Bax在D-半乳糖诱导的老化大鼠耳蜗中的作用

Effects of Bax on Cochleae in D-galactose-Induced Aging Rats

目的研究Bax介导的细胞凋亡在D-半乳糖诱导的老化大鼠耳蜗中的作用,探讨老年性耳聋外周听觉系统细胞凋亡的发生机制。方法 36只1月龄雄性Spragua-Dawley大鼠随机分成2组(各18只):1 D-半乳糖组:每日颈背部皮下注射D-半乳糖(500mg/kg),连续8周;2对照组:每日颈背部皮下注射同体积的生理盐水,连续8周。造模完成后,取两组大鼠耳蜗,利用实时定量PCR检测线粒体DNA(mitochondrial DNA,mt DNA)普遍缺失(common deletion,CD)的累积;利用western blot检测Bax蛋白的表达;利用免疫组织化学检测Cleaved caspase-3蛋白的表达;利用原位末端转移酶标记(terminal deoxynucleotidyl transferase(Td T)-mediated deoxyuridine triphosphate(d UTP)nick-endlabelling,TUNEL)染色检测大鼠耳蜗细胞凋亡发生的情况。所有实验数据采用两样本的t检验进行分析。结果和对照组的大鼠相比较,D-半乳糖诱导的老化大鼠mt DNA CD的累积在耳蜗组织中明显增多,差异有统计学意义(t值为6.631,P值<0.01);Bax和Cleaved caspase-3蛋白的表达在耳蜗组织中明显增强,差异有统计学意义(t值分别为20.914、20.043,P值均<0.01)。对照组的大鼠耳蜗没有检测到凋亡细胞,而D-半乳糖诱导的老化大鼠耳蜗底回血管纹检测到少量凋亡细胞。结论 Bax介导的细胞凋亡参与了D-半乳糖诱导的耳蜗老化过程,可能是导致老年性耳聋外周听觉系统细胞凋亡发生的重要原因。

Objective To investigate the effects of Bax on the cochlea in D-galactose-induced aging rats and the mech- anism of cell apoptosis in the peripheral auditory system in presbycusis. Methods Thirty-six 1-month male Sprague-Dawley rats were randomly divided into two groups （18 in each）： ① rats in D-galactose group received ally subcutaneous injection of D-gal at 500 mg/kg for 8 weeks, and ②rats in the control group received the same volume of the vehicle （0.9% saline） once a day for 8 weeks. After the experiment termination, cochlear tissues were harvested. The accumulation of mitochondrial DNA （mtDNA） common deletion （CD） was detected by real-time PCR. Protein levels of Bax and Cleaved caspase-3 were evaluated by western blot and immunohistochemical analysis. Apoptotic cells were determined by terminal deoxynucleotidyl transferase （TdT）-mediated deoxyuridine triphosphate （dUTP） nick-end-labelling （TUNEL） assay. All experimental data was analyzed using two-sample t-test. Results In comparison with the control group, the accumulation of mtDNA CD in the cochlea in rats of the D-galactose group was significantly increased （t = 6.631, P 〈 0.01）. Protein expressions of Bax and Cleaved caspase-3 in the cochlea were also significantly increased （t = 20.91d and 20.043, respectively, P 〈 0.01）. TUNEL-positive ceils were found only in the cochlea of D-gal-induced aging rats, with a small number of these cells limited to the stria vascularis of the basal turn. Conclusions Bax-dependent cell apoptosis may contribute to D-galactose-induced aging process in the cochlea and may be an important reason for the occurrence of cell apoptosis in the peripheral auditory system in presbycusis.