银杏苦内酯B对麻醉大鼠中枢神经元放电活动的影响

【作者】 林悦；

【导师】 武宇明；

【作者基本信息】 河北医科大学 ， 生理学， 2008， 硕士

【摘要】 银杏（Ginkgo biloba）在地球上已存在约2亿年,曾广泛分布于北半球的欧、亚、美洲,素有裸子植物“活化石”之称。其种子作为“白果”入药已有600多年的历史。银杏苦内酯（ginkgolides）是银杏提取物中的一种活性成分,包括银杏苦内酯A、B、C、M、J等,均为强血小板活化因子（Platelet activating factor, PAF）拮抗剂。其中银杏苦内酯B（Ginkgolide B,GB）活性最强,迄今对银杏苦内酯B的药理作用研究也最为集中。银杏苦内酯竞争性拮抗PAF与其受体的结合,从而使PAF不能通过G蛋白转导激活磷脂酶C、腺甘酸环化酶和酪氨酸蛋白激酶,从而阻断了PAF受体的信号转导,阻断了PAF的生物学效应。银杏苦内酯对中枢神经系统多种疾病有一定的防治作用。保护脑缺血、减少再灌注损伤,抗阿尔茨海默病（AD）,抑制神经细胞凋亡等诸多方面都表明银杏苦内酯在治疗中枢神经系统疾病方面有着光明的前景。关于银杏内酯B的中枢保护作用机制,特别是电生理不甚明确。海马结构简单,易于分离,且与心血管中枢相关核团有纤维联系,是神经电生理研究常用部位。下丘脑室旁核（paraventricular nucleus, PVN）是非常重要的心血管活动调控整合部位,接受来自海马的穹隆纤维。目前,有关银杏内酯B对大鼠中枢神经元电生理特性的影响尚未见报道。因此本论文对以上两部位进行了研究。Ⅰ银杏苦内酯B对大鼠海马CA1区神经元自发放电的影响目的:研究银杏苦内酯B（Ginkgolide B, BN52021）对静息状态下的海马脑片神经元活动的影响。方法:实验选用20±3天的雄性Sprague-Dawley大鼠。将大鼠快速断头,剥去颅骨和硬脑膜,取出脑组织,置于0-4℃预先用95% O₂和5% CO₂的混合气体饱和的人工脑脊液中,快速分离出一侧海马,切成厚度为350-500μm的脑片。然后将脑片移入人工脑脊液中,连续通以95% O₂和5% CO₂,室温下孵育60-90 min。应用细胞外记录单位放电技术,观察银杏苦内酯B对大鼠海马脑片神经元放电的影响。结果:（1）在43个CA1区神经元放电单位给予银杏苦内酯B（0.1,1,10μmol/L）2分钟,有42个放电单位（97.67%）放电频率明显降低,且呈剂量依赖性;（2）预先用0.2 mmol/L的L-glutamate （L-Glu）灌流海马脑片, 10个放电单位放电频率明显增加,表现为癫痫样放电,在此基础上灌流银杏苦内酯B（1μmol/L）2分钟,其癫痫样放电全部被抑制;（3）预先用L型钙通道开放剂Bay K 8644灌流8个海马脑片,8个单位（100%）全部放电增加,在此基础上灌流银杏苦内酯B（1μmol/L）2分钟,7个放电单位（87.5%）放电频率明显减低;（4）在8个CA1区神经元放电单位上,银杏苦内酯B（1μmol/L）的抑制效应可被广泛钾通道阻断剂（tetraethylammonium, TEA）1 mmol/L完全阻断。结论:银杏苦内酯B （Ginkgolide B, BN52021）可抑制海马神经元自发放电,并可抑制由L-glutamate诱发的神经元放电。提示银杏苦内酯B对中枢神经元通过降低其活动而具有一定程度的保护作用,这种作用可能与银杏苦内酯B抑制L型钙通道有关,而且可能与延迟整流型钾通道（delayed rectifier potassium channel, KDR）有关。Ⅱ银杏苦内酯B对大鼠下丘脑室旁核神经元自发放电的影响目的:研究银杏苦内酯B（Ginkgolide B, BN52021）对静息状态下的下丘脑脑片室旁核神经元活动的影响。方法:实验选用20±3天的雄性Sprague-Dawley大鼠。将大鼠快速断头,剥去颅骨和硬脑膜,取出脑组织,置于0-4℃预先用95% O₂和5% CO₂的混合气体饱和的人工脑脊液中,根据解剖学标志切得一块含有室旁核的下丘脑脑片,厚度为350-500μm,并置入人工脑脊液中,连续通以95% O₂和5% CO₂,室温下孵育60-90 min。应用细胞外记录单位放电技术,观察银杏苦内酯B对大鼠室旁核神经元放电活动的影响。结果:（1）在27个下丘脑室旁核神经元放电单位给予银杏苦内酯B（0.1,1,10μmol/L）2分钟,有26个放电单位（96.30%）放电频率明显降低,且呈剂量依赖性;（2）预先用0.2 mmol/L的L-glutamate （L-Glu）灌流下丘脑脑片, 8个放电单位放电频率明显增加,表现为癫痫样放电,在此基础上灌流银杏苦内酯B（1μmol/L）2分钟,其癫痫样放电全部被抑制;（3）预先用L型钙通道开放剂Bay K 8644灌流8个下丘脑脑片,8个单位（100%）全部放电增加,在此基础上灌流银杏苦内酯B（1μmol/L）2分钟,8个放电单位（100%）放电频率明显减低;（4）在8个下丘脑室旁核神经元放电单位上,银杏苦内酯B（1μmol/L）的抑制效应可被广泛钾通道阻断剂（tetraethylammonium, TEA）1 mmol/L完全阻断。结论:银杏苦内酯B （Ginkgolide B, BN52021）可抑制下丘脑室旁核神经元自发放电,并可抑制由L-glutamate诱发的神经元放电。提示银杏苦内酯B对心血管中枢神经元通过降低其活动而具有一定程度的保护作用,这种作用可能与银杏苦内酯B抑制L型钙通道有关,而且可能与延迟整流型钾通道（delayed rectifier potassium channel, KDR）有关。更多还原

【Abstract】 Background: Ginkgolide B is one of the major constituents of the terpenoid fraction of Ginkgo biloba extract (GbE). Previous investigations suggested that ginkgolide B is a potent platelet-activating factor receptor antagonist, which is also regarded as having neuroprotective effects in the CNS. Recent evidence suggests that GbE protects against neuronal death in global brain ischemia and in glutamate-induced excitotoxicity. The mechanisms underlying ginkgolide B’s beneficial effects on central neurons activity still need to be clarified as yet and the effect of ginkgolide B on spontaneous discharge of hippocampal neurons has not been reported.Aim: To examine the effects of Ginkgolide B (BN52021) on the discharges of neurons in CA1 area of hippocampal slices.Methods: Using extracellular recording technique.Results: (1) In response to the application of ginkgolide B (0.1, 1, 10μmol/L; n=43) into the perfusate for 2 min, the spontaneous discharge rates (SDR) of 42/43 (97.67%) neurons were significantly decreased in a dose-dependent manner. (2) Pretreatment with L-glutamate (L-Glu, 0.2mmol/L) led to a marked increase in the SDR of all 10 (100%) neurons in an epileptiform pattern. The increased discharges were suppressed significantly after ginkgolide B (1μmol/L) was applied into the perfusate for 2 min. (3) In 8 neurons, perfusion of the selective L-type calcium channel agonist, Bay K 8644 (0.1μmol/L), induced a significant increase in the discharge rate of 8/8 (100%) neurons. Ginkgolide B (1μmol/L) applied into the perfusate inhibited the discharges of 7/8 (87.5%) slices. (4) In 8 neurons, the broad potassium channels blocker, tetraethylammonium (TEA, 1mmol/L) completely blocked the inhibitory effect of ginkgolide B (1μmol/L).Conclusion: These results suggest that ginkgolide B can inhibit the electrical activity of CA1 neurons. The inhibitory effect may be related to the blockade of L-type voltage-activated calcium channel and may be concerned with delayed rectifier potassium channel (KDR),which indicated that ginkgolide B play a protective role on the central neurons. Background: Ginkgolide B is one of the major constituents of the terpenoid fraction of Ginkgo biloba extract (GbE). Previous investigations suggested that ginkgolide B is a potent platelet-activating factor receptor antagonist, which is also regarded as having protective effects in cardiovascular system and CNS. Recent evidence suggests that GbE protects against neuronal death in global brain ischemia and in glutamate-induced excitotoxicity. The mechanisms underlying ginkgolide B’s beneficial effects on central neurons activity still need to be clarified as yet.Aim: To study the central role of Ginkgolide B (BN52021) in regulating cardiovascular function of nervous center by examining the effects of GST on the electrical activity of rat paraventricular nucleus (PVN) neurons in hypothalamic slice preparation and to elucidate the mechanism involved.Methods: Using extracellular single-unit discharge recording technique.Results: (1) In response to the application of ginkgolide B (0.1, 1, 10μmol/L; n=27) into the perfusate for 2 min, the spontaneous discharge rates (SDR) of 26/27 (96.30%) neurons were significantly decreased in a dose-dependent manner. (2) Pretreatment with L-glutamate (L-Glu, 0.2mmol/L) led to a marked increase in the SDR of all 8 (100%) neurons in an epileptiform pattern. The increased discharges were suppressed significantly after ginkgolide B (1μmol/L) was applied into the perfusate for 2 min. (3) In 8 neurons, perfusion of the selective L-type calcium channel agonist, Bay K 8644 (0.1μmol/L), induced a significant increase in the discharge rate of 8/8 (100%) neurons. Ginkgolide B (1μmol/L) applied into the perfusate inhibited the discharges of 8/8 (100%) slices. (4) In 8 neurons, the broad potassium channels blocker, tetraethylammonium (TEA, 1mmol/L) completely blocked the inhibitory effect of ginkgolide B (1μmol/L).Conclusion: These results suggest that ginkgolide B can inhibit the electrical activity of paraventricular neurons. The inhibitory effect may be related to the blockade of L-type voltage-activated calcium channel and may be concerned with delayed rectifier potassium channel (KDR).更多还原