Caffeine Induces Autophagy And Apoptosis In Auditory Hair Cells Via The SGK1/HIF-1α Pathway

In our daily lives,caffeine is increasingly used in drinks,cosmetics,and medicine.The primary function of caffeine is as an efficient adenosine receptor antagonist to quickly stimulate the central nervous system,so it is also closely related to neurologic disease.According to previous studies,caffeine may be detrimental to hearing after acoustic trauma,suggesting the harmful effect of caffeine on hearing.Nevertheless,caffeine’s main mechanism for causing hearing loss remains unknown.Firstly,we explored the difference in auditory brainstem response(ABR),organ of Corti,stria vascularis,and spiral ganglion neurons between the control and caffeine-treated groups of C57BL/6 mice.RNA sequencing was conducted to profile mRNA expression differences in the cochlea of control and caffeine-treated mice.The results of RNA-Seq were verified by qRT-PCR and Western blotting in cochleas to find the differential genes that may play a vital role in this process.Then we use the HEI-OC1 cell line to verify it in vitro.A CCK-8 assay was used to evaluate the approximate concentration range of caffeine.Flow cytometry,TUNEL assay,immunocytochemistry,qRT-PCR,and Western blotting were performed to detect the effects of the differential gene in HEI-OC1 cells.Finally,the direct effect of caffeine on hair cells was further verified by the basilar membrane model in vitro.In vivo research showed that 120 mg/kg caffeine injection caused hearing loss by damaging the organ of Corti and spiral ganglion neurons.RNA-seq results suggested that SGK1 might play a vital role in ototoxicity.The low dose of caffeine has no apparent damage to hearing.The results of RNA-Seq suggested that SGK1 may play a key role in caffeine-induced ototoxicity and that caffeine directly increased the level of autophagy and apoptosis in the cochlea.Cell experiments in vitro confirmed the same results.The results of flow cytometry,TUNEL assay,immunocytochemistry,qRT-PCR,and Western blotting showed that caffeine caused autophagy and apoptosis via SGK1 pathway.We verified the interaction between SGK1 and HIF-la by co-IP.To confirm the role of SGK1 and HIF-1α,GSK650394 was used as an inhibitor of SGK1 and CoCl2 was used as an inducer of HIF1α.Western blotting analysis showed GSK650394 could inhibit the expression of HIF-1α,while no significant change was observed in the expression of SGK1 after CoCl2 treatment.Both GSK650394 and CoCl2 could relieve the caffeine-induced apoptosis and autophagy.When caffeine was directly applied to the basilar membrane model in vitro,we found that caffeine mainly damaged the inner hair cells of the basilar membrane in neonatal rats.In summary,these results indicated that caffeine induces autophagy and apoptosis in auditory hair cells via the SGK1/HIF-1α pathway,suggesting that high doses of caffeine may cause hearing loss.Additionally,our findings provided new insights into ototoxic drugs,demonstrating that SGK1 and its downstream pathways may be potential therapeutic targets for hearing research at the molecular level.