The study subjects were the workers who exposed to occupational noise in the factories. According to requests, all workers were requested to receive annual occupational health examination, such as general physical examination, peripheral blood collection and pure-tone audiometry (PTA) test. The questionnaire survey was performed to collect important information of subjects, including general family history of disease, personal disease history, smoking and alcohol consumption, and drug use history. In this study, the criteria for inclusion of study subjects were: (1) workers who exposed to occupational noise for more than one year; (2) workers only exposed to occupational noise; (3) Chinese Han workers. Nevertheless, the exclusion criteria were as follows: (1) workers who had family history of deafness; (2) workers who carried diseases that could affect normal hearing (e.g., otitis media, tinnitus, and skull trauma); (3) workers had recently treatment with ototoxic drugs (e.g., aminoglycosides, quinolones, aspirin) that damages the normal function of the inner ear; (4) workers had metabolic diseases (e.g., diabetes, hypertension, hyperlipidemia, etc.).
This study was approved by the Ethics Committee of Zhongda Hospital Affiliated to Southeast University and written informed consent was also acquired from all study subjects.
Noise exposure evaluation
Exposure level of noise was estimated according to equivalent continuous dB(A)-weighted sound pressure levels (LAeq, 8 h) with the TES 1350A sound level meter (TES, Taiwan). Cumulative noise exposure (CNE) was used to reflect the true individual noise exposure level based on LAeq.
Pure-tone audiometric examination
After stopping noise exposure for over 12 h, each study subject has to accept the PTA examination conducted by professional doctor using Voyager 522 audiometer (Madsen, Taastrup, Denmark) at frequencies of 0.5, 1, 2, 3, 4, and 6 kHz in a noise-proof room. Referring to GB/T7582-2004, the obtained raw data were adjusted by sex and age.
Selection of NIHL cases and normal hearing controls
Referring to the Chinese National Occupational Health Standard (GBZ49-2007), NIHL cases of this current study were define as occupational noise-exposed workers with binaural high frequencies (3, 4 and 6 kHz) hearing threshold level more than 25dB (A). Nevertheless, those with binaural high frequencies hearing threshold level less than 25dB (A) were included as the normal hearing controls. Totally, 124 subjects including 62 NIHL cases and 62 normal hearing controls were recruited in this study.
Chemicals and Reagents
Liquid chromatography grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). Both formic acid and ammonium formate were purchased from Thermo Fisher Scientific (Waltham, USA). Ultrapure water was prepared by Milli-Q water purify system (Millipore, USA).
Plasma sample collection and preparation
Considering food intake and drinking may result in the alteration of human metabolome, morning peripheral blood (collected after 12 h-fasting) is collected for plasma collection. Two milliliters of peripheral venous blood were collected from each study subject and transferred to tubes containing ethylenediaminetetraacetic acid. Plasma samples were isolated and centrifuged at 3500 rpm at room temperature for 15 min. Subsequently, all plasma samples were stored at -80 °C until metabolomics analysis.
All collected plasma samples were thawed at 4 °C and then vortexed for 20 s. A 100 μL aliquot of plasma sample was mixed using three times volume of methanol for protein precipitation. Then, the mixture was further vortexed for 30 s and was centrifuged at 13000 rpm at 4 °C for 20 min. The liquid supernatant was obtained for centrifugation at 13000 rpm at 4 °C for 20 min. Finally, a total of 20 μL aliquot of the supernatant was transferred into a sample vial for metabolomics analysis.
Chromatographic and mass spectrometric analysis
Metabonomic profiles of plasma samples from occupational noise-exposed workers was performed by ACQUITY Ultra Performance Liquid Chromatography (UPLC) (Waters, Milford, USA) system equipped with AB SCIEX Triple TOF 5600 System (AB SCIEX, Framingham, USA).
Chromatographic separation was implemented on an ACQUITY UPLC BEH C8 column (2.1 mm × 100 mm × 1.7 μm, Waters, Milford, USA). For positive ion mode, the mobile phase was composed of water with 0.1% formic acid (A) and acetonitrile (B). For negative ion mode, the mobile phase was 5 mM ammonium formate aqueous solution (C) and acetonitrile (D). The procedures of gradient elution were as follows: 5% solution B for 0-0.5 min, 5-20% solution B for 0.5-2 min, 20-25% solution B for 2-4 min, 25-60% solution B for 4-10 min, and 60-100% solution B for 10-15 min, 100% solution B for 15-16 min, and 5% solution B for 16-19 min. The delivery flow rate was set at 0.3 mL/min. Besides, the injection volume was 5 μL. All analyzed samples were kept at 4 °C, and the temperature of column was set at 40 °C.
Mass spectrometry (MS) was conducted with AB SCIEX Triple TOF 5600 System (AB SCIEX, Framingham, USA) coupled with electrospray ionization source. The parameters were as follows: collision energy, 35 V/-35 V; declustering potential, 80 V/-80 V; ion spray voltage, 5000 V/-4500 V; pressure of nebulizer gas, 50 psi; and curtain gas, 35 psi. Besides, the interface heater temperature was 500 °C and 550 °C, respectively. The scanning range of data acquisition was 50-1200 m/z.
Data processing and analysis
Firstly, the original data were processed using Progenesis QI software for baseline removing, peak identification, peak alignment and integration, and retention time adjustment. Then, a data matrix composed of retention time, mass-to-charge ratio, intensity of peak and information of sample was performed for further statistical analyses. For each plasma sample, the peak intensity corresponding to metabolite was further adjusted with the total intensity of peak of sample.
SIMCA-P 14.1 (Umetrics, Umea, Sweden) was applied for multivariate statistical analyses. Both principal component analysis (PCA) and orthogonal partial least square-discriminant analysis (OPLS-DA) were applied for distinguishing normal hearing controls and NIHL patients after unit variance (UV) scaling. The metabolites with variable importance of projection (VIP) values > 1 calculated by SIMCA-P were considered to be the potential biomarkers. Furthermore, a heatmap of metabolites was generated with Multi Experiment Viewer (MEV) software.
Some further analyses were also conducted in this study. MetaboAnalyst was applied for related metabolic pathway analysis (http://www.metaboanalyst.ca). Pathway plots were based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene Set Enrichment Analysis Software (GSEA, Broad Institute, Cambridge, MA, USA) was used to gene enrichment analysis. Furthermore, to characterize the gene functions, Human metabolome database (HMDB) was performed.
RNA extraction and RT-qPCR analysis
TRIZOL reagent (Invitrogen, Carlsbad, CA) was employed for RNA extraction. Double-stranded cDNA was synthesized with Takara Prime Script RT Reagent Kit (Takara Bio Inc., Clontech, Japan) referring to the instructions. Subsequently, RT-qPCR was conducted with SYBR Green real-time PCR kits (Toyobo, Osaka, Japan). The following primers were used for mRNA expression levels detection: PI3K, forward: 5’-CCACGACCATCATCAGGTGAA-3’, reverse: 5’-CCTCACGGAGGCATTCTAAAGT-3’; AKT, forward: 5’-GCAAGGTGATCCTGGTGAA-3’, reverse: 5’-TCGTGGGTCTGGAAAGAGTA-3’; ATG5, forward: 5’-AAAGATGTGCTTCGAGATGTGT-3’, reverse: 5’-CACTTTGTCAGTTACCAACGTCA-3’; and β-actin, forward: 5’-CTACCTCATGAAGATCCTCACCGA-3’, reverse: 5’-TTCTCCTTAATGTCACGCACGATT-3’.
Statistical analysis was carried out to ensure the potential metabolites significantly changed between NIHL cases and controls by using paired non-parametric test on MATLAB software (MathWorks). Otherwise, statistical analysis was conducted with the SPSS 23.0 software (SPSS, Chicago, Illinois, USA). The statistical significance criterion was set with a two-sided P value < 0.05.