Involvement Of The SIRT1/PGC-1α Signaling Pathway In Military Aviation Noise-induced Hidden Hearing Loss

Hidden hearing loss(HHL),a recently reported auditory disorder,exhibits a normal hearing threshold,but leads to hearing problems such as tinnitus and hyperacusis,and influences the ability to understand speech in the presence of loud background noise.The main damage of this type of auditory dysfunction lies on the ribbon synapse,which is composed of IHCs and SGNs.The synapse acts as an excitatory synapse connecting auditory receptors and plays a decisive role in the transmission and encoding of sound.The aircrew and ground crew of military helicopter are inevitably exposed to high intensity and low frequency noise environment,so the auditory system faces a higher risk of damage.No clinically applicable diagnostics or therapeutics for HHL have been approved yet,and the exact mechanism is still not revealed.The development of HHL might be associated with oxidative stress.As a sensor for regulating intracellular oxidative stress,SIRT1 plays a vital role in the redox system by deacetylating certain substrates.The aberrant expression of SIRT1 could result in oxidative stress in cochleae and hearing loss.We aim to build the animal model of military aviation noise-induced hidden hearing loss(NIHHL)by palying military helicopter noise,evaluate the dynamic changes in cochlear ribbon synapses and cochlear hair cell morphology,and observe the involvement of the SIRT1/PGC-1α signaling pathway in NIHHL,so as to reveal the pathogenesis of HHL and put forward the prevention strategy.Experiment 1.Effects of medium and low intensity military aviation noise on auditory function ObjectiveTo establish an animal model of military aviation noise-induced hidden hearing loss and evaluate it from a functional perspective,and provide a stable animal model for the morphological observation and molecular mechanism studies.Methods100d B(A)group,105 d B(A)group and 110 d B(A)group were three different noise intensity groups.Different noise intensity groups included the control group,1day(d),1week(w),2 w and 1 month(m)post noise exposure(PE)groups.Six guinea pigs were in each subgroup.Apart from the control group,the guinea pigs in each experimental group were stimulated by military helicopter noise with corresponding intensity for 2h.The auditory function was appraised with ABR and DPOAE.The hearing threshold,amplitudes of wave I and the latency of wave Ⅰ at 80 d B were analyzed by ABR test.The DPOAE amplitudes of 8 points including 500 Hz,750Hz,1k Hz,2k Hz,3k Hz,4k Hz,6k Hz and 8k Hz were analyzed by DPOAE test.ResultsFor 100 d B(A)group,the hearing threshold of guinea pigs could return to the normal level at 2w PE,amplitudes of wave I could also return to the normal level,the latency of wave Ⅰ showed no statistical differences.After 105 d B SPL military helicopter noise exposure for 2 hours,the hearing threshold of guinea pigs could return to the normal level at 2w PE,amplitudes of wave I could not return to the normal level at 1m PE,the latency of wave Ⅰ showed no statistical differences.After 110 d B SPL military helicopter noise exposure for 2 hours,the hearing threshold of guinea pigs could not return to the normal level at 1m PE,amplitudes of wave I could not return to the normal level at 1m PE,the latency of wave Ⅰ showed no statistical differences.After 100 d B SPL military helicopter noise exposure for 2 hours,DPOAE amplitudes could return to the normal level at 2w PE.After 105 d B SPL military helicopter noise exposure for 2 hours,DPOAE amplitudes in 500 Hz,750 Hz,1k Hz,2k Hz,3k Hz,6k Hz and8 k Hz returned to the normal level at 1w PE,and DPOAE amplitudes in 4k Hz could not return to the normal level at 1m PE.After 110 d B SPL military helicopter noise exposure for2 hours,DPOAE amplitudes in 500 Hz,750 Hz,1k Hz,2k Hz and 3k Hz returned to the normal level at 1w PE,and DPOAE amplitudes in 4k Hz,6k Hz and 8k Hz could not return to the normal level at 1m PE.ConclusionExposure to 105 d B SPL military helicopter noise for 2 hours causes a temporary threshold shift,and the decrease of ABR wave Ⅰ amplitude.From a functional point of view,the ideal stimulus parameters of the military aviation NIHHL model are determined,and a good animal model could be provided for further researches on military aviation NIHHL.Experiment 2.Effects of medium and low intensity military aviation noise on peripheral auditory structure ObjectiveTo evaluate the dynamic changes in cochlear ribbon synapses and cochlear hair cell morphology in an animal model of military aviation NIHHL and explore the characteristics of military aviation NIHHL in peripheral hearing system.MethodsThe control group and 1day(d),1week(w),2 w and 1 month(m)post noise exposure(PE)groups were five different groups,and six guinea pigs were in each group.The experimental groups received noise stimulation at 105 d B SPL for 2 h.Ribbon synapses were evaluated by inner ear basilar membrane preparation and immunofluorescence.The cochlear morphology was evaluated using scanning electron microscopy.ResultsThe count of ribbon synapses decreased significantly at 1d PE.Although it exhibited a trend of recovery,it remained lower at 1m PE.The basal turn was more vulnerable than the other three turn.The count of hair cells stayed almost intact until 1 month post noise exposure without significant loss.The stereocilia were chaotic after noise exposure and then returned to normal at 1m PE.ConclusionAfter 105 d B SPL military helicopter noise exposure for 2 hours,ribbon synapse density decreases,and the stereocilia are chaotic and then return to normal.The count of hair cells shows no loss.Experiment 3.Involvement of the SIRT1/PGC-1α signaling pathway in military aviation noise-induced hidden hearing loss ObjectiveTo observe the involvement of the SIRT1/PGC-1α signaling pathway in NIHHL and explore the molecular mechanism of military aviation NIHHL.MethodsThe control group and 1day(d),1week(w)and 2 w post noise exposure(PE)groups were four different groups,and 15 guinea pigs were in each group.The experimental groups received noise stimulation at 105 d B SPL for 2 h.Western blotting analysis was used to explore the SIRT1/PGC-1α signaling.The location of SIRT1 and PGC-1α was detected by immunofluorescence.The levels of SOD,MDA,CAT,ATP and SIRT1 activity were measured using a commercial testing kit.ResultsThe expression and activity of SIRT1 decreased at 1d PE,and could not return to the normal level at 2w PE.The expression of PGC-1α decreased at 1w PE,and could not return to the normal level at 2w PE.The expression of p53 showed no statistical differences,and the expression of ac-p53 increased to a level higher than that in the control group.The expression of SIRT1 and PGC-1α decreased significantly after noise exposure,which are mainly distributed in the organ of Corti and spiral ganglion.SOD,CAT and ATP were also influenced by noise exposure and were lower than those in the control group,but MDA showed no statistical differences compared with the control group.ConclusionAfter 105 d B SPL military helicopter noise exposure for 2 hours,the SIRT1/PGC-1αsignaling pathway and oxidative stress might mutually affect the pathological process of NIHHL.Experiment 4.Effects of resveratrol on military aviation noise-induced hidden hearing loss ObjectiveTo observe the effects of resveratrol on military aviation noise-induced hidden hearing loss and explore the involvement of the SIRT1/PGC-1α signaling pathway in NIHHL.MethodsGuinea pigs were randomly assigned into 3 groups: a control group,which received no noise exposure but vehicle(6.67% DMSO in PBS);a noise exposure group(NE),which received noise exposure and vehicle(6.67% DMSO in PBS);a resveratrol treatment group(RES),which received noise exposure and orally administered with resveratrol at the dose of 50 mg/kg body weight by using a gastric intubation once daily for 5 days until noise exposure.The animals in the NE group or the RES group were divided into four subgroups:1day(1d),1 week(1w),2 weeks(2w),and 1 month(1m)post-noise exposure(PE)groups.The experimental groups received noise stimulation at 105 d B SPL for 2 h.The auditory function was appraised with ABR.Ribbon synapses were evaluated by inner ear basilar membrane preparation and immunofluorescence.The cochlear morphology was evaluated using scanning electron microscopy.Western blotting analysis was used to explore the SIRT1/PGC-1α signaling.The location of SIRT1 and PGC-1α was detected by immunofluorescence.The levels of SOD,MDA,CAT,ATP and SIRT1 activity were measured using a commercial testing kit.ResultsAfter resveratrol treatment,SIRT1 expression and activity showed a significant increase after noise exposure,compared with the noise exposure group.In parallel,the PGC-1α and antioxidant proteins were also significantly altered after noise exposure,compared with the noise exposure group.The damage to the ribbon synapses and the stereocilia were attenuated by resveratrol as well.More importantly,the auditory function,especially ABR wave I amplitudes,was also promoted in the resveratrol treatment group.ConclusionResveratrol has an intervention effect on the occurrence and development of military aviation noise-induced hidden hearing loss and can protect the auditory system.The mechanism may be that resveratrol can activate SIRT1/PGC-1α signaling pathway,upregulate the expression of SIRT1/PGC-1α,improve the activity of antioxidant enzymes,and enhance the antioxidant capacity of cochlea.