川芎嗪对谷氨酸损伤海马神经元保护作用的研究
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摘要
海马是大脑中非常重要的结构,尤其是对脑的学习记忆功能。海马的损伤会导致瞬时记忆出现功能障碍,主要影响的是新的记忆形成而对以往的记忆影响不大。大脑缺氧时,海马是最易受损的器官之一。癫痫主要的病灶也是位于海马,慢性癫痫发作可损伤海马。在阿尔茨海默病、脑炎等疾病中海马也极易受到损伤。谷氨酸是大脑神经元释放的一种兴奋性神经递质,谷氨酸的过多释放可以引起神经元的过度兴奋,造成神经元损伤或者死亡。脑缺血、阿尔茨海默病、亨廷顿病等疾病的一个共同特点是谷氨酸产生的过度兴奋,这可能损伤到海马神经元。中药是治疗中枢神经系统疾病行之有效的方法之一,川芎嗪可以治疗某些中枢神经疾病如脑缺血,其机制有可能是通过拮抗谷氨酸的兴奋性毒性来发挥作用的,尚需进一步去验证。因此,本实验以体外培养海马神经细胞为研究对象,来探讨中药治疗神经系统疾病可能的作用和机制。
目的观察川芎嗪(Ligustrazine)对谷氨酸(Glu)损伤原代培养海马神经元生长的影响及促进神经元功能恢复的保护作用。
方法原代培养新生大鼠海马神经元,通过加入1mmol/L谷氨酸制作神经元损伤模型组;培养基中加入10μmol/L的盐酸川芎嗪溶液共同孵育12h后,加入1mmol/L谷氨酸损伤海马神经元20min,作为川芎嗪保护组。分别观察各组神经元生长过程中形态变化;采用MTT法检测细胞活性;测定培养液中乳酸脱氢酶(LDH )活力的变化;细胞免疫组化检测各组表达乙酰胆碱酯酶(AchE)情况。
结果川芎嗪能有效的提高原代培养的海马神经元的存活率,其存活率相对对照组提高了28.78%(P<0.01);川芎嗪的最适宜浓度是10μmol/L。以1mmol/L谷氨酸作用于培养的成熟海马神经元,细胞形态表现为突起减少,折光性下降;MMT显示细胞的存活率下降;应用10μmol/L川芎嗪后可改善因谷氨酸引起的神经元形态的改变,可显著提高谷氨酸损伤后海马神经元的活性(P<0.01),降低因谷氨酸损伤引起的细胞内LDH的过度释放(P<0.01);细胞AchE免疫组化结果:川芎嗪保护组的灰度值明显低于谷氨酸损伤组(P<0.01),与正常对照组无显著性差异(P>0.05)。
结论(1)川芎嗪对正常培养的海马神经元生长有促进作用,发现在一定浓度范围内可明显提高体外培养神经元的存活率,其最佳浓度是10μmol/L。(2)川芎嗪可明显拮抗谷氨酸兴奋性神经毒性作用,可改善因谷氨酸引起的细胞死亡,提高细胞的存活率。其可能的机制与川芎嗪可以稳定细胞膜,促进细胞膜完整,阻断谷氨酸的进一步损伤有关。(3)川芎嗪可拮抗因谷氨酸毒性作用引起的神经元功能受损,可改善神经元合成和分泌乙酰胆碱酯酶,改善损伤引起的神经元功能下降,对抗谷氨酸毒性引起的胆碱能神经元的死亡。
The hippocampus is critical for the formation of new autobiographical and fact memories. It may be function as a memory "gateway" through which new memories must pass before entering permanent storage in the brain. Hippocampal damage can result in anterograde amnesia: loss of ability to form new memories, although older memories may be safe. The hippocampus is especially sensitive to global reductions in oxygen level in the body. The hippocampus is also a common focus site in epilepsy, and can be damaged through chronic seizures. It is also sometimes damaged in diseases such as herpes encephalitis, and is one of the first brain areas to show damage in Alzheimer's disease. Glutamate is a powerful excitatory neurotransmitter which it is released by nerve cells in the brain. In many cases, cells activated by glutamate become overexcited. This overexcitation can lead to effects that can cause cell damage and/or death. The damage of glutamic overexcitation is a common course of many diseases such as cerebral ischemia, AD, HD, et al. The traditional medicine, Chinese herb is one of the most effective methods to treat the central nerve system disease, and the herb of Ligustrazine may be beneficial to the injury of neurons induced by the exitotoxicity of glutamate which is one of the key role in many disease courses.
Objective To observe the protective effect of ligustrazine on glutamate-induced injury in culture hippocampal neurons.
Methods Primarily cultured hippocampal neurons from newly born rat were incubated with Ligustrazine (10μmol/L) for 12 hours, then glutamate (1mmol/L) was added for 20 minutes to induce injury. Cell viability was detected by MTT assay, and the vigor of LDH was determined by biochemistry method. In the end, the protein of AchE in the cultured neurons was performed according to the protocol of immuocytochemistry.
Results After the pretreatment with the ligustrazine for 12 hours, the survival rate of cultured hippocampal neurons was increased 28.78% compared to the normal group(P<0.01), and the most suitable concentration of Ligustrazine is 10μmol/L. The neural processes were shorten and the refragibility of cultured cells desecened when neurons were injuryed by 1mmol/L glutamate. When the injury neurons induced by glutamate were treated by Ligustrazine the morphous of those cells were improved, survival rate of neurons was increased(P<0.01), and the ratio of LDH leakage from the injury kytoplasm was decreased(P<0.01). The expression of the AchE in the injury neurons was promoted by the pretreatment of the Ligustrazine.
Conclusion (1) Ligustrazine could significantly increase the survival rate of primary cultured hippocampal neuron and improve its growth. The most effective concentration is 10μmol/L. (2)The exitotoxicity of glutamate could be diluted by Ligustrazine, and the possible mechanism of the protective effection might involve in improving the stability and integrity of cytomembrane and obstructing the injury by glutamte. (3) Ligustrazine could inhibit the neural functional damage, improve the expression of protein AchE and decrease the neural death when the neural cells induced the injury with the glutamate.
目的观察川芎嗪(Ligustrazine)对谷氨酸(Glu)损伤原代培养海马神经元生长的影响及促进神经元功能恢复的保护作用。
方法原代培养新生大鼠海马神经元,通过加入1mmol/L谷氨酸制作神经元损伤模型组;培养基中加入10μmol/L的盐酸川芎嗪溶液共同孵育12h后,加入1mmol/L谷氨酸损伤海马神经元20min,作为川芎嗪保护组。分别观察各组神经元生长过程中形态变化;采用MTT法检测细胞活性;测定培养液中乳酸脱氢酶(LDH )活力的变化;细胞免疫组化检测各组表达乙酰胆碱酯酶(AchE)情况。
结果川芎嗪能有效的提高原代培养的海马神经元的存活率,其存活率相对对照组提高了28.78%(P<0.01);川芎嗪的最适宜浓度是10μmol/L。以1mmol/L谷氨酸作用于培养的成熟海马神经元,细胞形态表现为突起减少,折光性下降;MMT显示细胞的存活率下降;应用10μmol/L川芎嗪后可改善因谷氨酸引起的神经元形态的改变,可显著提高谷氨酸损伤后海马神经元的活性(P<0.01),降低因谷氨酸损伤引起的细胞内LDH的过度释放(P<0.01);细胞AchE免疫组化结果:川芎嗪保护组的灰度值明显低于谷氨酸损伤组(P<0.01),与正常对照组无显著性差异(P>0.05)。
结论(1)川芎嗪对正常培养的海马神经元生长有促进作用,发现在一定浓度范围内可明显提高体外培养神经元的存活率,其最佳浓度是10μmol/L。(2)川芎嗪可明显拮抗谷氨酸兴奋性神经毒性作用,可改善因谷氨酸引起的细胞死亡,提高细胞的存活率。其可能的机制与川芎嗪可以稳定细胞膜,促进细胞膜完整,阻断谷氨酸的进一步损伤有关。(3)川芎嗪可拮抗因谷氨酸毒性作用引起的神经元功能受损,可改善神经元合成和分泌乙酰胆碱酯酶,改善损伤引起的神经元功能下降,对抗谷氨酸毒性引起的胆碱能神经元的死亡。
The hippocampus is critical for the formation of new autobiographical and fact memories. It may be function as a memory "gateway" through which new memories must pass before entering permanent storage in the brain. Hippocampal damage can result in anterograde amnesia: loss of ability to form new memories, although older memories may be safe. The hippocampus is especially sensitive to global reductions in oxygen level in the body. The hippocampus is also a common focus site in epilepsy, and can be damaged through chronic seizures. It is also sometimes damaged in diseases such as herpes encephalitis, and is one of the first brain areas to show damage in Alzheimer's disease. Glutamate is a powerful excitatory neurotransmitter which it is released by nerve cells in the brain. In many cases, cells activated by glutamate become overexcited. This overexcitation can lead to effects that can cause cell damage and/or death. The damage of glutamic overexcitation is a common course of many diseases such as cerebral ischemia, AD, HD, et al. The traditional medicine, Chinese herb is one of the most effective methods to treat the central nerve system disease, and the herb of Ligustrazine may be beneficial to the injury of neurons induced by the exitotoxicity of glutamate which is one of the key role in many disease courses.
Objective To observe the protective effect of ligustrazine on glutamate-induced injury in culture hippocampal neurons.
Methods Primarily cultured hippocampal neurons from newly born rat were incubated with Ligustrazine (10μmol/L) for 12 hours, then glutamate (1mmol/L) was added for 20 minutes to induce injury. Cell viability was detected by MTT assay, and the vigor of LDH was determined by biochemistry method. In the end, the protein of AchE in the cultured neurons was performed according to the protocol of immuocytochemistry.
Results After the pretreatment with the ligustrazine for 12 hours, the survival rate of cultured hippocampal neurons was increased 28.78% compared to the normal group(P<0.01), and the most suitable concentration of Ligustrazine is 10μmol/L. The neural processes were shorten and the refragibility of cultured cells desecened when neurons were injuryed by 1mmol/L glutamate. When the injury neurons induced by glutamate were treated by Ligustrazine the morphous of those cells were improved, survival rate of neurons was increased(P<0.01), and the ratio of LDH leakage from the injury kytoplasm was decreased(P<0.01). The expression of the AchE in the injury neurons was promoted by the pretreatment of the Ligustrazine.
Conclusion (1) Ligustrazine could significantly increase the survival rate of primary cultured hippocampal neuron and improve its growth. The most effective concentration is 10μmol/L. (2)The exitotoxicity of glutamate could be diluted by Ligustrazine, and the possible mechanism of the protective effection might involve in improving the stability and integrity of cytomembrane and obstructing the injury by glutamte. (3) Ligustrazine could inhibit the neural functional damage, improve the expression of protein AchE and decrease the neural death when the neural cells induced the injury with the glutamate.
引文
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