Na-K-2Cl联合转运子1与钠钾交换ATP酶在C57BL/6J小鼠中的表达

Expression of NKCC1 and Na-K-ATPase in C57BL/6J mice with age-related hearing loss

目的内耳电解质紊乱与听力障碍有关,包括年龄相关性听力障碍。Na-K-2Cl联合转运子1(Na-K-2Cl co-transporter 1,NKCC1)和钠钾交换ATP酶(NaK-ATPase)的表达随年龄改变,由此分析与年龄相关性聋的发展关系。方法通过听性脑干反应(ABR)检测青年和老年C57BL/6J小鼠听力水平,反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RTPCR)和蛋白质印迹(wester nblot ting,W B)方法检测NKCC1和Na-K-ATPase在耳蜗的表达水平,使用NKCC1抑制剂呋塞米、Na-K-ATPase抑制剂乌巴因对C57BL/6J小鼠离子转运蛋白进行抑制。结果频率8、16和32 kHz时,老年C57BL/6J小鼠ABR反应阈值分别为(75±10)、(78±26)和(81±14)d BSPL。RT-PCR显示老年组的NKCC1和Na-K-ATPase的mRNA相对表达水平下降,分别是青年对照组0.52±0.06和0.35±0.04倍,两组分别比较差异有统计学意义(t=7.466和16.11,P均<0.05);WB显示老年组的NKCC1和Na-K-ATPase的蛋白相对表达水平下降,分别是青年对照组0.79±0.02和0.68±0.05倍,两组分别比较差异有统计学意义(t=8.857和6.771,P均<0.05)。对NKCC1和Na-KATPase进行抑制后,频率8、16和32 kHz时,给药组青年小鼠ABR阈值分别为(50±17)、(53±21)和(55±17)dB SPL以及(56±6)、(70±17)和(73±6)dB SPL。结论 C57BL/6J小鼠实验证明NKCC1和Na-K-ATPase可能与小鼠年龄相关性听力损失相关。

OBJECTIVE Disturbance of K^+ ion balance in inner ear is associated in age-related hearing loss. Our study is to investigate the role of NKCC1 and NaK-ATPase in cochlea and auditory function regulated by with different expression of NKCC1 and Na-K-ATPase. METHODS Auditory threshold of young or old C57 BL/6 J mice was measured by auditory brainstem response(ABR). The expression of NKCC1 and Na-K-ATPase in mice cochlea were evaluated by reverse transcription polymerase chain reaction(RT-PCR) and western blotting. Furosemide and Ouabain were applied in vivo to inhibit NKCC1 and Na-K-ATPase in C57 BL/6 J mice. RESULTS C57 BL/6 J mice developed hearing loss at 12 M by ABR threshold shif ting to(75±10),(78±26) and(81±14)d B SPL at frequencies of 8, 16 and 32 kHz;PCR showed that the relative expression of NKCC1 and Na-K-ATPase mRNA in the aged group decreased, which were 0.52±0.06 and 0.35±0.04 times higher than those in the young control group, the difference was statistically significant(t =7.466 and 16.11, all P<0.05). WB showed that relative expression of NKCC1 and Na-K-ATPase protein level in the aged group decreased by 0.79±0.02 and 0.68±0.05 times as much as that of the young control group, the difference was statistically significant(t=8.857 and 6.771, P all<0.05). After applied with Furosemide and Ouabain to suppress the two ion transporters, the ABR threshold increased to(50±17),(53±21),(55±17)dB SPL and(56±6),(70±17),(73±6)dB SPL at frequencies of 8, 16 and 32 kHz. CONCLUSION In vivo experiment of C57 BL/6 J suggested that NKCC1 and Na-K-ATPase might be related to age related hearing loss.