Oral Antioxidant Vitamins and Magnesium Limit Noise-Induced Hearing Loss by Promoting Sensory Hair Cell Survival: Role of Antioxidant Enzymes and Apoptosis Genes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Experimental Animals
2.2. Antioxidant (ACEMg) Supplementation
2.3. Noise Exposure Protocol
2.4. Auditory Brainstem Response (ABR) Recordings
ABR Data Analysis
2.5. Real Time-Quantitative Polymerase Chain Reaction (qPCR)
2.5.1. Cochlear Dissection and RNA Extraction
2.5.2. cDNA Synthesis and qPCR
2.6. Cochlear Whole-Mount Preparations
2.7. Cochlear Immunohistochemistry
2.8. Statistical Analysis
2.9. Preparation of Figures
3. Results
3.1. ACEMg Otoprotection Against NIHL: ABR Recordings
3.1.1. Auditory Thresholds and Threshold Shift after NIHL
3.1.2. Recovery of Auditory Thresholds and Threshold Shifts after Oral Administration of ACEMg
3.2. Loss of OHCs in the Cochlea after NIHL and ACEMg Otoprotection
3.2.1. Noise-Exposed, Untreated Rats
3.2.2. Oral Administration of ACEMg
3.3. Time Expression of Antioxidant Enzymes and Apoptosis Genes in the Cochlea after NIHL and ACEMg Otoprotection
3.3.1. Noise-Exposed, Untreated Rats
3.3.2. Oral Administration of ACEMg
3.4. Distribution of Immunostaining for Antioxidant Enzymes and Apoptosis-Related Proteins in the Cochlea after NIHL and ACEMg Otoprotection
3.4.1. Noise-Exposed, Untreated Rats
3.4.2. Oral Administration of ACEMg
4. Discussion
Author Contributions
Funding
Conflicts of Interest
References
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Gene | Accesion No. | Sequence (5’–3’) | Genomic Location (Exons; FW–RV) | Bp | PCR Efficiency | R2 |
---|---|---|---|---|---|---|
Hprt1 | NM_012583.2 | FW:TCCCAGCGTCGTGATTAGTGA RV:CCTTCATGACATCTCGAGCAAG | 1/2–3 a | 152 | 97.3% | 0.9996 |
Tbp | NM_001004198.1 | FW:CCCACATCACTGTTTCATGG RV:CCGTAAGGCATCATTGGACT | 1/2–3 | 215 | 99.2% | 0.9995 |
Bax | NM_017059 | FW: CGAGCTGATCAGAACCATCA RV:CTCAGCCCATCTTCTTCCAG | 5–6 | 91 | 98.4% | 0.9994 |
Bcl-2 | NM_016993 | FW:GAGCGTCAACAGGGAGATGT RV:CTCACTTGTGGCCCAGGTAT | 1–2 | 242 | 99.3% | 1.000 |
Casp3 | NM_012922 | FW:GGCCCTGAAATACGAAGTCA RV:GGCAGTAGTCGCCTCTGAAG | 4–5 | 209 | 97.6% | 0.9986 |
Cat | NM_012520 | FW:GAGGAAACGCCTGTGTGAGA RV:TTGGCAGCTATGTGAGAGCC | 11–13 | 201 | 98.8% | 0.9997 |
Gpx1 | NM_030826 | FW:GTTTCCCGTGCAATCAGTTC RV:CATTCCGCAGGAAGGTAAAG | 1–2 | 71 | 99.3% | 0.9972 |
Sod1 | NM_017050 | FW:CCACTGCAGGACCTCATTTT RV:CACCTTTGCCCAAGTCATCT | 3–5 | 216 | 99.1% | 0.9991 |
Primary Antibody | Immunogen | Host | Code/Clone | Dilution | Manufacturer |
---|---|---|---|---|---|
Catalase | C-terminus of catalase of mouse origin | Goat | SC-34285 | 1:100 | Santacruz, Biotechnology, Inc., Dallas, TX, USA |
GPX1 | Synthetic peptide conjugated to KLH derived from within residues 150 to the C-terminus of Human GPX1 | Rabbit | ab22604 | 1:100 | Abcam plc. Cambridge, UK |
SOD1 | C-terminus of SOD-1 of human origin | Goat | SC-8637 | 1:100 | Santacruz, Biotechnology |
Bcl-2 | N-terminus of Bcl-2 of human origin | Goat | SC-492 (N19) | 1:100 | Santacruz, Biotechnology |
Groups | Frequencies (kHz) | |||||||
---|---|---|---|---|---|---|---|---|
0.5 | 1 | 2 | 4 | 8 | 16 | 32 | ||
Threshold | ND-CTR | 48.5 ± 1.0 | 44.4 ± 1.1 | 37.9 ± 1.2 | 38.7 ± 1.4 | 37.3 ± 1.5 | 41.1 ± 1.7 | 43.9 ± 1.9 |
ND-1D | 77.8 ± 1.7 | 78.3 ± 1.2 | 78.3 ± 1.2 | 79.4 ± 0.6 | 80.0 ± 0.0 | 78.9 ± 0.7 | 78.3 ± 0.8 | |
ND-10D | 75.7 ± 1.4 | 76.1 ± 1.3 | 75.4 ± 1.9 | 78.2 ± 0.8 | 77.5 ± 1.0 | 76.8 ± 1.4 | 76.8 ± 1.0 | |
ND-30D | 80.0 ± 0.0 | 80.0 ± 0.0 | 80.0 ± 0.0 | 80.0 ± 0.0 | 80.0 ± 0.0 | 79.3 ± 0.7 | 79.3 ± 0.7 | |
ED-CTR | 47.4 ± 1.0 | 42.6 ± 1.1 | 37.1 ± 1.3 | 37.8 ± 1.3 | 37.4 ± 1.5 | 39.6 ± 1.3 | 42.4 ± 1.4 | |
ED-1D | 72.9 ± 2.9 | 70.7 ± 3.4 | 69.3 ± 4.0 | 74.3 ± 1.7 | 73.6 ± 4.0 | 72.9 ± 3.9 | 73.6 ± 2.1 | |
ED-10D | 62.0 ± 1.9 | 59.0 ± 2.1 | 54.0 ± 2.3 | 60.5 ± 2.3 | 60.0 ± 2.2 | 61.0 ± 1.9 | 60.5 ± 1.6 | |
ED-30D | 61.7 ± 2.1 | 62.5 ± 1.1 | 55.0 ± 5.3 | 59.2 ± 3.0 | 61.7 ± 1.1 | 65.0 ± 1.8 | 64.2 ± 2.7 | |
Anova: F(7,40) = | 46.9 (***) | 62.8 (***) | 29.2 (***) | 86.9 (***) | 66.8 (***) | 57.5 (***) | 84.3 (***) | |
0.5 | 1 | 2 | 4 | 8 | 16 | 32 | ||
Threshold Shift | ND-1D | 30.0 ± 1.7 | 36.7 ± 1.9 | 40.6 ± 1.9 | 44.4 ± 2.4 | 46.0 ± 1.8 | 42.8 ± 1.5 | 40.6 ± 1.3 |
ND-10D | 22.5 ± 2.7 | 26.0 ± 4.0 | 32.5 ± 4.4 | 35.0 ± 3.2 | 37.5 ± 3.0 | 32.0 ± 2.7 | 30.8 ± 3.9 | |
ND-30D | 25.5 ± 2.5 | 28.9 ± 3.6 | 35.5 ± 4.9 | 38.0 ± 2.8 | 40.0 ± 2.5 | 36.0 ± 3.2 | 31.8 ± 3.3 | |
ED-1D | 21.4 ± 3.0 | 25.0 ± 3.8 | 30.9 ± 5.4 | 33.6 ± 3.6 | 30.7 ± 3.8 | 30.0 ± 4.5 | 29.3 ± 3.0 | |
ED-10D | 15.5 ± 1.7 | 19.0 ± 2.4 | 20.0 ± 3.3 | 26.0 ± 1.9 | 25.5 ± 2.6 | 23.5 ± 2.8 | 23.0 ± 2.5 | |
ED-30D | 17.0 ± 2.5 | 20.0 ± 2.4 | 19.0 ± 2.2 | 25.5 ± 2.7 | 26.5 ± 3.0 | 24.0 ± 2.3 | 22.0 ± 2.1 | |
ANOVA: F (5,30 )= | 23.5 (***) | 5.6 (***) | 5.2 (***) | 8.6 (***) | 6.8 (***) | 5.8 (***) | 9.5 (***) |
Groups | Apical | Middle | Basal | |||||
---|---|---|---|---|---|---|---|---|
ND-CTR | 100.00 ± 0.00 | 100.00 ± 0.00 | 100.00 ± 0.00 | |||||
ND-1D | 92.04 ± 1.21 | 63.45 ± 1.68 | 73.12 ± 2.35 | |||||
ND-10D | 90.16 ± 2.12 | 53.79 ± 2.93 | 67.43 ± 0.97 | |||||
ND-30D | 81.73 ± 1.22 | 41.61 ± 2.20 | 61.28 ± 2.46 | |||||
ED-CTR | 100.00 ± 0.00 | 100.00 ± 0.00 | 100.00 ± 0.00 | |||||
ED-1D | 97.20 ± 1.23 | 75.12 ± 1.16 | 86.89 ± 2.26 | |||||
ED-10D | 96.73 ± 1.53 | 67.74 ± 2.46 | 80.80 ± 0.99 | |||||
ED-30D | 93.46 ± 1.49 | 64.29 ± 1.29 | 78.22 ± 1.11 | |||||
Anova: | F(7,48) = 13.8 (***) | F(7,48) = 97.9 (***) | F(7,48) = 41.9 (***) | |||||
Significance levels | ||||||||
Apical | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 1.000 | 0.311 | 0.989 | 0.025 | 0.971 | 0.000 | 0.406 | |
ED-CTR | 1.000 | 0.269 | 0.982 | 0.020 | 0.957 | 0.000 | 0.357 | |
ND-1D | 0.311 | 0.269 | 0.852 | 0.971 | 0.915 | 0.000 | 1.000 | |
ED-1D | 0.989 | 0.982 | 0.852 | 0.230 | 1.000 | 0.000 | 0.915 | |
ND-10D | 0.025 | 0.020 | 0.971 | 0.230 | 0.311 | 0.094 | 0.938 | |
ED-10D | 0.971 | 0.957 | 0.915 | 1.000 | 0.311 | 0.000 | 0.957 | |
ND-30D | 0.000 | 0.000 | 0.000 | 0.000 | 0.094 | 0.000 | 0.000 | |
ED-30D | 0.406 | 0.357 | 1.000 | 0.915 | 0.938 | 0.957 | 0.000 | |
Middle | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 1.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ED-CTR | 1.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ND-1D | 0.000 | 0.000 | 0.049 | 0.008 | 0.994 | 0.000 | 1.000 | |
ED-1D | 0.000 | 0.000 | 0.049 | 0.000 | 0.415 | 0.000 | 0.046 | |
ND-10D | 0.000 | 0.000 | 0.008 | 0.000 | 0.003 | 0.014 | 0.066 | |
ED-10D | 0.000 | 0.000 | 0.994 | 0.415 | 0.003 | 0.000 | 0.976 | |
ND-30D | 0.000 | 0.000 | 0.000 | 0.000 | 0.014 | 0.000 | 0.000 | |
ED-30D | 0.000 | 0.000 | 1.000 | 0.046 | 0.066 | 0.976 | 0.000 | |
Basal | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 0.992 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ED-CTR | 0.992 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ND-1D | 0.000 | 0.000 | 0.034 | 0.551 | 0.845 | 0.000 | 0.998 | |
ED-1D | 0.000 | 0.000 | 0.034 | 0.000 | 0.644 | 0.000 | 0.194 | |
ND-10D | 0.000 | 0.000 | 0.551 | 0.000 | 0.023 | 0.598 | 0.168 | |
ED-10D | 0.000 | 0.000 | 0.845 | 0.644 | 0.023 | 0.000 | 0.995 | |
ND-30D | 0.000 | 0.000 | 0.000 | 0.000 | 0.598 | 0.000 | 0.000 | |
ED-30D | 0.000 | 0.000 | 0.998 | 0.194 | 0.168 | 0.995 | 0.000 |
Groups | Apical | Middle | Basal | |||||
---|---|---|---|---|---|---|---|---|
ND-CTR | 100.00 ± 0.00 | 100.00 ± 0.00 | 100.00 ± 0.00 | |||||
ND-1D | 92.04 ± 1.21 | 63.45 ± 1.68 | 73.12 ± 2.35 | |||||
ND-10D | 90.16 ± 2.12 | 53.79 ± 2.93 | 67.43 ± 0.97 | |||||
ND-30D | 81.73 ± 1.22 | 41.61 ± 2.20 | 61.28 ± 2.46 | |||||
ED-CTR | 100.00 ± 0.00 | 100.00 ± 0.00 | 100.00 ± 0.00 | |||||
ED-1D | 97.20 ± 1.23 | 75.12 ± 1.16 | 86.89 ± 2.26 | |||||
ED-10D | 96.73 ± 1.53 | 67.74 ± 2.46 | 80.80 ± 0.99 | |||||
ED-30D | 93.46 ± 1.49 | 64.29 ± 1.29 | 78.22 ± 1.11 | |||||
Anova: | F(7,48) = 13.8 (***) | F(7,48) = 97.9 (***) | F(7,48) = 41.9 (***) | |||||
Significance levels | ||||||||
Apical | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 1.000 | 0.311 | 0.989 | 0.025 | 0.971 | 0.000 | 0.406 | |
ED-CTR | 1.000 | 0.269 | 0.982 | 0.020 | 0.957 | 0.000 | 0.357 | |
ND-1D | 0.311 | 0.269 | 0.852 | 0.971 | 0.915 | 0.000 | 1.000 | |
ED-1D | 0.989 | 0.982 | 0.852 | 0.230 | 1.000 | 0.000 | 0.915 | |
ND-10D | 0.025 | 0.020 | 0.971 | 0.230 | 0.311 | 0.094 | 0.938 | |
ED-10D | 0.971 | 0.957 | 0.915 | 1.000 | 0.311 | 0.000 | 0.957 | |
ND-30D | 0.000 | 0.000 | 0.000 | 0.000 | 0.094 | 0.000 | 0.000 | |
ED-30D | 0.406 | 0.357 | 1.000 | 0.915 | 0.938 | 0.957 | 0.000 | |
Middle | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 1.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ED-CTR | 1.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ND-1D | 0.000 | 0.000 | 0.049 | 0.008 | 0.994 | 0.000 | 1.000 | |
ED-1D | 0.000 | 0.000 | 0.049 | 0.000 | 0.415 | 0.000 | 0.046 | |
ND-10D | 0.000 | 0.000 | 0.008 | 0.000 | 0.003 | 0.014 | 0.066 | |
ED-10D | 0.000 | 0.000 | 0.994 | 0.415 | 0.003 | 0.000 | 0.976 | |
ND-30D | 0.000 | 0.000 | 0.000 | 0.000 | 0.014 | 0.000 | 0.000 | |
ED-30D | 0.000 | 0.000 | 1.000 | 0.046 | 0.066 | 0.976 | 0.000 | |
Basal | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 0.992 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ED-CTR | 0.992 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ND-1D | 0.000 | 0.000 | 0.034 | 0.551 | 0.845 | 0.000 | 0.998 | |
ED-1D | 0.000 | 0.000 | 0.034 | 0.000 | 0.644 | 0.000 | 0.194 | |
ND-10D | 0.000 | 0.000 | 0.551 | 0.000 | 0.023 | 0.598 | 0.168 | |
ED-10D | 0.000 | 0.000 | 0.845 | 0.644 | 0.023 | 0.000 | 0.995 | |
ND-30D | 0.000 | 0.000 | 0.000 | 0.000 | 0.598 | 0.000 | 0.000 | |
ED-30D | 0.000 | 0.000 | 0.998 | 0.194 | 0.168 | 0.995 | 0.000 |
Groups | Bax | Bcl-2 | Casp3 | |||||
---|---|---|---|---|---|---|---|---|
ND-CTR | 0.95 ± 0.04 | 1.02 ± 0.03 | 0.95 ± 0.03 | |||||
ND-1D | 0.99 ± 0.04 | 1.00 ± 0.03 | 1.30 ± 0.05 | |||||
ND-10D | 1.63 ± 0.09 | 1.55 ± 0.07 | 1.71 ± 0.07 | |||||
ND-30D | 1.14 ± 0.04 | 1.13 ± 0.04 | 1.24 ± 0.07 | |||||
ED-CTR | 1.02 ± 0.03 | 1.01 ± 0.04 | 1.00 ± 0.01 | |||||
ED-1D | 0.89 ± 0.02 | 0.92 ± 0.04 | 1.22 ± 0.04 | |||||
ED-10D | 1.42 ± 0.05 | 1.90 ± 0.08 | 1.32 ± 0.04 | |||||
ED-30D | 0.86 ± 0.02 | 1.05 ± 0.04 | 1.06 ± 0.02 | |||||
Anova: | F(7,274) = 35.7 (***) | F(7,254) = 45.8 (***) | F(7,266) = 35.8 (***) | |||||
Significance levels | ||||||||
Bax | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 0.922 | 0.999 | 0.984 | 0.000 | 0.000 | 0.161 | 0.886 | |
ED-CTR | 0.922 | 1.000 | 0.346 | 0.000 | 0.000 | 0.793 | 0.162 | |
ND-1D | 0.999 | 1.000 | 0.896 | 0.000 | 0.000 | 0.675 | 0.722 | |
ED-1D | 0.984 | 0.346 | 0.896 | 0.000 | 0.000 | 0.014 | 1.000 | |
ND-10D | 0.000 | 0.000 | 0.000 | 0.000 | 0.249 | 0.000 | 0.000 | |
ED-10D | 0.000 | 0.000 | 0.000 | 0.000 | 0.249 | 0.006 | 0.000 | |
ND-30D | 0.161 | 0.793 | 0.675 | 0.014 | 0.000 | 0.006 | 0.005 | |
ED-30D | 0.886 | 0.162 | 0.722 | 1.000 | 0.000 | 0.000 | 0.005 | |
Bcl-2 | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 1.000 | 1.000 | 0.954 | 0.000 | 0.000 | 0.946 | 1.000 | |
ED-CTR | 1.000 | 1.000 | 0.952 | 0.000 | 0.000 | 0.886 | 1.000 | |
ND-1D | 1.000 | 1.000 | 0.994 | 0.000 | 0.000 | 0.917 | 1.000 | |
ED-1D | 0.954 | 0.952 | 0.994 | 0.000 | 0.000 | 0.373 | 0.823 | |
ND-10D | 0.000 | 0.000 | 0.000 | 0.000 | 0.009 | 0.003 | 0.000 | |
ED-10D | 0.000 | 0.000 | 0.000 | 0.000 | 0.009 | 0.000 | 0.000 | |
ND-30D | 0.946 | 0.886 | 0.917 | 0.373 | 0.003 | 0.000 | 0.988 | |
ED-30D | 1.000 | 1.000 | 1.000 | 0.823 | 0.000 | 0.000 | 0.988 | |
Casp3 | ND-CTR | ED-CTR | ND-1D | ED-1D | ND-10D | ED-10D | ND-30D | ED-30D |
ND-CTR | 0.981 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.486 | |
ED-CTR | 0.981 | 0.000 | 0.004 | 0.000 | 0.000 | 0.003 | 0.951 | |
ND-1D | 0.000 | 0.000 | 0.955 | 0.000 | 1.000 | 0.997 | 0.010 | |
ED-1D | 0.000 | 0.004 | 0.955 | 0.000 | 0.745 | 1.000 | 0.222 | |
ND-10D | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | |
ED-10D | 0.000 | 0.000 | 1.000 | 0.745 | 0.000 | 0.949 | 0.002 | |
ND-30D | 0.000 | 0.003 | 0.997 | 1.000 | 0.000 | 0.949 | 0.149 | |
ED-30D | 0.486 | 0.951 | 0.010 | 0.222 | 0.000 | 0.002 | 0.149 |
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Alvarado, J.C.; Fuentes-Santamaría, V.; Melgar-Rojas, P.; Gabaldón-Ull, M.C.; Cabanes-Sanchis, J.J.; Juiz, J.M. Oral Antioxidant Vitamins and Magnesium Limit Noise-Induced Hearing Loss by Promoting Sensory Hair Cell Survival: Role of Antioxidant Enzymes and Apoptosis Genes. Antioxidants 2020, 9, 1177. https://doi.org/10.3390/antiox9121177
Alvarado JC, Fuentes-Santamaría V, Melgar-Rojas P, Gabaldón-Ull MC, Cabanes-Sanchis JJ, Juiz JM. Oral Antioxidant Vitamins and Magnesium Limit Noise-Induced Hearing Loss by Promoting Sensory Hair Cell Survival: Role of Antioxidant Enzymes and Apoptosis Genes. Antioxidants. 2020; 9(12):1177. https://doi.org/10.3390/antiox9121177
Chicago/Turabian StyleAlvarado, Juan C., Verónica Fuentes-Santamaría, Pedro Melgar-Rojas, María C. Gabaldón-Ull, José J. Cabanes-Sanchis, and José M. Juiz. 2020. "Oral Antioxidant Vitamins and Magnesium Limit Noise-Induced Hearing Loss by Promoting Sensory Hair Cell Survival: Role of Antioxidant Enzymes and Apoptosis Genes" Antioxidants 9, no. 12: 1177. https://doi.org/10.3390/antiox9121177
APA StyleAlvarado, J. C., Fuentes-Santamaría, V., Melgar-Rojas, P., Gabaldón-Ull, M. C., Cabanes-Sanchis, J. J., & Juiz, J. M. (2020). Oral Antioxidant Vitamins and Magnesium Limit Noise-Induced Hearing Loss by Promoting Sensory Hair Cell Survival: Role of Antioxidant Enzymes and Apoptosis Genes. Antioxidants, 9(12), 1177. https://doi.org/10.3390/antiox9121177