大鼠星状神经节快兴奋性突触后电位变化与三七总皂甙的影响

背景:有研究显示,三七总皂甙能明显增强大鼠的学习与记忆能力,但其对外周神经系统的作用仍需进一步观察。目的:观察三七总皂甙对大鼠星状神经节快兴奋性突触后电位的作用。设计:观察对照实验。单位:广西医科大学药理学教研室。材料:实验于2005-01/2006-02在广西医科大学实验中心药理实验室完成。选用30只健康雄性清洁级SD大鼠,体质量(220±20)g,由广西医科大学实验动物中心提供。SEN-7203数字式三通道刺激器、MEZ8301型微电极放大器为日本NIHONKOHDEN公司产品,玻璃微电极拉制仪、微电极操纵仪均为日本Narishige公司产品。三七总皂甙(为云南昆明雅阁臣药业公司产品批号020802)氯化乙酰胆碱(Ach)为美国Sigma公司产品。方法:大鼠麻醉后迅速处死,打开胸壁,在显微镜下将星状神经节离体并将标本迅速移至灌流浴槽,剥去神经节外层结缔组织膜,用金属细针固定其边缘。用含体积分数0.95O2与体积分数0.05CO2混合气体和pH为(7.4±0.05)的Kreb's持续、恒温(34.0±0.5℃)灌流神经节,同时采用质量浓度范围0.08～0.16g/L三七总皂甙对神经节进行灌流和培养。①用内充3mmol/LKCl的玻璃微电极穿刺离体星状神经节神经元,记录细胞内突触后膜除极反应的幅度。②选用三七总皂甙可逆性抑制快兴奋性突触后电位的最高浓度0.16g/L直接灌流,观察三七总皂甙对外源性Ach(1mmol/L,1min)引起的突触后膜除极反应幅度和时程的影响。③选用三七总皂甙可逆性抑制快兴奋性突触后电位的最高浓度0.16g/L直接灌流,观察三七总皂甙对膜性质(膜电阻和膜电位)的影响。主要观察指标:①细胞内突触后膜除极反应的幅度。②最高浓度0.16g/L三七总皂甙对外源性Ach引起的突触后膜除极反应幅度和时程及对膜性质(膜电阻和膜电位)的影响。结果:纳入大鼠30只均进入结果分析。①三七总皂甙对快兴奋性突触后电位的影响:三七总皂甙在0.10～0.16g/L范围内可逆性抑制快兴奋性突触后电位,使快兴奋性突触后电位幅度减小,或使顺行动作电位变成快兴奋性突触后电位,三七总皂甙质量浓度越高,对快兴奋性突触后电位的抑制作用越明显。抑制作用多在三七总皂甙灌流3～10min内出现,0.16g/L三七总皂甙起效最快,通常在3～4min内就使快兴奋性突触后电位明显下降。停止三七总皂甙灌流后,用正常Kreb's冲洗15～20min可使快兴奋性突触后电位基本恢复至给药前的对照水平。②三七总皂甙对外源性Ach引起膜除极反应的影响:用0.16g/L三七总皂甙灌流前后Ach除极反应的幅度和时程分别为(15.5±2.4)mV,(256.1±21.5)s,与给三七总皂甙后无明显差异[(14.3±1.9)mV,(228.6±24.5)s,P>0.05]。③三七总皂甙对膜性质的影响:给药前后平均膜电位差异不明显[-(55.5±12.1),-(54.3±10.4)mV,P>0.05]。平均膜电阻亦无明显差异[(53.9±5.1),(55.1±4.8)MΩ,P>0.05]。结论:三七总皂甙对大鼠星状神经节快兴奋性突触后电位有可逆性抑制作用,抑制可能是通过突触前机制产生。

Effects of Panax notoginseng saponins on the fast-excitatory postsynaptic potential in rat stellate ganglion

BACKGROUND: Panax notoginseng saponins (PNS) could significantly improve the learning and memory ability of rats, but its influence to peripheral nervous system still needs further investigation.OBJECTIVE: To observe the effects of PNS on the fast-excitatory postsynaptic potential (f-EPSP) in stellate ganglion (SG) of rats.DESIGN: Observation and controlled trial.SETTING: Pharmacological Laboratory of Guangxi Medical University.MATERIALS: The experiment was carried out at the Pharmacological Laboratory of the Experimental Center of Guangxi Medical University from January 2005 to February 2006. Thirty healthy male SD rats of clean grade and (220±20) g, provided by the Experimental Animal Center of Guangxi Medical University; SEN-7203 digital three track strip stimulator, microelectrode amplifier (MEZ8301, Japan NIHON KOHDEN COMPANY); glass microelectrode puller, and microelectrode manipulator, both the products of Narishige Company, Japan; PNS, provided by Kunming Jacobson Pharmaceutical Co., Ltd, and acetylchloride chline (Ach), the product of Sigma, U.S.A.METHODS: After the animals were executed acutely, their chest wall was opened to isolate SG rapidly under microscope, which was transferred to the perfusion chamber, and fixed with wire needles after peeling the connective tissue membrane. The ganglia were perfused continuously with the mixture of volume fraction 0.95 O2 and 0.05 CO2 plus Krebs solution with pH (7.4±0.05). Meanwhile, 0.08-0.16 g/L PNS was employed to perfuse and culture SG.①The glass microelectrode filled with 3 mmol/L KCI was used to puncture the isolated SG and record the amplitude and duration of depolarizing reaction of postsynaptic membrane.②PNS with the maximum concentration of 0.16 g/L, which could inhibit the f-EPSPs, was perfused to observe the effect of PNS on the amplitude and duration of depolarizing reaction of postsynaptic membrane induced by exogenous Ach (1 mmol/L, 1 minute).③PNS with the maximum concentration of 0.16 g/L, which could inhibit the f-EPSPs, was perfused to observe the effect of PNS on membrane resistance and membrane potential.MAIN OUTCOME MEASURES:①Amplitude of depolarizing reaction of postsynaptic membrane; ②Effect of PNS on the amplitude and duration of depolarizing reaction of postsynaptic membrane induced by exogenous Ach, and membrane resistance and membrane potential.RESULTS: Thirty rats were involved in the result analysis. ①PNS ranged 0.08 to 0.16 g/L could reversibly depress the f-EPSPs amplitude of, or change the forward active potential into f-EPSP; the higher the concentration of PNS, the more obvious the inhibition was. The depression appeared in 3-10 minutes after PNS perfusion, and the effect reached the peak at 0.16 g/L; f-EPSP was decreased evidently in 3 to 4 minutes. The inhibition nearly recovered to the control level after washing the ganglia with Krebs solution for 15 to 20 minutes. ②Effect of PNS on exogenous ACh-induced depolarization: The amplitude and duration of the Ach-induced depolarization did not significantly change before and 5 minutes after 0.16 g/L PNS perfusion [before: (15.5±2.4) mV, (256.1±21.5) seconds; after: (14.3±1.9) mV, (228.6±24.5) seconds, P＞0.05].③Effects of PNS on membrane potential and membrane resistance: The mean membrane potential and membrane resistance were not significantly changed after PNS perfusion [before:-(55.5±12.1) mV, (53.9±5.1) MΩ; after: -(54.3±10.4) mV, (55.1±4.8)MΩ, P＞0.05].CONCLUSION: PNS could reversibly depress the fast-excitatory postsynaptic potential in stellate ganglion of rats by presynaptic mechanism.