【作者】 郑桃；

【导师】 彭成；

【作者基本信息】 成都中医药大学 ， 药理学， 2011， 硕士

【摘要】 目的：研究乌头碱对大鼠大脑皮质神经细胞的最小毒性浓度和毒作用机制,并探索人参皂苷Rg1对乌头碱神经毒性是否具有拮抗作用。方法：采用均匀设计及MTT比色法研究不同浓度乌头碱作用不同时间对神经细胞存活情况的影响,从而确定乌头碱对神经细胞的最小毒性浓度和临界作用时间点；采用MTT法测定乌头碱与人参皂苷Rgl以不同比例配伍混合使用对神经细胞存活情况的影响；采用光学显微镜和电子显微镜观察乌头碱、人参皂苷Rg1及两者配伍对细胞形态的影响；采用细胞酶学方法、比浊法、酶联免疫吸附法等方法测定乌头碱、人参皂苷Rg1及两者配伍对细胞生物膜、内环境、能量代谢及部分神经递质的影响,从而揭示乌头碱对神经细胞的毒性作用机制,并探索人参皂苷Rg1对乌头碱神经毒性是否有拮抗作用。结果：乌头碱对神经细胞的最小毒性浓度为0.2%,临界作用时间点为0.5min、5min和30min。0.2%乌头碱作用于神经细胞30min后,可使细胞突起明显断裂、减少,胞体胀大,细胞膜不完整,胞浆出现大量空泡,细胞器大量丢失,细胞核固缩,核内染色质出现严重的积聚、边集现象；同时培养介质中LDH活力增高,细胞内ACP活力及MDA含量升高,SOD活力降低；细胞内[K+]、[Ca2+]及Na+,K+-ATP酶活力升高,[Na+]、[Mg2+]及Ca2+,Mg2+-ATP酶活力降低；细胞内糖原含量及细胞色素C氧化酶活力降低；神经递质中谷氨酸及乙酰胆碱24h释放量降低,Y-氨基丁酸、儿茶酚胺、P物质和阿片肽释放量升高。乌头碱与人参皂苷Rg1以不同比例配伍合用后对乌头碱所致上述改变具有拮抗作用。结论：0.2%乌头碱作用30min对神经细胞有显著毒性,其对细胞形态及细胞内多种细胞器有直接的损伤,并可导致细胞生物膜的损伤、内环境紊乱、能量代谢障碍以及神经递质分泌异常。乌头碱对神经细胞的毒性作用机制可能为：乌头碱损伤生物膜,代偿性增强Na+,K+-ATP酶活力,细胞内外离子浓度改变,导致能量代谢障碍、生物电位及电信号的改变,同时还导致神经递质释放异常；同时乌头碱降低Ca2+,Mg2+-ATP酶活力,导致细胞内钙超载,从而造成细胞形态和功能的损伤以及神经递质释放量的改变。多种病理情况相互影响,又导致损伤进一步加剧,最终在机体上表现为各种中毒症状。人参皂苷Rg1对乌头碱神经毒性具有拮抗作用,以两者1：1配伍应用效果最佳,其作用机制可能与保护细胞生物膜、抗氧化、抑制细胞内钙超载、改善能量代谢并调节神经递质的释放等有关。更多还原

【Abstract】 Objective:To research the minimum toxic concentration and the toxic mechanism of aconitine on cortex neuron cells, and whether the Ginsenoside Rgl can antagonise toxicity of aconitine or not.Methods:Uniform design and MTT method were used to study how aconitine of different concentrations influence on neuron cells at different time, and to find out the minimum toxic concentration and the critical role of time; the MTT method was used to determine how the mixture of aconitine and Ginsenoside Rgl in different proportions infect the survival of nerve cell; optical microscopy and electron microscopy were used to observe the influence on cell morphology by aconitine Ginsenoside Rgl and the mixture; enzyme method, turbidimetric method and ELISA were used to determine the influence on cell membrane, internal enviroment, energy metabolism and some of neurotransmitters, and to reveal the toxic mechanism of aconitine on nerve cells, then to explore whether the Ginsenoside Rgl can antagonise toxicity of aconitine or not.Results:The minimum toxic concentration of aconitine on nerve cell was 0.2%, and the critical role of the times effect on nerve cells were 0.5min,5min and 30min. After 30min,0.2% aconitine lead to the obvious broken and reduction of neurites, swelling of the cell body, incomplete of the membrane, emerging a large number of cytoplasmic vacuoles, loss of organelles, nuclear condensation, serious accumulation and margination of nuclear chromatin. The activity of LDH in culture medium was increased. In the cells, the activity of ACP, Na+, K+-ATPase and the contents of MDA, [K+] and [Ca2+] were increased, the activity of SOD and Ca2+,Mg2+-ATPase and the contents of [Na+] and [Mg2+] were decreased. The content of intracellular glycogen and the activity of COX were reduced. Meanwhile, the release of Glu and Ach within 24h was decreased, and the release of y-GABA, CA, SP and OP was increased. The mixture of aconitine and Ginsenoside Rgl in different proportions could lighten the changes caused by aconitine.Conclusions:0.2% aconitine had significant toxicity. It directly damaged the cell morphology and cell organelles, and also could cause damage of cell membrane, disturbance of internal enviroment, obstacle of energy metabolism and abnormal secretion of neurotransmitters. The toxicity mechanism of aconitine on nerve cells probably conducted in following way:aconitine damaged the biomembrane, and compensatory increased the activity of Na+, K+-ATPase. Ion concentrations between the inside and outside of cells were changed, which lead to obstacle of energy metabolism, changes of bioelectric potential and electrical signal, and abnormal release of neurotransmitters. Meanwhile, aconitine reduced the activity of Ca2+,Mg2+-ATPase, which lead to overload of intracellular calcium. As a result, cell morphology and function were damaged, and release of neurotransmitters was changed. Interaction of a variety of pathological conditions has led to aggravated injury, and ultimately manifested in the body for a variety of symptoms of poisoning.Ginsenoside Rgl could antagonise the toxicity of aconitine, the antagonism was probably to protect the cell membrane, inhibit the overload of intracellular calcium, improve the energy metabolization and regulated the release of neurotransmitters. Besides, the mixture of the two in equal proportion can gain the best effect.更多还原