苯并[a]芘对大鼠脑组织中神经细胞凋亡及Bcl-2和Bax基因表达的影响

目的 观察苯并[a]芘(B[a]P)染毒大鼠神经细胞凋亡及凋亡相关基因Bcl-2、Bax蛋白的表达,以探讨B[a]P对大鼠神经毒性及其作用机制.方法 32只SD大鼠,按体重随机分为4组,每组8只,染毒组分为高、中、低3个剂量组(126.2、63.1、31.5 μg/kg的B[a]P溶液),并设溶剂对照组(50 μl/kg橄榄油),均采用侧脑室微量注射染毒处理,每周1次,连续3周;在染毒3周后采用原位末端缺口标记(TUNEL)法和免疫组织化学法分别对各组大鼠脑组织凋亡细胞的分布及Bcl-2、Bax免疫反应阳性细胞进行观察和检测.结果 TUNEL染色结果显示,在大鼠大脑皮质及海马区的细胞核中可以见到棕黄色的凋亡小体,而且随着染毒剂量的增高,出现凋亡小体的细胞数逐渐增多.与溶剂对照组相比,高剂量组大鼠大脑皮质及海马神经细胞凋亡指数(AI)均明显升高,差异均有统计学意义(P＜0.01或P＜0.05).免疫组化染色结果显示,与溶剂对照组相比,中、高剂量染毒组大鼠大脑皮质及各剂量组海马区神经细胞的Bcl-2蛋白表达均明显下降,Bax蛋白表达均明显升高,各组大鼠皮质及海马神经细胞的Bcl-2/Bax比值均下降,差异均有统计学意义(P＜0.05或(P＜0.01).大鼠大脑皮质及海马区神经细胞AI与Bel-2呈负相关(r=-0.927,P＜0.01;r=-0.934,P＜0.01),AI与Bax呈正相关(r=0.858,P＜0.01;r=0.874,P＜0.01),AI与Bcl-2/Bax比值呈负相关(r=-0.939,P＜0.01;r=-0.942,P＜0.01).结论 B[a]P可引起大鼠大脑皮质及海马区神经元细胞的凋亡,且调控凋亡相关基因Bcl-2、Bax蛋白的表达,在B[a]P致神经细胞凋亡的过程中发挥了重要的作用.

Effects of benzo(a)pryene on apoptosis of neuronal cells and expression of Bcl-2 and Bax proteins in rat brain tissue

Objective To observe the effects of Benzo(a)pryene (BaP) on apoptosis of neuronal cells and expression of Bcl-2 and Bax proteins and to explore the mechanism of neurotoxicity induced by BaP in rats.Methods A total of 32 SD rats were divided randomly into 4 groups,i.e.3 BaP ( 126.2,63.1 and 31.5 μg/kg)groups and a solvent control (50 μg/kg olive oil) group.All rats were exposed to BaP or olive oil by lateral cerebral ventricle micro-injection 1 time a week for 3 weeks.The apoptosis of neuronal cells was detected with TdT-mediated dUTP-biotin nicked labeling (TUNEL) assay and the expression levels of Bcl-2 and Bax were measuredwith SABC immunohistochemistry in the cerebral cortex and hippocampus tissues of rats.Results The results of TUNEL assay showed that apoptosis bodies on the surface of the neurons in the cerebral cortex and hippocampus were clearly observed and the number of apoptosis bodies increased with BaP.Apoptosis indexes (Als) of the rat cerebral cortex and hippocampus in high exposure group were significantly higher than those in control group (P＜0.05 or P＜0.01 ).The analysis of immunohistochemistry showed that the Bcl-2 expression levels significantly decreased,the Bax expression levels obviously increased and the ratio of Bcl-2 to Bax decreased in the rat cerebral cortex and hippocampus of medium and high exposure groups,as compared with control group (P＜0.05 or P＜0.01 ).In the rat cerebral cortex and hippocampus,there were the negative correlation (r=-0.927,P＜0.01; r=-0.934,P＜0.01 ) between AI and Bcl-2,the positive correlation (r=0.858,P＜0.01; r=0.847,P＜0.01 ) between AI and Bax and the negative correlation (r=-0.939,P＜0.01; r=-0.942,P＜0.01 ) between AI and Bcl-2/Bax.Conclusion BaP could induce the apoptosis of neuronal cells in the rat cerebral cortex and hippocampus.Bcl-2 and Bax protein expression may play an important role in the apoptosis of neuronal cells induced by BaP.