| Objective: To explore the changes of the proteome, the effects and mechanisms of electroacupuncture at auricular points on the proteome in auditory cortex(AC), and the changes of the expressions of Prohibition and Endophilin-A1 in AC and inferior colliculus(IC) of guinea pig following chronic kanamycin-induced deafness before and after electroacupuncture.Methods:1. Animal groups.105 healthy adult guinea pigs were randomly divide into three groups, including control group(CG, n=7), kanamycin treatment group(KTG, n=49)and kanamycin sulfate+electroacupuncture at auricular points(KEG, n=49). Guinea pigs were treated with saline at 500mg/kg per day for 7 days in CG. Guinea pigs were treated with kanamycin by subcutaneous injection at 500mg/kg per day for 7 day in KTG and KEG. Moreover, guinea pigs were electroacupunctured at Ting gong and Yi feng for 15 minutes half an hour of kanamycin treatment in KEG. Depending on the time of specimen collection, the KTG and KEG were divided into 7 groups, including 1, 7, 14, 28, 56, 70 and 140 days group. 2. Establishment of the proteomic maps and identification of differentially expressed proteins in AC. The cells were lysed and concentration was determined in AC by Bradford method. The electrophorograms were obtained in AC of CG, KTG and KEG using Two-dimensional electrophoresis, and twenty one differentially expressed proteins were selected. Then the peptide fingerprints were picked up by MALDI-TOF-MS, andproteins were identified using the Mascot matching software and by retrieving the NCBI databases.3.Expressions of Prohibition and Endophilin-A1 in AC and IC. The expressions of Prohibition and Endophilin-A1 in AC and IC were detected by Western blots. 4. Statistical Methods. Data are expressed by x ±s, and the statistical software is SPSS20.0.Theresults were analyzed by ANOVA, P<0.05 means statistical significance, and T-test was selected to analyze the differences between two groups. Results: 1. Twenty one protein spots where there were 2 times higher in gray scale were identified, and there were eighteen proteins such as 14-3-3protein, Hippocalcin-like protein 1, Visinin-like protein 1, Gamma-soluble NSF attachment protein, 78 k Da glucose-regulated protein, ATP synthase subunit alpha, Stathmin, Keratin, type Ⅱ cytoskeletal 1, Calretinin, Endophilin-A1, Pyridoxal phosphate phosphatase, Spectrin, Heterogeneous nuclear ribonucleo protein C, Creatine kinase B-type, Keratin, type Ⅰ cytoskeletal 10,Heat shock 70 k Da Protein cognate 3, Prohibitin,Zinc finger and BTB Domain-containing protein. 2. Changes in Prohibitin and Endophilin-A1 expression in AC: The expressions of Prohibitin were 1.11±0.13 in CG, 1.05±0.14, 1.01±0.10, 0.92±0.09, 0.88±0.16, 0.85±0.14, 0.94±0.15 and 1.08±0.13 in KTG, and 0.82±0.11, 1.32±0.14, 0.97±0.13, 1.08±0.12, 1.01±0.11, 1.20±0.16 and 1.09±0.07 in KEG respectively at 1, 7, 14, 28, 56, 70 and 140 days. There were no effects of kanamycin(F=2.185, P=0.075), and significant effects of elecroacupuncture(F=7.490, P<0.001) on the expression of Prohibitin in AC. The expression of Prohibitin declined in KTG at 14, 28 and 56 days(P<0.05, compared with the CG) and increased in KEG at 7 and 70 days(P<0.05, compared with the KTG). Moreover, the expressions of Endophilin-A1 were 0.42±0.10 in CG, 0.64±0.02, 0.44±0.04, 0.58±0.10, 0.52±0.03, 0.45±0.04, 0.42±0.02 and 0.42±0.04 in KTG, and 0.56±0.06, 0.47±0.04, 0.46±0.02, 0.42±0.06, 0.35±0.02, 0.43±0.06 and 0.43±0.06 in KEG respectively at 1, 7, 14, 28, 56, 70 and 140 days. There were significant effects of kanamycin(F=12.823, P<0.001) and elecroacupuncture(F=6.421, P=0.001) on the expression of Endophilin-A1 in AC. The expression of Endophilin-A1 increased in KTG at 1 and 14 days(P<0.05, compared with the CG) and declined in KEG at 14, 28 and 56 days(P<0.05, compared with the KTG). 3. Changes in Prohibitin and Endophilin-A1 expression in IC: The expressions of Prohibitin were 1.29±0.07 in CG, 0.92±0.08, 1.02±0.14, 0.78±0.10, 0.81±0.07, 0.76±0.08, 0.98±0.08 and 1.33±0.10 in KTG, and 1.02±0.09, 1.30±0.13, 0.79±0.08, 1.17±0.14, 1.42±0.13, 1.04±0.17 and 1.33±0.10 in KEG respectively at 1, 7, 14, 28, 56, 70 and 140 days. There were significant effects of kanamycin(F=17.210, P<0.001) and elecroacupuncture(F=12.544,P<0.001) on the expression of Prohibitin in IC. There was a decrease in the expression of Prohibitin in KTG at 1, 7, 14, 28, 56 and 70 days(P<0.05), and an increase in KEG at 7, 28 and 56 days(P<0.05) respectively in comparison with the CG and KTG. Moreover, the expressions of Endophilin-A1 were 0.95±0.05 in CG, 0.97±0.09, 0.98±0.08, 1.52±0.14, 1.14±0.11, 0.95±0.08, 0.99±0.10 and 0.96±0.11 in KTG, and 0.96±0.08, 0.99±0.10, 0.95±0.10, 0.98±0.09、0.92±0.02, 0.72±0.06 and 0.97±0.10 in KEG respectively at 1, 7, 14, 28, 56, 70 and 140 days. There were effects of elecroacupuncture(F=5.476, P<0.05) and kanamycin(F=15.679, P<0.001) on the expression of Endophilin-A1 in IC. There was an increase of the expression of Endophilin-A1 in KTG at 14 and 28 days(P<0.05), and a decline in KEG at 14 and 70 days(P<0.05) respectively in comparison with the CG and KTG. Conclusions: 1. The eighteen proteins such as 14-3-3protein may be involved in the occurrence and development of the chronic kanamycin-induced deafness, and also participate in inhibiting the process of chronic kanamycin-induced deafness by electroacupuncturing at Tinggong and Yifeng in guinea pig. 2. The Prohibitin may play a role in the process of chronic kanamycin-induced deafness by regulating the function of mitochondrial, conducting the generation of ROS, and adjusting the susceptibility to damage of neurons. Electroacupuncture at Tinggong and Yifeng may enhance the recovery of function of AC and IC by increasing the expression of Prohibitin. 3. Endophilin-A1 may participate in the process of chronic kanamycin-induced deafness by adjusting the signal transduction pathways, and electroacupuncture at auricular points may inhibiting the occurrence of the chronic kanamycin-induced deafness by reducing the expression of Endophilin-A1 in AC and IC. |
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