硫酸镁对大鼠脑弥漫性轴索损伤海马区Bcl-2和Bax蛋白表达影响的研究

The effect of magnesium sulfate on expression of bax and bcl-2 proteins after brain diffuse axonal injury in rats

摘要:

目的 研究脑弥漫性轴索损伤后神经细胞迟发性死亡的机制，探讨镁离子对大鼠脑弥漫性轴索损伤后神经元的保护作用。方法 采用Marmarou方法制备大鼠重度脑弥漫性轴索损伤模型，分为创伤组（n=25）、生理盐水组（n=40）和硫酸镁组（n=40）。硫酸镁组伤后半小时给予25%硫酸镁（750 μmol/kg），生理盐水组在相同时间给予等量的生理盐水腹腔注射，于伤后6小时、24小时、3天、5天及7天5个时相点处死。采用HE染色、免疫组织化学技术动态观察大鼠海马区的组织病理改变，Bcl-2和Bax蛋白的表达情况，以及使用硫酸镁干预后对上述表达的影响。结果 ①Bcl-2蛋白的表达：假手术组大鼠海马区仅见极少量Bcl-2阳性细胞，着色淡。在伤后6小时即有大量的Bcl-2阳性细胞，随时间渐增，24小时达到高峰，3～7天逐渐减少。②Bax蛋白的表达：在DAI后大鼠海马区有大量Bax阳性细胞，在伤后6小时就有所增加，24小时显著增加，3天达到高峰。Bcl-2/Bax值在损伤后随时间逐渐上升。③硫酸镁组中，海马区的Bax表达与对照组相比有所减少，而Bcl-2相应增加，Bcl-2/Bax比值也是上调的，均有统计学意义（P<0.05）。结论 大鼠脑弥漫性轴索损伤后，海马区神经元存在有迟发性细胞死亡即凋亡现象。Bax与Bcl-2参与细胞凋亡过程。硫酸镁可通过抑制Bax蛋白，上调Bcl-2蛋白，减少神经细胞凋亡，对促进神经细胞修复和功能重塑有益。

Abstract:

Objective The purpose is to clarify the protection of magnesium ions to rat neurone after diffuse axonal injury (DAI), and explore the mechanism of delayed neurone death after DAI on the level of molecular biology.Methods The rat model of severe DAI was prepared according to the method described by Marmarou.All survivors were randomized into 3 groups: control group (n=25)、normal saline (NS) group(n=40)、magnesium sulfate (MgSO₄) group (n=40).Each group were divided into 5 subgroups according to the time of injury at 6 h、24 h、72 h、120 h and 168 h respectively. The rat in MgSO₄ group and NS group when after injury in 30 minutes were treated with MgSO₄ (750 μmol/kg) and NS by intraperitoneal respectively, then were killed by decapitation at above mentioned time.Pathologic changes in hippocampus and the expression of bcl-2 and bax proteins after DAI were determined by HE and immunohistochemistry staining. Analyze the effects after using MgSO₄.Results 1.The expression of bcl-2: bcl-2 nearly expression in hippocampus of non-sugery group, but had higher expression in hippocampus after 6 hours of DAI, reached maximum 24 hours after injury, and declined obviously on days 3-7 after injury.2.The expression of Bax: Bax had a little expression in non-sugery group, and had higher expression in hippocampus after 24 hours of DAI, reached maximum 3 days after, and restore normal level 7 days after.3. The ratio of bcl-2/bax was raised gradually after injury.4.Compared with NS groups, the expression of Bax in hippocampus were lower in MgSO₄ groups, whereas the bcl-2 level and the ratio of bcl-2/bax were raised. There was highly significant difference in the level of bcl-2、Bax、bcl-2/bax in MgSO₄ groups and NS groups (P<0.05).Conclusions After DAI in rats the phenomena of delayed cell death existed in the neurons in hippocampus, neuronal apoptosis play an important role in the course of delayed cell death. The rising expressions of bax and bcl-2 proteins may participate in the regulation of the course of apoptosis after DAI. The expressions of bax and bcl-2 proteins related with the time after DAI.4.The up-regulation of bcl-2 and down-regulation of bax expression maybe contribute to neuro-protective effect of magnesium sulfate. Magnesium sulfate may have a potential as therapeutic agent for the treatment of DAI. Magnesium sulfate may suppress apoptosis of neural cells and can stimulate cell rehabilitation.