栝楼桂枝颗粒抗缺血性脑卒中大鼠神经元及原代海马神经元凋亡研究

Anti-apoptosis Effect of Gualou Guizhi Granule on Neurons in Ischemic Stroke Rats and Primary Hippocampal Neurons

目的:观察栝楼桂枝颗粒(GLGZG)抗缺血性脑卒中大鼠神经元及原代海马神经元凋亡的作用,探讨其作用机制,为其治疗脑卒中提供依据。方法:建立谷氨酸诱导原代海马神经元兴奋性毒性损伤模型、大鼠脑缺血再灌注损伤模型,采用MTT、LDH法测定GLGZG对原代海马神经元细胞活性和LDH活性的影响,采用Western Blot、Real-time PCR检测GLGZG对原代海马神经元及大鼠海马组织中Caspase-3、Bcl-2、Bax m RNA及蛋白表达水平的影响。结果:用不同浓度(100μg/m L、200μg/m L、300μg/m L)的GLGZG分别作用于原代海马神经元24 h,3个剂量组细胞存活率较谷氨酸组细胞存活率升高,其中GLGZG 200μg/m L、300μg/m L 2组细胞活力显著升高(P<0.05,P<0.01);3个剂量组LDH活力较谷氨酸组显著降低,其中GLGZG 200μg/m L、300μg/m L 2组可显著抑制谷氨酸损伤神经元LDH的释放(P<0.05,P<0.01)。GLGZG 200μg/m L、300μg/m L 2组干预后,与谷氨酸组比较可显著降低原代海马神经元Caspase-3 m RNA、Bax m RNA的表达(P<0.05,P<0.01),升高Bcl-2 m RNA的表达(P<0.05),蛋白表达的趋势与m RNA相似;GLGZG组与模型组比较,可降低大鼠海马组织Caspase-3、Bax蛋白的表达(P<0.05或P<0.01),升高Bcl-2蛋白的表达(P<0.01)。结论:GLGZG对缺血性脑卒中大鼠及原代海马神经元有一定的保护作用,其保护作用与其抗细胞凋亡作用有关。

Objective:To investigate the anti-apoptosis effect of Gualou Guizhi granule(GLGZG) on neurons in ischemic stroke rats and primary hippocampal neurons, and then explore the involved protective mechanism of GLGZG, thus providing a theoretical basis for the treatment of stroke. Methods:Glutamate-induced primary hippocampal neurons excitotoxicity injury model and focal cerebral ischemic-reperfusion injury model were established. MTT and LDH assay were used to detect the effect of GLGZG on primary hippocampal neurons and LDH activity. At the same time, Western Blot and Real-time PCR were used to detect the effect of GLGZG on gene expressions of Caspase-3, Bcl-2 and Bax m RNA in primary hippocampal neurons as well as the protein expressions in hippocampus tissues of rats. Results:The hippocampal neuron cells were treated with different concentrations of GLGZG(100μg/m L, 200 μg/m L, 300 μg/m L) for 24 hours, the cell viability increased(P<0.05,P<0.01) and the LDH activity significantly reduced(P<0.05,P<0.01) in the above three GLGZG groups in comparison with that in the glutamate group. The cell viability were significantly improved, while the release of glutamate-induced injured neurons LDH could be significantly suppressed in the group at a dose of 200 μg/m L and 300 μg/m L(P<0.05,P<0.01). After the treatment with GLGZG, the down-regulated expression of Caspase-3, Bax m RNA level(P<0.05,P<0.01) and up-regulated expression of Bcl-2 m RNA(P<0.05) were observed in primary hippocampal neurons in the above three GLGZG grous in comparison with that in the glutamate group. In addition, the protein expression is similar with m RNA, the down-regulated expression of Caspase-3, Bax protein(P<0.05,P<0.01), and up-regulated expression of Bcl-2 protein(P<0.01) were observed in hippocampal tissues of rats. Conclusion:GLGZG has protective effects on neurons in ischemic stroke rats and primary hippocampal neurons and the potential mechanism of GLGZG may be related to its anti-apototic function.