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
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摘要
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.
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.
引文
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[3]Liu C,Glowatzki E,Fuchs PA.Unmyelinated type II afferent neurons report cochlear damage.Proc Natl Acad Sci USA,2015,112(47):14723-14727.
[4]Schermuly L,Vossieck T,Klinke R.Furosemide has no effect on endocochlear potential and tuning properties of primary afferent fibres in the pigeon inner ear.Hear Res,1990,50(1-2):295-298.
[5]Xiong H,Chu H,Zhou X,et al.Simultaneously reduced NKCC1and Na,K-ATPase expression in murine cochlear lateral wall contribute to conservation of endocochlear potential following a sensorineural hearing loss.Neurosci Lett,2011,488(2):204-209.
[6]Ding B,Walton JP,Zhu X,et al.Age-related changes in Na,K-ATPase expression,subunit isoform selection and assembly in the stria vascularis lateral wall of mouse cochlea.Hear Res,2018,367:59-73.
[7]Kikuchi T,Adams JC,Miyabe Y,et al.Potassium ion recycling pathway via gap junction systems in the mammalian cochlea and its interruption in hereditary nonsyndromic deafness.Med Electron Microsc,2000,33(2):51-56.
[8]黄秋红,黄子真,叶咏怡,等.B细胞淋巴瘤-2蛋白在年龄相关性聋小鼠耳蜗中的表达.中国耳鼻咽喉头颈外科,2017,24(6):305-308.
[9]Zhao J,Guo X,Du Y,et al.Correlative study of peripheral ATP1A1 gene expression level to anxiety severity score on major depressive disorder patients.J Basic Clin Physiol Pharmacol,2016,27(6):563-567.
[10]Wangemann P.K(+)cycling and its regulation in the cochlea and the vestibular labyrinth.Audiol Neurootol,2002,7(4):199-205.
[11]郑旭青,代天星,林晓敏,等.KCNJ10通道蛋白在C57BL/6J小鼠耳蜗的年龄相关性表达及与听力关系.中国耳鼻咽喉头颈外科,2013,20(8):409-413.
[12]Liu Y,Chu H,Chen J,et al.Age-related change in the expression of NKCC1 in the cochlear lateral wall of C57BL/6J mice.Acta Otolaryngol,2014,134(10):1047-1051.
[13]Yoshida T,Nin F,Ogata G,et al.NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K+transport that controls the electrochemical properties in the mammalian cochlea.Pflugers Arch,2015,467(7):1577-1589.
[14]Roy M,Sivan-Loukianova E,Eberl DF.Cell-type-specific roles of Na+/K+ATPase subunits in Drosophila auditory mechanosensation.Proc Natl Acad Sci U S A,2013,110(1):181-186.
[15]Halonen J,H inton A S,Frisina R D,e t a l.L ong-term t reatment with aldosterone slows the progression of age-related hearing loss.Hear Res,2016,336:63-71.
[16]Ponce-Coria J,Markadieu N,Austin TM,et al.A novel Ste20-related proline/alanine-r ich k inase(SPA K)-independent pathway involving calcium-binding protein 39(Cab39)and serine threonine kinase with no lysine member 4(WNK4)in the activation of Na-K-Cl cotransporters.J Biol Chem,2014,289(25):17680-17688.
[17]Grat ton MA,Smyth BJ,Lam CF,et al.Decline i n the endocochlear potential corresponds to decreased Na,K-ATPase activity in the lateral wall of quiet-aged gerbils.Hear Res,1997,108(1-2):9-16.
[2]Wilms V,Kopl C,Sofgen C,et al.Molecular bases of K+secretory cells in the inner ear:shared and distinct features between birds and mammals.Sci Rep,2016,6:34203.
[3]Liu C,Glowatzki E,Fuchs PA.Unmyelinated type II afferent neurons report cochlear damage.Proc Natl Acad Sci USA,2015,112(47):14723-14727.
[4]Schermuly L,Vossieck T,Klinke R.Furosemide has no effect on endocochlear potential and tuning properties of primary afferent fibres in the pigeon inner ear.Hear Res,1990,50(1-2):295-298.
[5]Xiong H,Chu H,Zhou X,et al.Simultaneously reduced NKCC1and Na,K-ATPase expression in murine cochlear lateral wall contribute to conservation of endocochlear potential following a sensorineural hearing loss.Neurosci Lett,2011,488(2):204-209.
[6]Ding B,Walton JP,Zhu X,et al.Age-related changes in Na,K-ATPase expression,subunit isoform selection and assembly in the stria vascularis lateral wall of mouse cochlea.Hear Res,2018,367:59-73.
[7]Kikuchi T,Adams JC,Miyabe Y,et al.Potassium ion recycling pathway via gap junction systems in the mammalian cochlea and its interruption in hereditary nonsyndromic deafness.Med Electron Microsc,2000,33(2):51-56.
[8]黄秋红,黄子真,叶咏怡,等.B细胞淋巴瘤-2蛋白在年龄相关性聋小鼠耳蜗中的表达.中国耳鼻咽喉头颈外科,2017,24(6):305-308.
[9]Zhao J,Guo X,Du Y,et al.Correlative study of peripheral ATP1A1 gene expression level to anxiety severity score on major depressive disorder patients.J Basic Clin Physiol Pharmacol,2016,27(6):563-567.
[10]Wangemann P.K(+)cycling and its regulation in the cochlea and the vestibular labyrinth.Audiol Neurootol,2002,7(4):199-205.
[11]郑旭青,代天星,林晓敏,等.KCNJ10通道蛋白在C57BL/6J小鼠耳蜗的年龄相关性表达及与听力关系.中国耳鼻咽喉头颈外科,2013,20(8):409-413.
[12]Liu Y,Chu H,Chen J,et al.Age-related change in the expression of NKCC1 in the cochlear lateral wall of C57BL/6J mice.Acta Otolaryngol,2014,134(10):1047-1051.
[13]Yoshida T,Nin F,Ogata G,et al.NKCCs in the fibrocytes of the spiral ligament are silent on the unidirectional K+transport that controls the electrochemical properties in the mammalian cochlea.Pflugers Arch,2015,467(7):1577-1589.
[14]Roy M,Sivan-Loukianova E,Eberl DF.Cell-type-specific roles of Na+/K+ATPase subunits in Drosophila auditory mechanosensation.Proc Natl Acad Sci U S A,2013,110(1):181-186.
[15]Halonen J,H inton A S,Frisina R D,e t a l.L ong-term t reatment with aldosterone slows the progression of age-related hearing loss.Hear Res,2016,336:63-71.
[16]Ponce-Coria J,Markadieu N,Austin TM,et al.A novel Ste20-related proline/alanine-r ich k inase(SPA K)-independent pathway involving calcium-binding protein 39(Cab39)and serine threonine kinase with no lysine member 4(WNK4)in the activation of Na-K-Cl cotransporters.J Biol Chem,2014,289(25):17680-17688.
[17]Grat ton MA,Smyth BJ,Lam CF,et al.Decline i n the endocochlear potential corresponds to decreased Na,K-ATPase activity in the lateral wall of quiet-aged gerbils.Hear Res,1997,108(1-2):9-16.