Effects Of Electroacupuncture At Tinggong And Yifeng On Proteome In Auditory Center Of Guinea Pig With Age-related Hearing Loss

Objective: To explore the effects of electroacupuncture at Tinggong andYifeng on proteome in auditory center of guinea pig with age-relatedhearing loss (AHL) induced by D-galactose. Methods:1. Groups andmodel building. There were four groups.30adult guinea pigs wererandomly divided into three groups including the D-galactose group (DG,n=10), D-galactose and electroacupuncture group (DEG, n=10), andcontrol group (CG, n=10). The elderly group (EG) included1018-month-old guinea pigs. After raised for one week, guinea pigs in theD-galactose group (DG) have been subcutaneously injected D-galactose(300mg/kg·day) for6weeks. The control group (CG) received the samedose of physiological saline as D-galactose. Moreover, guinea pigs in theD-galactose and electroacupuncture group (DEG) were subcutaneouslyinjected D-galactose (300mg/kg·day), and electroacupunctured atTinggong and Yifeng for15minutes half an hour later. Finally, guineapigs in18months old group were conventionally raised.2. Audiologytest. After the experiment, the auditory brainstem response (ABR) andauditory steady state response (ASSR) were tested.3. Establishment ofproteome maps and identification of differential proteins in auditory cortex. The proteins were extracted after cell lysed and theconcentrations were determined by Bradford method. Theelectrophorograms were obtained in auditory cortex of guinea pig ofCG, DG and DEG using Two-dimensional electrophoresis (2-DE), and tendifferentially expressed proteins were selected. Then the peptidef i n g e r p r i n t i n g s w e r e o b t a i n e d b y m a t r i x a s s i s t e d l a s e rdesorption/ionization mass spectrometry (MALDI-TOF-MS), andmatched them with database from NCBI using Mascot software.4. Expressions of COMT and β-catenin in auditory cortex andinferior colliculus. The expressions of COMT and β-catenin in auditorycortex and inferior colliculus of guinea pigs were detecded by Westernblots.5. Statistical Methods. The results were analysed by the one-wayANOVA using SPSS13.0software, and P<0.05means statisticalsignificance. Results:1. Changes in weight. There was no significantchanges in the weight among guinea pigs of CG, DG and DEG (P>0.05).2. Changes in ABR. There was a significant increase in the latent periodof the third wave of ABR in the DG and18months old group (P<0.05,compared with CG) and a significant decrease in the DEG (P<0.05,compared with DG).There was no significant difference between the DGand18months old group (P>0.05).3. Changes in thresholds of ASSR.There was significant difference only between the DG and CG at the4kHz (P<0.05).4. Differentially expressed proteins in auditory cortex. Ten protein spots where there were2times higher in gray scale wereidentified, and there were eight proteins such as guanine deaminase, ATPsynthase subunit beta, alpha-enolase, pyruvate dehydrogenase E1component subunit beta, creatine kinase, tubulin beta-2Achain, triosephosphate isomerase and phosphoglycerate kinase.5. Changes in β-catenin in auditory cortex and inferior colliculus.There was a significant decrease in β-catenin expression in auditorycortex and inferior colliculus of the DG and18months old group (P<0.05,compared with CG) and a significant increase in the DEG (P<0.05,compared with DG). There was no significant difference between the DGand18months old group (P>0.05).6. Changes in COMT in auditorycortex and inferior colliculus. There was a significant decrease inCOMT expression in auditory cortex and inferior colliculus of the DGand EG (P <0.05, compared with CG), and a significant increase in theDEG (P <0.05, compared with DG). There was no significant differencebetween the DG and18months old group in auditory cortex (P>0.05).Conclusions:1. It is an effective method to build a guinea pig modelwith AHL by subcutaneously injecting D-galactose (300mg/kg·day) for6weeks.2. It is useful to electroacupuncture at Tinggong and Yifeng forinhibiting the process of AHL.3. The above eight proteins such asguanine deaminase may be involved in the occurrence and developmentof AHL in guinea pig, and also participate in inhibiting the process of AHL by electroacupuncturing at Tinggong and Yifeng.4.The COMT may play a role in the aging process induced by D-galactoseof the auditory center, and delaying the occurrence of AHL by regulatingthe metabolism of catecholamines.5. Beta-catenin may participate inthe aging process of induced by D-galactose of the auditory center, andinhibiting the occurrence of AHL by regulating cell growth, apoptosis anddifferentiation by Wnt/beta-catenin signal pathway.