Abstract Background Previous studies have shown that the traditional Chinese medicine (TCM) called “compound healthy ear agent” (CHEA) had anti-apoptosis effects in cochlear hair cells and spiral ganglion neurons, and could protect mice hearing against presbycusis or age-related hearing loss (AHL), as well as aminoglycoside antibiotic-induced ototoxicity. Because its mechanisms of action are still unclear, we investigated the mechanism of action of CHEA against AHL in mice using proteomics techniques. Methods Eighteen C57BL/6J mice at 1 month of age were randomly divided into three groups: (A) drinking water until 2 months of age, K2M); (B) drinking water until 7 months of age to induce AHL, K7M; (C) drinking water containing CHEA daily until 7 months of age as treatment group, Z7M. At 2 or 7 months mice were sacrificed and their cochleae were removed for proteomics analysis. Results The numbers of proteins with a false discovery rate (FDR) < 1% were respectively 5873 for qualitative and 5492 for quantitative statistics. The numbers of proteins with differential enrichment at least 1.5-fold (p < 0.05) were respectively 351 for K7M vs K2M groups, 52 for Z7M vs K7M groups, 264 for Z7M vs K2M groups. The differentially expressed proteins in the Z7M group were involved in synaptic molecular transmission, energy metabolism, immune response, antioxidant defenses, and anti-apoptosis. Conclusion The TCM CHEA played a protective role against AHL in mice by regulating the expression of specific proteins and genes in cochlear hair cells and spiral ganglion neurons. Besides the pathways expected to be involved (antioxidant and anti-apoptosis), proteins related to immune response is a new finding of the present study. Keywords: Proteomics, C57BL/6J mice, Presbycusis, Traditional Chinese medicine, Cochlear hair cell degeneration 1. Introduction Age-related hearing loss (AHL) or presbycusis is a type of deafness that presents as a bilateral, symmetrical hearing loss (HL) which begins with the inability to hear high frequencies [[37]1]. Pathologically it involves progressive, bilateral and symmetrical degeneration of the inner ear structures [[38]2]. AHL affects >50% of adults by age 75 years, and nearly all adults over 90 years of age [[39]3]. The etiology and pathology of AHL are quite complex, and many aspects are still unclear. A series of previous studies have shown that the traditional Chinese medicine “compound healthy ear agent” (CHEA) had significant anti-apoptotic effects in cochlear hair cells and spiral ganglion neurons, and could protect the hearing of mice with AHL [[40][4], [41][5], [42][6], [43][7], [44][8]]. CHEA also known as “Jian Er”, is an herbal preparation mainly containing Radix puerariae, Salvia miltiorrhiza, Astragalus membranaceus, and Drynaria fortune. Further studies found that its mechanisms of action were related to enhancing antioxidant enzymes, reducing the formation of reactive oxygen species (ROS), protecting mitochondrial function, and increasing the expression of nerve growth factors [[45]9,[46]10]. For over a decade, we have repeatedly studied the C57BL/6J mouse model of AHL in China and found that the loss of hair cells had occurred at the hook region and its vicinity in the cochlear basal turn by 3 months of age. By 7 months of age, additional hair cells were missing in the front and middle segments of the cochlear basal turn, and high-frequency hearing loss could be detected. The spiral ganglion cells and nerve fibers were also damaged in the cochlear basal turn by 7 months of age. However, in 2-month-old mice the hair cells were not damaged, and the mouse hearing thresholds remained within the normal range. As shown by observation from 3 months to 24 months of age, the missing hair cells progressively increased in number from the basal turn to the apical turn, The hearing loss also progressed from high frequency to low frequency over this period. Therefore 7-month-old mice were chosen as a relatively short period of time to provide alterations in hair cell morphology and audiology findings, as we have reported in our previous drug interventional research [[47]11]. This choice could also shorten the observation time and save manpower and resources. Therefore, animals of 2, 3 and 7 months of age were considered to be suitable models for studying traditional Chinese medicine in the treatment of AHL [[48]12]. In order to further investigate the etiology and pathology of AHL and understand the mechanism of the pharmacodynamic effects of CHEA in the prevention and treatment of AHL, we employed 7 months old C57BL/6J mice as an animal model of AHL as described in previous studies. After 7 months of treatment with CHEA in drinking water, we removed the cochleae and analyzed them using proteomics technology. The goal was to explore the biological effects of CHEA on the up-regulation or down-regulation within a wide range of specific proteins. 2. Materials and methods 2.1. Experimental animals Eighteen SPF 1-month-old newly weaned healthy C57BL/6J mice, half male and half female, weighing 16–18 g, were provided by Beijing Weitong Lihua Laboratory Animal Technology Co., LTD. (SCXK Beijing 2012-000), and kept in a SPF animal laboratory (SYXK Guangxi 2019-000). All operations were in accordance with the ethical requirements for experimental animals of Guangxi University of Traditional Chinese Medicine (Approval No.: IACUC 20161015). 2.2. Experimental groups 18C57BL/6J mice were selected from the same batch of 60 animals, and were randomly divided into three groups of six mice each with half female and half male. Six animals in the first group drank tap water from weaning (1 month of age) until 7 months of age as the AHL group (K7M group); six animals in the second group [49]drank tap water containing CHEA daily, from 1 month until 7 months of age as the treatment group (Z7M group). In addition, an additional six untreated C57BL/6J mice drank tap water until 2 months of age (K2M group) and were used as a control group before disease development. Mice were sacrificed at 2 months of age for K2M group and at 7 months of age for K7M and Z7M groups, by decapitation under deep ketamine/xylazine anesthesia. Cochleae were prepared and evaluated as described in detail in previous publications [[50]6,[51][13], [52][14], [53][15], [54][16]]. The temporal bones were removed and the cochleae were surgically removed (2 cochlear samples per animal). The tissue samples were snap frozen in liquid nitrogen and stored at −80 °C. Three animals (2 male and 1 female) were randomly selected from each group to be used for initial measurement of proteomics. The remaining three animals (1 male and 2 female) per group were used to repeat the proteomics assays as a quality control. The second repetition of the proteomics assay was in close agreement with the first determination, and the data are not reported separately. 2.3. Preparation of CHEA The traditional Chinese medicine CHEA is patented in China (Patent No. ZL2013104661403) for the prevention and treatment of sensorineural deafness. CHEA is composed mainly of the following herbs: Radix puerariae, Salvia miltiorrhiza, Astragalus membranaceus, and Drynaria fortune. The chemical components of CHEA are comprised mainly of isoflavones, lipid-soluble diterpenoid quinones, water-soluble phenolic acids, astragalus polysaccharide, saponins, flavonoids, triterpenes, phenolic acids, etc. CHEA was manufactured in the Pharmaceutical Factory of Ruikang Medical College in Guangxi University of Chinese Medicine. These botanical materials were successively treated by cooking in water, filtering, concentrating, drying, pulverizing, and passing through a mesh to produce a powder according to the methods described in the Patent previously used to prepare this herbal medicine in capsules. One gram of the final CHEA powder was equal to 6.63 g of the raw herbal materials. By applying the rule of dosage equivalence between a mouse and an adult human [[55]17], the dosage for mice was calculated to be 1.83 g/kg/day. Powder (0.05 g) was added to 5 mL of water, and then provided to the animals in their water bottles from 1 month of age to 4 months of age. After that, mice were daily administered the calculated dosage (per body weight) by gavage needle, from 4 months of age to 7 months of age. These animals received the calculated dosage in drinking water each day without any other source of water. 2.4. Reagents and instruments The main reagents employed in the experiments are listed in [56]Table 1. Table 1. Main reagents. Reagent Name Producer Product number Batch number TMT10 labeling kit ThermoScientific 90110 N-TD264171 Hydroxylamine ThermoFisher 90115 PD199190 BCA Kit ThermoScientific 23227 UB276927 Mass spectrometry grade acetonitrile ThermoScientific A955-4 168772 Mass spectrometry water ThermoScientific W6-4 180416 Lysis solution Biyuntian Biotechnology Research Institute p0013g 092918190103 PMSF SangonBiotech A610425-0005 E809BA0022 Na2HPO4▪12H2O SangonBiotech A501725-0500 E719BA0012 Na2H2PO4▪H2O SangonBiotech A100823-0100 E329BA0012 NaCl SangonBiotech A501218-0001 E502BA0012 40% Acr-Bis SangonBiotech B546014-0500 EB02KA0062 Tris-HCl/pH6.8/pH8.8 SangonBiotech B546020-0250/B546019-0250 E227KA7816/EA18KA9854 APS SangonBiotech A600072-0025 DC20BA1005 TEMED SangonBiotech A100761-0025 C106BA0030 Tris SangonBiotech A600194-0500 F117BA0002 DTT SangonBiotech A620058-0005 C729BA0004 Glycerin SangonBiotech A100854-0500 BA19BA0001 Bromophenol blue SangonBiotech A100449-0005 EC05BA0011 TFA SangonBiotech TS4295-013 13020696 Urea SangonBiotech A600148-0002 BB06BA0025 IAA SangonBiotech A600539-0005 EA16BA0005 G-250 SangonBiotech A600077-0025 E809BA0014 Glycine Sinopharm Chemical Reagent 17-1323-01 171204107 SDS Sinopharm Chemical Reagent 17-1313-01 15J070011 Phosphate buffer Sinopharm Chemical Reagent 10015418 LOT: 20150706 TEAB SIGMA T7408-500 mL BCBX6381 Absolute ethyl alcohol GENERAL-REAGENT G73537B P1346753 Isopropyl alcohol ThermoScientific A451-4 182060 Trypsin Beijing hualishi Technology HLS try001c no20190820c Formic acid CNW Technologies GmbH CAS:64-18-6 172433 [57]Open in a new tab The main instruments employed in the experiments are listed in [58]Table 2. Table 2. Main instruments. Instrument Name Manufacturer Model number Q Exactive Mass spectrometer ThermoFisher IQLAAEGAAPFALGMBDK Easy-nLC 1200 Liquid phase system ThermoFisher Easy-nLC 1200 High performance liquid chromatograph Agilent Agilent 1100 series Orbitrap Fusion ThermoFisher IQLAAEGAAPFADBMBCX Q Exactive HF ThermoFisher IQLAAEGAAPFALGMBFZ Desktop refrigerated centrifuge Shanghai Luxianyi centrifuge instrument TGL-16A Ultrasonic cell disruptor Ningbo Xinzhi Biotechnology JY98-IIIN SDS-PAGE Gel electrophoresis apparatus Beijing six One Instrument Factory DYY-6C Enzyme-labeled instrument Shanghai Kehua Experimental System ST-360 ImageScanner EPSON ES-1000G Electronic balance Shanghai Yueping Scientific Instrument FA2004B Freeze drying apparatus Ningbo Xinzhi Biotechnology SCIENTZ-10 N [59]Open in a new tab 2.5. Proteomics methods The TMTTM (Tandem Mass Tags) in vitro labeling technology developed by ThermoFisher was used for the proteomics study. The brief steps were as follows. * (1) Determination of protein concentration. The frozen sample was added to 300 μL of sample lysis buffer containing the protease inhibitor PMSF, at a final concentration of 1 mM. The samples were subjected to ultrasonic tissue disruption on ice at 80 W power, 1 s ultrasound bursts were applied 90 times in 3 min. The supernatant was obtained by centrifugation at 12000 rpm for 10 min at room temperature and centrifuged again. The supernatant contained the total protein contents of the sample, which was measured for protein concentration and then stored at −80 °C for later use. The protein concentration was determined by the BCA method (bicinchoninic acid). Under alkaline conditions when BCA binds to proteins, Cu2+ is reduced to a Cu+ chelate with two BCA molecules, and the working reagent forms a purple complex from the original apple green. The water-soluble complex shown a good linear relationship of absorbance at 562 nm and protein concentration over a wide range. According to the instructions of the BCA reagent kit, the required volume of buffer solution was prepared with buffers A: B (50:1 v/v). The protein solution was diluted with ultrapure water to within the working range of the standard curve. A clean 96 well plate was aliquoted with a gradient of BSA: 0, 1, 2, 4, 8, 12, 16, 20 μL of a 1 mg/mL solution. The corresponding volume of ultrapure water was added until the total volume was 20 μL. Samples of the protein test solution were adjusted to 20 μL and added to the wells in triplicate. Then 200 μL chromogenic solution was added to each well and reacted at 37 °C for 30 min. The absorbance values were measured at 562 nm using a microplate reader. The protein concentration was obtained using the standard curve. * (2) SDS-polyacrylamide gel electrophoresis (12% SDS-PAGE) was carried out with 10 μg protein taken from each sample. The gel was stained by Coomassie brilliant blue, and quantified using an ImageScanner in full-color scanning mode and an optical density value of 300 dpi. * (3) Trypsin enzymatic hydrolysis and labeling. After proteins were quantified, 100 μg of each sample in an ultrafiltration tube plus 120 μL reducing agent buffer (10 mM DTT, 8 M urea, 100 mM TEAB, pH 8.0) was incubated at 60 °C for 1 h. Trypsin hydrolysis and TMT labeling was performed according to the manufacturer's protocol. 8 μL of 5% hydroxylamine was used to terminate the reaction for 15 min, and then the samples were lyophilized and stored at −80 °C. * (4)Reverse phase chromatographic separation was performed using an Agilent 1100 HPLC chromatographic column: Agilent Zorbax Extend C18 narrow diameter column, 2.1 × 150 mm, 5 μm. Detection wavelength: UV 210 nm and 280 nm. Mobile phase A: ACN-H2O (2:98, v/v). Mobile phase B: ACN-H[2]O(90:10, v/v). Flow rate: 300 μL/min. Gradient elution conditions: 0–8 min, 98% A; 8∼8.01 min, 98%–95% A; 8.01–48 min, 95%–75% A; 48–60 min, 75∼60% A; 60∼60.01 min, 60∼10% A; 60.01–70 min, 10% A; 70∼70.01 min, 10–98% A; 70.01–75 min, 98% A. Samples were collected each minute over 8–60 min. * (5)After collection, the samples were lyophilized and stored for LC-MS. Chromatographic conditions: flow rate of 300 nL/min to Acclaim PepMap 100 100 μm × 2 cm column (RP-C18, Thermo Fisher), and then followed by an analytical column Acclaim PepMap RSLC, 75 μm × 15 cm (RP-C18, Thermo Fisher). Mobile phase A: H[2]O-FA (99.9:0.1, v/v); Mobile phase B: ACN–H[2]O–FA (80:19.9:0.1, v/v/v); Gradient elution conditions: 0–40 min, 5–30% B; 40–54 min, 30–50% B; 54–55 min, 50–100% B; 55–60 min, 100%B. Mass spectrometry conditions: resolution ratio of Class A MS was set at 70000, and the automatic gain control value was set as 1e6. The mass spectrum was scanned over a m/z range of 300–1600, and MS/MS scanning was performed on 10 of the highest peaks. Collection of all MS/MS spectra used energetic collision and splitting using data-dependent positive ion mode. The collision energy was set at 32 eV, the resolution ratio of MS/MS was set at 35000, the automatic gain control was set at 2e5, the maximum ion accumulation time was 80 ms, the dynamic exclusion time was set at 30 s. 2.6. Data analysis The Proteome Discoverer™ 2.2 (Thermo Company, USA) software was used for analysis. We employed the mouse database of UniProt. The false discovery rate (FDR) for peptide identification was calculated by dividing the number of expected false positive identifications by the total number of target identifications and was always less than 1%. The retrieved results were screened for credible proteins according to the criteria of Score Sequest HT > 0, unique peptide ≥1, and the blank values were removed. Based on the screened credible proteins (multiple repetitions were analyzed on the basis of proteins that could be identified), the fold change (FC) values were calculated and p-values compared by a two-tailed Students t-test. With a FC > 1.5 or a FC < 0.66 and a p-value <0.05 as the criteria, significant proteins were identified. The cloud platform of OmicsBean ([60]http://www.omicsbean.cn/) was used for omics data integration and analysis, and Gene Ontology (GO, [61]http://geneontology.org/) for functional annotation, enrichment and KEGG pathway analysis ([62]https://www.genome.jp/kegg/pathway.html) on the differentially expressed proteins. The obtained data were uploaded to iProx ([63]https://www.iprox.cn/). 3. Results We used PCA analysis for quality control of the data. Each point in [64]Fig. 1 represents a repetition of a group experiment, with different colors and shapes distinguishing different groups. The protein quantification standard curve is shown in [65]Fig. 2. The molecular weights of the proteins were determined by SDS-PAGE [[66]Fig. 3]. The relative amounts of the peptides were obtained by calculating the peak area ratios from HPLC chromatograms. After LC-MS/MS detection and the database search, the number of qualitatively different proteins was 5873. Among these, the number of proteins with FDR<1% and Abundance>1 detected was 5492. The qualitative and quantitative analysis was performed with the Shotgun proteomics method, which first used a protease to cleave the protein into peptide segments, and then used chromatography tandem mass spectrometry to detect the peptides. Through matching the peptide segment map with the theoretical map in the database, the proteins were qualitatively identified, and then the proteins were relatively quantified by the peak intensity in mass spectrometry. Fig. 1. [67]Fig. 1 [68]Open in a new tab PCA analysis for quality control of data. The protein expression in three groups was analyzed by principal component analysis (PCA). Each point in the figure represents a repetition within each group. Different colors and shapes represent different groups. 95% confidence ellipse using Hotelling T-squared. M1 (PCA-X) is a standard parameter generated by Proteome DiscovererTM 2.2 software. (For interpretation of the references to color in this figure legend, the reader is referred