乙苯对大鼠脑组织氧化损伤和超微结构及凋亡相关基因表达的影响

目的 观察亚慢性接触乙苯对大鼠脑组织氧化损伤、超微结构及凋亡相关基因表达的影响.方法 SD大鼠40只,随机分为4组,每组20只.暴露于0.0、433.5、4335.0和6500.0mg/m3的乙苯,6h/次,5次/周,共13周,建立大鼠乙苯亚慢性染毒模型,检测脑组织乙酰胆碱酯酶(AChE)活力、还原型谷胱甘肽(GSH)和丙二醛(MDA)含量,电子显微镜观察脑组织超微结构,实时定量聚合酶链反应(Real-time PCR)法检测脑组织B细胞淋巴瘤-2相关联X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)、细胞色素C(Cyt C)、细胞凋亡蛋白酶(Caspase-3)、半胱天冬酶-9(Caspase-9)等基因的表达.结果 4335.0和6500.0 mg/m3剂量组大鼠脑组织内MDA含量(2.03±0.56)、(4.17±1.31)nmol/mg pro]明显高于对照组(1.08±0.26)nmol/mg pro],差异有统计学意义(P<0.05),各剂量组大鼠脑组织AChE活力(0.321±0.066)、(0.276±0.031)、(0.202±0.041)U/mg]和GSH含量(35.19±15.08)、(33.42±15.32)、(27.99±7.53)mg/g pro]均明显低于对照组AchE活力(0.583±0.125)U/mg,GSH含量(76.38±18.41)mg/g pro],差异有统计学意义(P<0.05,P<0.05).6500.0mg/m3乙苯处理组大鼠脑组织电镜下观察可见核仁呈半月形,胞质线粒体减少,受损的神经纤维内含高电子密度的髓磷体结构,为膜性结构脂质过氧化损伤所形成.4335.0和6500.0 mg/m3剂量组Bax基因表达水平明显高于对照组和433.5 mg/m3剂量组;与对照组相比,各剂量组Cyt C、caspase-9、caspase-3基因表达水平皆明显升高,差异有统计学意义(P<0.05);各剂量组Bcl-2的基因表达水平显著低于对照组,差异有统计学意义(P<0.05).结论 乙苯可诱导大鼠脑组织氧化损伤,发生凋亡,其机制可能乙苯可导致Bax、Cyt C、caspase-9、caspase-3基因的上调和抑制Bcl-2的表达有关.

Effects of ethylbenzene on oxidative damage, ultrastructure and expressions of apoptosis-related genes in rat brain tissues

Objective To investigate the influence of ethylbenzene on oxidative damage, ultrastructure and the expressions of apoptosis-related genes in the rat brain tissues. Methods Four groups of 10 males of Sprague-Dawley rats were allocated randomly, and inhaled daily with different doses of ethylbenzene: 0,433.5 mg/m3, 4335.0 mg/m3, and 6500.0 mg/m3 6 h daily, 5 days per week for 13 weeks. The contents of glutathione (GSH) and malondialdehyde (MDA) and activity of acetylcholinesterase (AChE) were assayed, respectively. The ultrastructure of brain tissues was observed via electron microscope. The gene expression levels of Bax, Bcl-2, cytochrome C, caspase-9 and caspase-3 in brain tissues were measured by real-time polymerase chain reaction(PCR), respectively. Results The contents of MDA(2.03±0.56), (4.17±1.31) nmol/mg pro] in the brain tissues of 4335.0 mg/m3 and 6500.0 mg/m3 ethylbenzene-treated groups were significantly higher than that(1.08±0.26) nmol/mg pro] in the control group (P<0.05),while AChE activities(0.321 ±0.066),(0.276±0.031),(0.202±0.041) U/mg] and GSH contents( 35.19±15.08 ),( 33.42±15.32 ),( 27.99±7.53 ) mg/g pro] in all ethylbenzene-treated groups were remarkably depressed (P<0.05, P<0.05, respectively). After 6500.0 mg/m3 ethylbenzene inhalation, the nucleolus exhibit demilune with decreased mitochondria. Electrondense of myelin occurred in the injured nerve, ascribing to lipid peroxidationed membrane. The gene expression level of Bax in brain tissue of 4335.0 mg/m3 and 6500.0 mg/m3 ethylbenzene-treated group was significantly higher than that in the control group (P<0.05). Compared with the control group, the gene expression levels of cytochrome C, caspase-9 and caspase-3 in all ethylbenzene-treated groups were enhanced (P<0.05, P<0.05, respectively), while bcl-2 gene expression levels in all ethylbenzene-treated groups were decreased (P<0.05). Conclusion Ethylbenzene can induce oxidative damage and apoptosis in brain tissues. The apoptotic mechanism might be involved with up-regulation of Bax, cytochrome C, caspase-9 and caspase-3, as well as restraint of Bcl-2.