【摘要】 目的本研究通过应用多种剂量的水杨酸长期慢性注射方法,来防治年龄相关老年性聋DBA/2J小鼠的听力损失,拟选择有效的水杨酸剂量,达到防治老年性聋的目的。方法选用快速老化的年龄相关老年性聋DBA/2J小鼠,各组随机选取10只小鼠,从4周龄开始用100、150、200和250mg/Kg的水杨酸腹腔连续注射30天,对照组用同剂量的生理盐水注射,在试验前和治疗后测定动物的ABR反应阈值。在治疗30天后,动物麻醉处死,3只动物耳蜗分离取出,耳蜗行全基底膜免疫组化prestin表达标记;3只动物行扫描电镜观察和硝酸银染色全耳蜗铺片毛细胞计数观察;另3只动物行耳蜗蛋白提取Western Blotting蛋白印痕测定和全耳蜗RNA提取RT-PCR测定。结果水杨酸长期慢性注射治疗30天后,200mg治疗组的效果最好,ABR反应阈值较治疗前没有差异,而其它各组的ABR反应阈值较治疗前均有上升,其ABR反应阈值治疗前与治疗后的差值依次分别为:200mg治疗组-0.37dBSPL,100mg治疗组11.15dBSPL,250mg治疗组12.68dBSPL,150mg治疗组16.72dBSPL。全耳蜗毛细胞计数结果为:200mg治疗组的毛细胞存活百分率最好,表现为耳蜗顶回基本存活,中回大部分毛细胞存活,基底部仍有少量毛细胞存活。其次为100mg治疗组,而150mg和250mg治疗组较差,毛细胞损失最严重的是盐水对照组。各组的ABR反应阈值与毛细胞计数结果基本一致。耳蜗基底膜行免疫组化prestin表达标记可见,200mg治疗组的prestin表达荧光最强,其次是100mg治疗组,150mg治疗组较弱,而250mg治疗组和盐水组更弱。Western Blotting蛋白印痕测定和全耳蜗RNA提取RT-PCR测定亦证明同样的结果。结论本研究证明200mg的水杨酸长期慢性注射可以有效的防治老年性聋的发展进程,合适剂量的水杨酸可以上调耳蜗毛细胞prestin的表达,达到补偿因老年退化导致的prestin表达下降,使耳蜗毛细胞的损失减少,听功能减退明显减缓,但要彻底阻止老年性聋的发展,尚需联合其它方法进行更广泛和深入的研究。更多还原

【Abstract】 Objective Long-term administration of salicylate has been shown to increase prestin expression, although high-doses of salicylate can be ototoxic and cause hearing loss and tinnitus. In this research, effects of different doses of salicylates on age-related hearing loss(ARHL) in DBA/2 J mice were investigated. Methods Four weeks old DBA/2 J mice(15 to 18 g) were divided into four groups(10 in each group) to receive intraperitoneally(i.p.) injected salicylate dissolved in sterile saline at 100, 150, 200 or 250 mg/kg for 30 consecutive days. Control animals received equivalent volume of saline. Auditory brainstem responses(ABR) thresholds were recorded before and after each treatment.All animals were killed by decapitation upon ending drug treatment and final ABR testing, and the cochlea removed for prestin immunostaining(n=3 in each group), Western Blotting and RT-PCR for detection of Bcl-2 and caspase-3 genes(n=3 in each group), and light microscopy observation of hair cells loss(n=3 in each group). Results Post-treatment ABR thresholds showed no significant shifts in the 200 mg/kg(-0.37 dB) groups, but were elevated in the other dose groups(11.15 dB for the 100 mg/kg group, 12.68 dB for the 250 mg/kg group and 16.72 dB for the 150 mg/kg group). Cochlear hair cells count showed best cell survival in the 200 mg/kg group with minimal loss in the apical turn, mild loss in the middle turn and still some survival in the basal turn, but significant hair cell loss in the 150 mg/kg and 250 mg/kg groups, although the worst cell loss was seen in animals receiving normal saline, consistent to ABR threshold results.The strongest in situ OHC prestin immunostaining on whole-mount cochlear preparation was seen in the 200 mg/kg group, followed by the 100 mg/kg and 150 mg/kg groups, with the lowest seen in the 250 mg/kg and control groups.However, Bcl-2 expression in the inner hair cell was strong in the 150 mg/kg group, and only at moderate levels in the100 mg/kg and 200 mg/kg groups. Significant reduction of Bcl-2 expression in the cochlea was associated with elevated hearing thresholds and hair cell loss in the 250 mg/kg group. Caspase-3 expression was strong in the control group and decreased salicylate doses increased. Conclusion This study suggests that salicylate at appropriate doses(200 mg/kg)appears to delay ARHL development in mice, probably by up-regulating OHC prestin expression mechanisms, although its effect may be limited and its combination with other therapeutic approaches needs to be further investigated.更多还原