| Age-related hearing loss(ARHL)is one of the most common chronic health problems that faces an aging population.Inflammation is an important pathological characteristic in ARHL,but the molecular mechanisms that cause inflammatory responses in this disease are unclear.Recent studies have shown that abnormal activation of cyclic GMP-AMP synthase(cGAS)-interferonstimulated gene(STING)pathway is closely related to neuroinflammation in various neurodegenerative diseases,however,whether they drive the inflammatory response in ARHL is unclear.Melatonin is an endogenous free radical scavenger synthesized by neuronal mitochondria,whose synthesis decreases with aging and neurodegeneration.Insufficient levels of melatonin impair mitochondrial homeostasis,leading to the release of mitochondrial DNA(mtDNA)into the cytosol,inducing cGAS-STING pathway activation to promote type I interferon response and inflammatory cytokine expression.Study shows melatonin supplementation helps delay ARHL progression,however,the mechanism of treatment remains unclear.In conclusion,we propose the following hypothesis:As the auditory system ages,mtDNA release increases and the cGASSTING pathway is activated,thereby promoting the inflammatory responses of ARHL.Melatonin may delay ARHL by reducing mtDNA release,inhibiting the cGAS-STING pathway and inflammatory responses of the aging auditory pathway.To test these hypotheses,we used C57BL/6J mice of different months of age and mice supplemented with long-term melatonin to evaluate the auditory function of each mouse by using the auditory brainstem response(ABR)technique and the levels of cytosolic mtDNA,cytokines,and cGAS-STING signaling in mouse cochlea,inferior colliculus,and auditory cortex by quantitative real-time polymerase chain reaction(qRT-PCR),western blotting and immunohistochemical techniques.Results showed that the ABR thresholds of C57BL/6J mice increased with age,and cytosolic mtDNA,inflammatory cytokines(IFNβ,Cxcl10,Ifit3,IL-6,TNF-α),and cGAS/STING/interferon regulatory factor 3(IRF3)expression were upregulated in the cochlea,inferior colliculus,and auditory cortex tissue of aging mice.Melatonin treatment significantly decreased cytosolic mtDNA levels,reduced the expression of inflammatory cytokines IL-6,TNF-α,IFN-β,Ifit3,and Cxc110,down-regulated cGAS/STING/IRF3 expression in the cochlea,inferior colliculus,and auditory cortex of aging mice,and delayed ARHL progression.These results suggest that cytosolic mtDNA-mediated activation of cGAS-STING pathway may be a potential mechanism of increased inflammation in the auditory pathway that leads to ARHL.Melatonin may delay ARHL progression by inhibiting cytosolic mtDNA and cGAS-STING pathway in the auditory pathway.PartⅠ Age-related changes of cGAS-STING signaling in the mouse auditory pathway and presbycusisBackground ARHL is mainly associated with nerve cell degeneration in the peripheral and/or central auditory systems.During aging,the auditory system shows mitochondrial dysfunction and increased inflammatory responses.Mitochondrial dysfunction promotes leakage of mtDNA into the cytosol,which activates the cGAS-STING pathway to induce type I interferon and inflammatory responses.However,whether this pathway is involved in the occurrence and development of ARHL is unknown.Objective To evaluate whether there are age-related changes in the expression of cytosolic mtDNA and cGAS-STING pathway in the mouse auditory pathway,and to preliminarily explore their relationship with ARHL.In order to further determine the related genes of auditory aging and explore the molecular mechanism of ARHL pathogenesis to provide experimental basis.Methods Sixty C57BL/6J mice of different months were divided into the following four groups:3-,6-,9-,and 12-month groups.Auditory function was evaluated by measuring the ABR thresholds,and cytosolic mtDNA,cytokines(IFNβ,Cxc110,Ifit3,IL-6,TNF-α),and cGAS/STING/IRF3 expression level in the cochlea,inferior colliculus and auditory cortex of mice of different age groups were detected by qRT-PCR and western blotting.The expression and localization of cGAS in the cochlea,inferior colliculus and auditory cortex were detected by immunohistochemical techniques.Results ABR results showed that the ABR thresholds of C57BL/6J mice increased with age.The hearing thresholds at 12 kHz,20 kHz,and 36 kHz and clicks were significantly higher in 6-month-old mice compared with 3-month-old mice,but not for 6 kHz.ABR thresholds were significantly higher among 9-and 12-month-old mice compared with 3-month-old mice at all frequencies tested.Immunohistochemical results showed that cGAS expression was present both on the membrane and in the cytoplasm around the nucleus of the spiral ganglion neurons,organ of Corti,stria vascularis of the cochlea,throughout all three subdivisions of the inferior colliculus,and the entire auditory cortex.The expression of cytosolic mtDNA,cGAS,STING,IRF3 and cytokines IFNβ,Cxcl10,Ifit3,IL-6,TNF-α mRNA in the cochlea,inferior colliculus,and auditory cortex of 6-,9-,and 12-month-old mice were higher than of 3-month-old mice.The protein expression levels of cGAS,STING and p-IRF3 in the cochlea,inferior colliculus and auditory cortex were significantly increased in 6-,9-,and 12month-old mice compared with 3-month-old mice.Conclusions Increased mtDNA release,cGAS-STING pathway activation and enhanced inflammatory responses in aging auditory pathways.PartⅡ Melatonin inhibits cGAS-STING signaling and delays the development of presbycusis in miceBackground Inflammation is an important pathological characteristic in ARHL,The overactivation of cGAS-STING pathway leads to neuroinflammation and contributes to the progression of neurodegenerative diseases.Activation of cGASSTING pathway and enhanced inflammatory responses were also detected in the aging auditory pathway.We also detected cGAS-STING pathway activation and enhanced inflammatory response in the aging auditory pathway,and found increased cytosolic mtDNA,we speculate that cytosolic mtDNA may play an important role in the pathological process of ARHL by activating cGAS-STINGmediated type I interferon and inflammatory response,and may be the underlying cause of ARHL.It is currently known that melatonin can inhibit the release of mtDNA in neurons,inhibit the cGAS-STING pathway and the expression of proinflammatory cytokines,thereby delaying central nervous system diseases.At the same time,there was an inverse association between plasma levels of melatonin and ARHL,and melatonin supplementation could help delay ARHL progression.Objective To investigate whether melatonin can delay the progression of ARHL by inhibiting cGAS-STING signaling and inflammatory factors in the auditory pathway,thereby reducing the inflammatory response.Methods Sixty 3-month-old C57BL/6J mice were divided into the following four groups:younger group(3 months old),elder group(12 months old),melatonin group,and vitamin C group,15 mice in each group.The elder group received oral ethanol vehicle solution until the age of 12 months,the melatonin group received oral melatonin 10 mg/kg/d until the age of 12 months,and the vitamin C group received oral vitamin C 150 mg/kg/d until the age of 12 months.ABR thresholds were measured at clicks and at 6 kHz,12 kHz,20 kHz,and 36 kHz at 3,6,9,and 12 months of age,respectively.After the last ABR test,the cochlea,inferior colliculus,and auditory cortex were taken,and the levels of cytosolic mtDNA,cGAS,STING,IRF3,and cytokines(IFNβ,Cxcl10,Ifit3,IL6,TNF-α)were investigated by qRT-PCR,The protein levels of cGAS,STING,IRF3 and p-IRF3 were analyzed by western blotting technique.Results The ABR test results showed no differences in ABR thresholds between groups of animals at 3 months of age.At 6 months of age,no statistically significant differences were found in hearing thresholds at all frequencies tested between the melatonin group,the vitamin C group,and the elder group.At 9 months of age,the ABR thresholds at 6 kHz and 36 kHz and clicks were significantly lower in melatonin group and vitamin C group compared with elder group.At 12 months of age,mice in the melatonin and vitamin C groups had significantly lower hearing thresholds than the elder group at all frequencies tested.qRT-PCR results showed that compared with the younger group,the cytosolic mtDNA levels in the cochlea,inferior colliculus and auditory cortex tissue of the elder group increased,cGAS,STING,IRF3 and cytokines IFNβ,Cxcl10,Ifit3,IL-6,TNF-α mRNA expression increased,and melatonin or vitamin C treatment prevented these changes.Western blot results showed that the expression levels of cGAS,STING and p-IRF3 proteins in the cochlea,inferior colliculus and auditory cortex tissue of the elder mice were significantly higher than those of the younger mice,and melatonin or vitamin C treatment significantly reduced the expression levels of these proteins.Conclusion Melatonin has a protective effect on hearing impairment in aging,and its possible mechanisms may be through reducing the release of mtDNA,inhibiting the cGAS-STING pathway,and reducing the inflammatory response in the auditory pathway. |
