动作电位及豚鼠心室肌单细胞钙电流的协同作用

采用标准微电极技术和膜片钳记录技术,分别研究了粉防己碱(Tet)和颅通定(rotundine)对家兔窦房结动作电位及豚鼠心室肌单细胞钙电流的影响。结果表明:Tet1～200μmol·L^-1,rotundine3～300μmol·L^-1能浓度依赖性地降低兔窦房结动作电位的APA,Vmax和SP₄,延长SCL。Tet0.3μmol·L^-1与rotundine1μmol·L^-1合用亦有作用。在膜片钳实验中Tet0.1μmol·L^-1和rotundine1μmol·L-1合用能有效抑制豚鼠心室肌单细胞钙电流,抑制率为19.2%。提示,两种植物源性抗钙剂有良好的协同效果。     

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。}     

前胡丙素对培养大鼠心肌细胞内游离Ca2+的影响

用Fura-2/AM技术直接观察前胡丙素(Pra-C)对培养大鼠心室肌细胞内游离钙的影响。结果显示Pra-C浓度为0.1～1.0μmol·L^-1可明显抑制CaCl₂,高K⁺和Bay K 8644引起[Ca²⁺]i增加,并且有剂量—效应关系,对ouabain引起的[Ca²⁺]i增加无明显作用。结果提示Pra-C降低心肌细胞[Ca²⁺]i的作用与抑制电压依赖性钙通道有关。     

杨梅素对血小板活化因子拮抗的作用

目的研究杨梅素对血小板活化因子（PAF）的拮抗作用。方法以放射配体结合试验观察［³H］PAF与家兔血小板受体特异性结合的作用；以分光光度法测定PAF诱发血小板粘附的强度；以Fura-2荧光分光光度法测定兔多形核白细胞（PMNs）内钙离子浓度。结果杨梅素可浓度依赖地抑制［³H］PAF与血小板受体的特异性结合，其IC₅₀分别为 34.8，85.7和118.6 μmol·L^-1；该药可明显抑制PAF诱发的血小板粘附及PMNs内游离钙浓度的升高，且呈明显的量效关系，其抑制血小板粘附的IC₅₀为 13.1 μmol·L^-1。结论杨梅素具有抗PAF作用，为一新的PAF受体拮抗剂。     

PAF and LTB4 biosynthesis in the human neutrophil: effects of putative inhibitors of phospholipase A2 and specific inhibitors of 5-lipoxygenase.

The effect of several putative phospholipase A2 (PLA2) inhibitors on [3H]-acetate incorporation into platelet-activating factor (PAF) upon calcium ionophore A23187 stimulation of purified human neutrophils (PMN) were evaluated in vitro. PLA2 inhibitors such as p-bromophenacyl bromide (pBPB), ellagic acid, aristolochic acid and gossypol were without effect or only weakly inhibited PAF biosynthesis. Luffariellolide, a potent PLA2 inhibitor isolated from the marine sponge Luffariella sp., dose-dependently inhibited PAF production (IC50 = 5 microM). Due to the relationship between PAF and LTB4 biosynthesis, the effect of inhibiting LTB4 production on PAF biosynthesis was investigated. At concentrations which reduce LTB4 production by greater than 95%, Wy-50,295 tromethamine and A-64,077, specific 5-lipoxygenase (5-LO) inhibitors, did not significantly effect PAF production. In contrast, L-663,536, the 5-LO translocation inhibitor, was a potent inhibitor of PAF production (IC50 = 1 microM). This activity of L-663,536 may contribute to its pharmacological profile at higher doses. These data also suggest that PAF biosynthesis in human PMNs is not dependent on the formation or continued presence of leukotrienes.     

蛋白酪氨酸磷酸化在血小板激活因子诱导血小板信号传导中的调节 …

AIM: To study the role of protein tyrosine phosphorylation (PTP) in platelet activating factor (PAF)-induced platelet signal transduction cascade. METHODS: Washed rabbit platelets were used to test the inhibitory effect of genistein (Gen) on platelet aggregation and serotonin secretion. Intracellular Ca2+ ([Ca2+]i) and pH (pHi) were measured by a dual wavelength fluorophotometer with Fura 2-AM and BCECF-AM. PTP was determined with a specific anti-phosphotyrosine monoclonal antibody by Western blotting. RESULTS: Pretreatment with Gen (100 and 200 mumol.L-1) inhibited PAF (20 nmol.L-1)-stimulated platelet serotonin release by 23.7% +/- 2.0% and 41% +/- 8%, respectively. Similar inhibitory effects of Gen were observed on PAF-evoked increase of [Ca2+]i and intracellular alkalization. PAF also elicited a pronounced increase in PTP of several bands with M(r) 70,000, 60,000, 50,000, 42,000/40,000, and 34,000, which were suppressed markedly by Gen 200 and 400 mumol.L-1. Pretreatment with staurosporine (Sta) 20 nmol.L-1, BAPTA 200 mumol.L-1, and egtazic acid 2 mmol.L-1 to inhibit PKC activation, [Ca2+]i elevation, and Ca2+ influx respectively, also showed an inhibitory effects on the formation of PTP. CONCLUSION: PTP is involved in multiple signal transduction pathways induced by PAF, on which PKC activation and calcium mobilization play a regulatory role.     

Mechanisms of the influence of magnolol on eicosanoid metabolism in neutrophils

We have demonstrated that magnolol suppressed thromboxane B2 (TXB2) and leukotriene B4 (LTB4) formation in A23187-stimulated rat neutrophils. Maximum inhibition was obtained with about 10 microM magnolol. Magnolol was more effective in the inhibition of cyclooxygenase (COX) activity than in the inhibition of 5-lipoxygenase (5-LO) activity as assessed by means of enzyme activity determination in vitro and COX and 5-LO metabolic capacity analyses in vivo. Magnolol alone stimulated cytosolic phospholipase A2 (cPLA2) phosphorylation and the translocation of 5-LO and cPLA2 to the membrane, and evoked arachidonic acid (AA) release. Recruitment of both 5-LO and cPLA2 to the membranes was suppressed by EGTA. Arachidonyl trifluoromethyl ketone (AACOCF3), a PLA2 inhibitor, bromoenol lactone (BEL), a Ca2+-independent PLA2 (iPLA2) inhibitor, and EGTA suppressed the magnolol-induced AA release. However, none of the follows affected magnolol-induced AA-release: 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), a MAPK kinase (MEK) inhibitor, or 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X), a protein kinase C (PKC) inhibitor. In addition, magnolol at 30 microM did not stimulate the p38 MAPK and extracellular signal-regulated kinase 2 (ERK2) enzyme activities. These results indicated that magnolol inhibits the formation of prostaglandins and leukotrienes in A23187-stimulated rat neutrophils, probably through a direct blockade of COX and 5-LO activities. The stimulatory effects of magnolol at high concentration on the membrane association of 5-LO and cPLA2 are attributable to the elevation of [Ca2+]i, and on the AA release is likely via activation of cPLA2 and iPLA2.     

血小板活化因子诱导血小板P—选择素表达及相关信号传导机制

AIM: To study the intracellular signal transduction mechanisms of platelet activating factor (PAF)-induced platelet P-selectin expression. METHODS: Human blood platelets were used to test the effect of PAF-induced P-selectin expression using flow cytometry. RESULTS: PAF 20 nmol.L-1 elicited a moderate upregulation of P-selectin expression [(47.5 +/- 1.3)% vs control (3.8 +/- 0.9)%, P < 0.01]. Pretreatment with egtazic acid (EGTA) 2 mmol.L-1 and 5,5'- dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetracetic acid (BAPTA) 200 mumol.L-1 to block Ca2+ influx or chelate the intracellular calcium, respectively, reduced P-selectin expression in response to PAF [(13.3 +/- 0.9)% and (16.8 +/- 1.9)% vs (47.5 +/- 1.3)% of PAF group, P < 0.01]. Inhibition of Na+/H+ exchange with amiloride (Ami) 400 mumol.L-1 resulted in an inhibition of P-selectin expression [(37.5 +/- 2.1)% vs (47.5 +/- 1.3)% of PAF group, P < 0.01]. Genistein (Gen) 300 mumol.L-1 to inhibit protein tyrosine phosphorylation showed similar effect [(29 +/- 4)% vs (47.5 +/- 1.3)% of PAF group, P < 0.01]. CONCLUSION: Multiple signal transduction pathways, including protein tyrosine phosphorylation, Na+/H+ exchange, and Ca2+ mobilization, mediated PAF-induced P-selectin expression.     

普鲁托品对兔血小板内钙的影响

AIM: To study the influence of protopine (Pro) on the cytoplasmic free Ca2+ concentration ([Ca2+]i) in rabbit platelets. METHODS: Measurement of [Ca2+]i of platelets in vitro by Fura 2-AM fluorescence technique. RESULTS: In the presence of CaCl2 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 attenuated the rise in [Ca2+]i evoked by ADP from (420 +/- 57) to (320 +/- 26), (264 +/- 21), and (180 +/- 14) nmol.L-1, respectively, by arachidonic acid (AA) from (280 +/- 36) to (210 +/- 17), (184 +/- 21), and (143 +/- 16) nmol.L-1, respectively, and by platelet-activating factor (PAF) from (350 +/- 42) to (282 +/- 31), (223 +/- 30), and (165 +/- 15) nmol.L-1, respectively. In the presence of egtazic acid 1 mmol.L-1, Pro 10, 20, and 40 mumol.L-1 reduced the Ca2+ release induced by ADP, AA, and PAF, respectively. Pro 10, 20, and 40 mumol.L-1 also decreased ADP-, AA-, and PAF-induced Ca2+ influx. CONCLUSION: Pro inhibited not only Ca2+ release but also the influx of Ca2+.     

TMB-8对去甲肾上腺素和BHQ引起新生大鼠单个脑细胞内游离钙增高的影响

应用AR-CM-MIC阳离子测定系统,研究TMB-8对体外新生SD大鼠单个脑细胞内游离钙的抑制作用及其机制。结果表明,在无细胞外钙情况下,静息[Ca²⁺]i为79±13nmol·L^-1。TMB-810,30μmol·L^-1能明显降低静息[Ca²⁺]i。TMB-8100μmol·L^-1对高钾去极化引起的[Ca²⁺]i显著增高无明显影响。在细胞外钙为1.3mmol·L^-1时,去甲肾上腺素诱导的细胞内[Ca²⁺]i升高可部分被TMB-8抑制;TMB-8(30μmol·L^-1)对BHQ引起的[Ca²⁺]i的升高无明显抑制作用。而当细胞外液[Ca²⁺]i为0时,TMB-8几乎完全抑制了去甲肾上腺素和BHQ的作用。提示TMB-8降低脑细胞内游离钙的作用机制是通过促使细胞内钙进入肌浆网以抑制内钙的释放,并通过饱和肌浆网内Ca²⁺间接地阻滞细胞膜钙通道。     

丁基苯酞对大鼠血栓形成及血小板功能的影响

目的研究消旋、左旋和右旋丁基苯酞(dl-,l-和d-NBP)对血栓形成及血小板功能的影响。方法利用半体外血栓形成术及比浊法,观察dl-,l-和d-NBP及阿司匹林(Asp)对大鼠血栓湿重和血小板聚集率的影响,并用放免法、荧光分光光度法测定其对血小板内cAMP和TXB2的水平以及血小板5-HT释放率的影响。结果ip,dl-NBP和l-NBP可剂量依赖性地抑制大鼠血栓形成,且l-NBP作用与Asp相似,d-NBP对半体外血栓形成无显著作用;dl-,d-和l-NBP可显著抑制胶原、ADP、花生四烯酸诱导的血小板聚集。结论NBP有抗血栓作用,l-NBP作用最强,dl-NBP作用较弱,其抗栓作用与升高血小板内cAMP的含量及抑制5-HT释放有关。