Expressions Of TMPRSS3,ENaC-α In The Cochlea Of Rats With Age-related And Kanamycin-induced Hearing Loss

Backgrounds TMPRSS3 (transmembrane protease serine 3) is a member of transmembraneⅡserine proteases(TTSPs). TMPRSS3 is the first-found protease that its mutation could lead to deafness and is mutated in non-syndromic autosomal recessive deafness(DFNB8/10).The 14 pathogenic changes are found to date. However, it is not clear about the function of TMPRSS3.Objective On the basis of researching rats with age-related and kanamycin-induced hearing loss, to investigate the expression of TMPRSS3 in the SD rats cochlea, and to explore the roles of TMPRSS3 in sensorineural deafness.Methods SD rats which were used for kanamycin-induced hearing loss were 3-month-old and were divided into four groups in this study. GroupⅠ(control), treated with normal saline for 14 days. GroupⅡ(KN3d), treated with kanamycin sulfate for 3 days. GroupⅢ(KN7d), treated with kanamycin sulfate for 7 days. GroupⅣ(KN14d), treated with kanamycin sulfate for 14 days. Rats used for age-related hearing loss were divided into three groups:3-,12- and 24- month-old. The auditory function was evaluated by evoked auditory brainstem responses (ABR). Paraffin-embedded immunohistochemistry was used for evaluation of TMPRSS3 expression in the cochlea. The protein was extracted from the cochlea tissues, and TMPRSS3 protein levels in the cochlea were detected by Western blot assay. The RNA was extracted from the cochlea tissues and TMPRSS3 mRNA levels in the cochlea were measured by reverse transcription-polymase chain reaction (RT-PCR).Results 1. Rats with kanamycin-induced hearing loss:(1) Auditory function:The order of the ABR thresholds from high to low was KN14d > KN7d> KN3d. (2) Immunohistochemistry:A positive reaction for TMPRSS3 staining was found in the cochlea of each group. The expression of TMPRSS3 immunoreactivity prominently decreased following the increase of injection days. (3) Western blot assay showed that the levels of TMPRSS3 in the cochlea of SD rats decreased with injection days. (4) RT-PCR assay:RT-PCR demonstrated that the mRNA expression of TMPRSS3 decreased with injection days.2. Rats with. age-related hearing loss:(1) Auditory function:ABR thresholds increased with aging at 16 kHz. There were significant differences between each two groups. At 8 kHz, ABR thresholds of 24-month-old rats were higher than 3-and 12-month-old rats, there wasn't significant differences between 3-and 12-month-old rats. (2) Immunohistochemistry:The expression of TMPRSS3 immunoreactivity prominently decreased following age increase. (3) Western blot assay: TMPRSS3 protein in the cochlea of SD rats decreased with aging. (4) RT-PCR assay:RT-PCR demonstrated that the mRNA expression of TMPRSS3 decreased with aging.Conclusions Positive reaction for TMPRSS3 staining was found in the cochlea of normal SD rats, mainly observed in the spiral ganglion and organ of Corti. The protein and mRNA of TMPRSS3 decreased with kanamycin administration and aging, and were basically consistant with the auditory function. These results indicated that TMPRSS3 may take part in the processes of hearing loss. Backgrouds ENaC (epithelial sodium channel) is a member of ENaC/Deg, and hasα,β,γ.subunits. ENaC is expressed in the distal nephron,colon,lung,cochlea and so on. In the Xenopus oocyte expression system, proteolytic processing of TMPRSS3 was associated with increased ENaC mediated currents. TMPRSS3 mutants failed to undergo proteolytic cleavage and activate ENaC. However, it is not clear about ENaC protein's function in regulating the metabolism of endolymph and maintenance of normal hearing.Objective To investigate the expression of ENaC-αin the SD rats with age-related and kanamycin-induced hearing loss, and to explore the roles of ENaC-a in the two kinds of hearing loss, whether it has correlation with TMPRSS3.Methods Group dividing is as same as PartⅠ. Paraffin-embedded immunohistochemistry was used for evaluation of ENaC-a expression in the cochlea. The protein was extracted from the cochlea tissues, and ENaC-a protein levels in the cochlea were detected by Western blot assay. The RNA was extracted from the cochlea tissues and ENaC-a mRNA levels in the cochlea were measured by reverse transcription-polymase chain reaction (RT-PCR).Results 1. Rats with kanamycin-induced hearing loss: (1)Immunohistochemistry:A positive reaction for ENaC-a staining was found in the cochlea of each group. The expression of ENaC-a immunoreactivity prominently decreased in the KN14d group compared with KN6d,KN3d and control groups, but there wasn't significant differences among KN6d,KN3d and control groups. (2)Western blot assay showed that the levels of ENaC-a protein decreased in the KN14d group compared with KN3d,KN7d and control groups, but there wasn't significant differences among KN3d,KN7d and control groups. (3)RT-PCR assay:There wasn't significant differences among KN3d,KN7d and control groups, ENaC-a decreased in the KN14d group compared with KN3d,KN7d and control groups.2. Rats with age-related hearing loss:(1)Immunohistochemistry:The expression of ENaC-a immunoreactivity prominently decreased following age increase. (2)Western blot assay showed that ENaC-a protein in the cochlea of SD rats decreased with aging. (3)RT-PCR assay: RT-PCR demonstrated that the mRNA expression of ENaC-a in the cochlea of SD rats decreased with aging.Conclusions Positive reaction for ENaC-αstaining was found in the cochlea of normal SD rats, mainly observed in the the spiral ganglion and organ of Corti. The protein and mRNA of ENaC-αdecreased with injection days and aging. These results has some correlation with TMPRSS3 and indicated that TMPRSS3 may regulate the expression of ENaC.