TMT-Based Quantitative Proteomics Reveals Cochlear Protein Profile Alterations in Mice with Noise-Induced Hearing Loss

Abstract

Noise-induced hearing loss (NIHL) is a global occupational disease affecting health. To date, genetic polymorphism studies on NIHL have been performed extensively. However, the proteomic profiles in the cochleae of mice suffering noise damage remain unclear. The goal of this current study was to perform a comprehensive investigation on characterizing protein expression changes in the cochlea based on a mouse model of NIHL using tandem mass tag (TMT)-labeling quantitative proteomics, and to reveal the potential biomarkers and pathogenesis of NIHL. Male C57BL/6J mice were exposed to noise at 120 dB SPL for 4 h to construct the NIHL mouse model. The levels of MDA and SOD, and the production of proinflammatory cytokines including TNF-α and IL-6 in the mice cochleae, were determined using chemical colorimetrical and ELISA kits. Moreover, differentially expressed proteins (DEPs) were validated using Western blotting. The mouse model showed that the ABR thresholds at frequencies of 4, 8, 12, 16, 24 and 32 kHz were significantly increased, and outer hair cells (HCs) showed a distinct loss in the noise-exposed mice. Proteomics analysis revealed that 221 DEPs were associated with NIHL. Bioinformatics analysis showed that a set of key inflammation and autophagy-related DEPs (ITGA1, KNG1, CFI, FGF1, AKT2 and ATG5) were enriched in PI3K/AKT, ECM-receptor interaction, and focal adhesion pathways. The results revealed that the MDA level was significantly increased, but the activity of SOD decreased in noise-exposed mice compared to the control mice. Moreover, TNF-α and IL-6 were significantly increased in the noise-exposed mice. Western blotting revealed that the expression levels of ITGA1, KNG1, and CFI were upregulated, but FGF1, AKT2, and ATG5 were significantly downregulated in noise-exposed mice. This study provides new scientific clues about the future biomarkers and pathogenesis studies underlying NIHL. Furthermore, the findings suggest that the validated DEPs may be valuable biomarkers of NIHL, and inflammation and autophagy may be pivotal mechanisms that underlie NIHL.

Keywords: autophagy; cochlea; inflammation; noise-induced hearing loss; proteomics.

Figure 1

The flow chart of this study design.

Figure 2

Noise-induced hearing loss (NIHL) mouse model was constructed under 120 dB SPL for 4 h condition. (a) Comparison of mean auditory brainstem response (ABR) thresholds at frequencies of 4, 8, 12, 16, 24 and 32 kHz between the control group (n = 18) and noise group (n = 18). (b) Myosin7a (red) and phalloidin (green) immunofluorescence staining of mice cochleae in the control group (n = 3) and noise group (n = 3). (c) Quantification of outer hair cells (OHCs) in the apex, middle and base segments of cochlea in the control group (n = 3) and noise group (n = 3). Scale bars = 20 μm. Data are represented as mean ± SEM. ** p < 0.01, *** p < 0.001.

Figure 3

Heat map of protein abundance differences between control group and noise group. Red color indicates high abundance, and purple color indicates low abundance.

Figure 4

Bioinformatics analysis of identified differentially expressed proteins (DEPs). (a) The top 30 Gene Ontology (GO) terms of DEPs based on biological process, cellular component, and molecular function. (b) Protein–protein interaction (PPI) network analysis of DEPs involving in the significantly enriched signaling pathways.

Figure 5

Measurement of expression levels of DEPs, oxidative stress markers and proinflammatory cytokines production. (a) Validation of representative inflammation and autophagy-related DEPs by western blots in the control group (n = 3) and noise group (n = 3). (b) Comparison of MDA level and SOD activity in cochlea between the control group (n = 3) and noise group (n = 3). (c) Comparison of proinflammatory cytokines TNF-α and IL-6 in cochlea between the control group (n = 3) and noise group (n = 3). Data are represented as mean ± SD. * p < 0.05, ** p < 0.01.