ABSTRACT
BACKGROUND: Excessive oxidative stress of the inner ear as a result of high, intense noise exposure is regarded as a major mechanism underlying the development of noise-induced hearing loss (NIHL). The present study was designed to explore the effect and mechanism of activated transcription factor 3 (ATF3) in reduction/oxidation homeostasis of NIHL. METHOD: In vitro and in vivo assays were performed to investigate the functional role of ATF3 in the inner ear. Mice hearing was measured using auditory brainstem response. ATF3 short hairpin RNA (shRNA) was transfected into House Ear Institute-Organ of Corti 1 (HEI-OC1) cells to decrease ATF3 expression. Western blotting and quantitative real-time polymerase chain reaction (RT-qPCR) were performed to quantify ATF3, NRF2, HO-1 and NQO1 expression. Glutathione (GSH) assay was performed to detect intracellular GSH levels. ATF3 immunofluorescence analysis was carried out in cochlear cryosectioned samples and HEI-OC1 cells to localize ATF3 expression. Cell counting kit 8 assay and flow cytometry were performed to analyze cell viability. RESULT: ATF3 was upregulated in noise-exposed cochleae and HEI-OC1 cells treated with H2O2. NRF2 is a key factor regulated by ATF3. NRF2, HO-1, NQO1, and GSH expression was significantly downregulated in shATF3 HEI-OC1 cells. ATF3 silencing promoted reactive oxygen species accumulation and increased apoptosis and necrosis with H2O2 stimulus. CONCLUSION: ATF3 functions as an antioxidative factor by activating the NRF2/HO-1 pathway.
Subject(s)
Activating Transcription Factor 3 , Hearing Loss, Noise-Induced , NF-E2-Related Factor 2 , Activating Transcription Factor 3/metabolism , Animals , Apoptosis , Disease Models, Animal , Heme Oxygenase-1 , Hydrogen Peroxide/pharmacology , Membrane Proteins , Mice , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Reactive Oxygen Species/metabolism , Signal TransductionABSTRACT
Survival of cochlear sensory epithelial cells may be regulated by inhibitor of differentiation-1 (Id1) and the N-methyl-D-aspartic acid (NMDA) receptor. However, it is unclear whether Id1 and the NMDA receptor are involved in the radiation-mediated survival of rat cochlear sensory epithelial cells. Here, we show that the percentage of apoptotic cells increased, the percentage of cells in the S phase decreased, Id1 mRNA and protein expression decreased and the NMDA receptor subtype 2B (NR2B) mRNA and protein level increased in OC1 cells after radiation. Cells infected with the Id1 gene exhibited higher Id1 mRNA and protein levels and lower NR2B mRNA and protein levels than the control cells. In contrast, after transfection of the Id1 siRNA into OC1 cells, Id1 mRNA and protein expression decreased and NR2B mRNA and protein expression increased relative to that of the control group. Additionally, treatment with ifenprodil for 24 h before radiation reduced apoptosis and increased the percentage of cells in the S phase. Our results suggest that Id1 and NR2B might regulate the survival of OC1 cells following radiation.
Subject(s)
Epithelial Cells/radiation effects , Inhibitor of Differentiation Protein 1/radiation effects , Organ of Corti/radiation effects , RNA, Messenger/radiation effects , Receptors, N-Methyl-D-Aspartate/radiation effects , Animals , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Line , Cell Survival/drug effects , Cell Survival/radiation effects , Cochlea/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Inhibitor of Differentiation Protein 1/genetics , Inhibitor of Differentiation Protein 1/metabolism , Organ of Corti/cytology , Organ of Corti/drug effects , Organ of Corti/metabolism , Piperidines/pharmacology , RNA, Messenger/metabolism , RNA, Small Interfering , Rats , Receptors, N-Methyl-D-Aspartate/genetics , Receptors, N-Methyl-D-Aspartate/metabolism , S Phase/drug effects , S Phase/radiation effects , TransfectionABSTRACT
Age-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K(+) circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.
Subject(s)
Aging/genetics , Potassium Channels, Inwardly Rectifying/genetics , Presbycusis/genetics , RNA, Messenger/genetics , Spiral Ligament of Cochlea/metabolism , Aging/metabolism , Animals , Cations, Monovalent , Fluorescent Antibody Technique , Gene Expression Regulation , Ion Transport , Mice , Mice, Inbred C57BL , Microtomy , Potassium/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Presbycusis/metabolism , Presbycusis/physiopathology , RNA, Messenger/metabolism , Spiral Ligament of Cochlea/physiopathology , Spiral Ligament of Cochlea/ultrastructure , Kir5.1 ChannelABSTRACT
CONCLUSION: The large conductance Ca(2+)-activated K(+ )channels (BK) expression is decreased in the cochleae of age-related hearing loss (AHL) mice. BK channel may be associated with AHL. OBJECTIVE: AHL is the most common among elderly persons. BK channels act as sensors for membrane voltage and intracellular Ca(2+ )and are essential for hearing. To investigate the distribution of BK channel in the cochleae of C57BL/6J mice, and the relationship between the expression of BK channel and the etiology of AHL. METHODS: BK expression was studied in the cochleae of C57BL/6J mice at various ages (4, 12, 26, 52 weeks). The expressions of BK at the protein and mRNA levels were detected by immunofluorescence technique, western blot and quantitative real time PCR. RESULTS: In comparison with 4-week-old mice, BK expressions in the cochleae at 12, 26 and 52 weeks of age were significantly and gradually decreased at both the protein and the mRNA levels. The immunofluorescence technique showed the BK channel was located in the hair cells and cells of the spiral ganglion, spiral ligament and stria vascularis and its expression also decreased with aging.
Subject(s)
Cochlea/metabolism , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Presbycusis/etiology , Aging/metabolism , Animals , Hearing , Mice, Inbred C57BL , Presbycusis/metabolism , RNA, Messenger/metabolismABSTRACT
CONCLUSIONS: Na(+)-K(+)-2Cl(-) co-transporter isoform 1 (NKCC1) mRNA and protein decrease with increasing age in the cochlear lateral wall of C57BL/6J (C57) mice. The down-regulation of NKCC1 may influence the K(+) transport efficiency and the homeostasis of ion transport cells, and cause the irreversible damage of cochlear cells in old C57 mice. Our results indicate that NKCC1 may play an important role in the pathogenesis of age-related hearing loss (AHL). OBJECTIVES: The aim of the present study was to investigate the relationship between the functional expression of NKCC1 transporter and the etiology of AHL. METHODS: C57 mice were used and randomly divided into four groups according to age (4 weeks, 14 weeks, 26 weeks, and 52 weeks). Immunofluorescence technique, quantitative real-time PCR, and western blot were applied to detect the expression of NKCC1 in the cochlear lateral wall of C57 mice at the various ages. RESULTS: In all four groups, the expression of NKCC1 was observed in the stria vascularis and type II fibrocytes of the spiral ligament. Also, the expression of NKCC1 appeared to decrease with age at both the transcriptional level and the protein level.
