Bax在D-半乳糖诱导的老化大鼠耳蜗中的作用

摘要

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）：①D-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, mtDNA）普遍缺失（common dele-tion, 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 (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labelling, 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mtDNA 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):①rats in D-galactose group received aily subcutaneous injection of D-gal at 500 mg/kg for 8 weeks, and②rats 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=20.914 and 20.043, respectively, P<0.01). TUNEL-positive cells 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.