Functional enrichment analyses of GSRd and SIRT1 via bioinformatics analyses
We retrieved essential information of GSRd from the Encyclopedia of Traditional Chinese Medicine (ETCM) . The gene ontology (GO) enrichment analysis of GSRd pharmacological effects and the single gene enrichment analysis of SIRT1 were conducted by using the STITCH database .
Forty-eight male guinea pigs, weighing approximately 250–300 g, were purchased from the Experimental Animal Center of Air Force Medical University (Xi’an, China). All animals had a sensitive auricle reflex, normal tympanic membrane, and no history of noise contact. They were housed in an environment with natural light, room temperature of 20–25ºC, air relative humidity of 60–65%, and free access to food and water. Adaptive feeding was administered for 5 d before the start of the experiment. All experimental procedures were approved by the Animal Ethics Committee of our university. Ginsenoside Rd (Tai-He Biopharmaceutical, Guangzhou, China) was kindly provided by the Department of Neurology in Xijing Hospital (Xi’an, China).
Guinea pigs were randomly assigned to one of four groups, each containing 12 animals: the control group (Con), which received no noise stimulation nor treatment; the noise group (NE), which was exposed to military helicopter noise but did not receive drug treatment; the GSRd treatment group (Rd), which was exposed to military helicopter noise and injected intraperitoneally with 30 mg/kg GSRd dissolved in glycerol; and the experimental control group (Vehl), which was exposed to military helicopter noise and injected intraperitoneally with just glycerol at 30 mg/kg. GSRd/glycerol was injected from 5 days before noise stimulation to the end of noise stimulation for a total of 10 days.
Noise stimulation and procedures
The ambient noise of a military helicopter was collected and input to a speaker (Soundtop SF-12; Jia-sheng Audio Equipment, Co., Ltd., Guangzhou, China) through a power amplifier (Soundtop QA-700; Jia-sheng Audio Equipment) for cyclic playback. Noise stimulation was conducted in a soundproof room with an air-conditioned fan to moisten the air and strengthen local ventilation. The guinea pigs from the experimental groups were placed in a rat cage on which a speaker was placed. The noise intensity was measured by an A-weighted sound level (Heng-sheng Electronics, Jiaxing, China) to ensure that the difference in the sound pressure level in the activity range of guinea pigs was less than 3 dB. The animals were exposed to 115 dB (A) noise stimulation for 4 hours daily for 5 consecutive days . The Con group was not exposed to noise stimulation. The background noise in the cage was less than 20 dB, and the other conditions were the same as those in the experimental groups.
After detecting the hearing threshold levels , the animals were euthanized. The bilateral temporal bones of guinea pigs were removed, and the bilateral cochleae were separated immediately. There were 24 cochlear specimens in each group of guinea pigs. The specimens were distributed as shown in Table 2.
The cochlear tissues used in the surface preparation of the basilar membrane and section staining were removed and then soaked in 4% paraformaldehyde, and those used for scanning electron microscopy (SEM) were soaked in 2.5% glutaraldehyde. The stapes were removed with microscopic forceps under an anatomical microscope. A small hole was created at the tip of the cochlea by a syringe needle. The corresponding fixed fluid was slowly injected into the cochlea through the perforated cochlear tip and the oval window more than three times. The specimens were then transferred into an Eppendorf tube containing the fixed solution overnight at 4ºC. Beginning on the second day, the 10% EDTA solution was replaced for decalcification for 14 days. The freshly decalcified solution was replaced every day until the cochlear bone softened. The decalcified cochlear tissues were used for surface preparation of the basilar membrane or were embedded in paraffin to prepare 5 µm sections parallel to the direction of the modiolus for terminal dUTP nick end labeling (TUNEL) and immunofluorescence staining. The rest of the specimens were stored at -80ºC.
Phalloidin staining was conducted, as described by Qi and colleagues . The spiral case and spiral ligament were removed with microscopic forceps under an anatomical microscope. The basilar membrane was separated step-by-step from the tip to the bottom of the cochlea and was moved into a 96-well plate. The removed basilar membranes were rinsed three times with 0.1M phosphate-buffered saline (PBS) for 1 min each time. A 100 µL of 1% Triton X-100 (MP Biomedicals, Solon, OH, USA) was subsequently added to each hole for 10 min. Specimens were rinsed three times with 0.1M PBS for 5 min each time. A phalloidin dilution (1:1000; ab176753; Abcam, Cambridge, MA, USA) was added to each well for 30 min without light. The specimens were rinsed three times with 0.1M PBS and 4,6-diamino-2-phenylindole (DAPI) (1:1000; ab104139; Abcam) and staining was conducted for 8 min without light to display the nucleus. The specimens were then rinsed with 0.1M PBS three times for 5 min each time. The basilar membrane was drawn onto the glass slide by using a pipette. The positive and negative sides of the basilar membrane were observed under a microscope (SMZ745; Nikon Corporation, Tokyo, Japan). The slides were carefully covered with an 80% glycerin seal to prevent fluorescence quenching. A confocal microscope (LSM 800; Zeiss, Oberkochen, Germany) was used to observe the experimental results.
The experimental steps of SEM corresponded to those proposed in articles by Sung et al.  and Santi et al. . The basilar membrane was soaked overnight in 2.5% glutaraldehyde at 4ºC. The specimens were rinsed three times with 0.1M PBS for 15 min each, and then postfixed in 1% osmium tetroxide for 2 h at a room temperature of 20°C–25°C. The specimens were then dehydrated with 30%, 50%, 70%, 80%, and 90% gradient ethanol and anhydrous ethanol; critical-point-dried by using liquid carbon dioxide; and then sputter-coated with gold-palladium for 30 s. The results were observed under a scanning electron microscope (S-3400; Hitachi, Tokyo, Japan).
TUNEL staining, as described by Liu and colleagues , was carried out on sections of the cochlea by using the In Situ Cell Death Detection Kit (11684795910; Roche, Basel, Switzerland), based on the manufacturer's instructions. Sections of the cochlea were deparaffinized using xylene, hydrated with anhydrous, 90%, 80%, and 70% gradient ethanol. After rinsing with distilled water, the sections were covered with proteinase K solution for 15 min. They were subsequently rinsed with 0.1M PBS and distilled water for 6 min each and fixed with 3% methanol-hydrogen peroxide solution for 20 min. Following three rinses with 0.1M PBS for 6 min each, the sections were covered with 3% bovine serum albumin-PBS solution for blocking, followed by incubation with the TUNEL reaction mixture in the dark for 1 h at a room temperature of 20°C–25°C. Finally, the sections were rinsed with PBS-Tween (PBST) buffer solution and 0.1M PBS for 6 min each. The results were examined and photographed under fluorescence microscopy (DP71; Olympus, Tokyo, Japan).
Immunofluorescence staining for 4-hydroxy-4-hydroxynonenal and 3-nitrotyrosine
Immunofluorescence staining was carried out, as described by Tian and colleagues . Sections of cochlea were deparaffinized using xylene; hydrated with anhydrous, 90%, 80%, and 70% gradient ethanol; and then rinsed three times with 0.1M PBS for 10 min each. A 1% Triton X-100 (MP Biomedicals) was added for 10 min. Three rinses were carried out using 0.1M PBS. The sections were incubated with 5% BSA (MP Biomedicals) for 1 h. The antibodies used for immunofluorescence staining included rabbit polyclonal anti-4-hydroxy-4-hydroxynonenal (anti-4-HNE) (1:1000; ab46545; Abcam) and mouse monoclonal anti-3-nitrotyrosine (anti-3-NT) (1:1000; ab61392; Abcam). Antibodies were diluted and were added to the sections. The sections were placed in a black wet box and stored overnight in a refrigerator at 4ºC. They were removed on the second day and reheated at a room temperature of 20°C–25ºC for 1 h before the three washes with 0.1M PBS. The secondary antibodies of cy3-labeled goat antirabbit immunoglobulin G (IgG) (red, 1:1000; Zhuangzhi Biotechnology, Xi’an, China) and 488-labeled goat anti-mouse IgG (green, 1:1000; Zhuangzhi Biotechnology) were added and the sections were incubated at room temperature at 20°C–25°C without light for 2 h. Another three rinses with 0.1 M PBS were applied and 8 min of DAPI (1:1000, ab104139, Abcam, USA) staining was applied without light to display the nucleus. Glycerin (80%) was used to prevent fluorescence quenching. A confocal microscope (LSM 800; Zeiss) was used for observation.
Quantitative real-time polymerase chain reaction analysis
The protocols used in this study were conducted, as described by Chen et al . RNAiso (TaKaRa, Kyoto, Japan) was added to the homogenized cochlear specimens to extract total ribonucleic acid (RNA), based on the manufacturer’s instructions. The extracted RNA was diluted to 300–500 ng/µL, and then combined with 2 µL 5 × Primer Script RT Master Mix (TaKaRa). The 10 µL reverse transcription reaction system was filled with diethyl pyrocarbonate water. The mixture was placed in a real-time system (Applied Biosystems, Waltham, MA, USA) at 37ºC for 15 min and then at 85ºC for 5 s to synthesize the complementary DNA (cDNA) template. Two microliters of the cDNA template were mixed with 20 µL of SYBR Premix Ex Taq™ II (2×) (TaKaRa), 14 µL of diethyl pyrocarbonate water, and 2 µL of the forward and reverse primers (Table 3; Sangon, China). The configured reaction system was fully blown and mixed and then was added to an octagonal tube with 9 µL per hole. The parameters were set as follows: 95ºC for 30 s, 40 cycles of 95ºC for 5 s and 60ºC for 34 s, and 65ºC for 15 s. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control and the 2−ΔΔCt method was used to calculate the relative expression of the target gene.
The protocols used in this study were conducted as described by Su et al . The left and right cochlea of the same guinea pig were placed together in a tissue homogenizer. A 200 µL protein extraction reagent (78505; Thermo Scientific, Waltham, MA, USA) containing 2mM phenylmethylsulfonyl fluoride was used as the protein lysate to extract the total cochlear proteins. The total protein concentration was calculated using the BCA Protein Assay Kit (23250; Thermo Scientific). A total of 30 µg of each protein sample was denatured, separated on 4–12% Bis-Tris PAGE gels, and then transferred to polyvinylidene fluoride membranes (0.45 µm; Millipore, Darmstadt, Germany). The membranes were blocked in 5% fat-free milk powder for 2 h at a room temperature of 20°C–25°C and were then incubated with rabbit polyclonal antibody against Bax (1:500; WL01637; Wanleibio, Shenyang, China), Bcl-2 (1:500; WL01556; Wanleibio, China), SIRT1 (1:500; WL02995; Wanleibio), PGC-1α (1:500; WL02123; Wanleibio), or β-actin (1:1000; WL01845; Wanleibio) overnight at 4ºC. After six rinses with PBST for 5 min each, the membranes were incubated in PBST with horseradish peroxidase-conjugated goat antirabbit IgG (1:5000; WLA023; Wanleibio) for 1 h at a room temperature of 20°C–25°C and were detected using enhanced chemiluminescence detection reagents (Millipore) in a Gel Image Analyzing System (Tanon Science & Technology, Shanghai, China). The band intensity was measured using Image J v1.51 (National Institutes of Health, Bethesda, MD, USA), and the values were normalized to β-actin.
Detection of superoxide dismutase activity, malondialdehyde level, and glutathione peroxidase level
The left and right cochlea of the same guinea pig were placed in the same Eppendorf tube, and the samples were prepared with 0.9% normal saline as a 10% homogenate. Superoxide dismutase (SOD) assay kit (WST-1 method; A001-3; Jiancheng Biotechnology, Nanjing, China), malondialdehyde (MDA) assay kit (TBA method; A003-1; Jiancheng Biotechnology), and glutathione peroxidase (GSH-Px) assay kit (colorimetric method; A005-1; Jiancheng Biotechnology) were used to detect SOD activity, MDA levels, and GSH-Px levels, based on the manufacturer’s instructions. Each group’s chromaticity was assessed using a microplate reader (Thermo Fisher) at 450 nm for SOD activity, 532 nm for the MDA level, and 412 nm for the GSH-Px level.
The results are presented as mean ± the standard error. The hearing level; immunofluorescence staining results; mRNA and protein expression; and SOD, MDA, and GSH-Px activities were statistically analyzed using one-way analysis of variance. Dunnett’s t-test was used to compare the experimental and control groups. Differences were significant when P < 0.05. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA) and SPSS 23.0 (IBM Corporation, Armonk, NY, USA).