Mechanism Of Erlong Zuoci Wan Inhibiting Age-Related Hearing Loss Through Regulation Of Autophagy And Apoptosis Via MiR-34a/SIRT1/p53 Pathway

Objective:To explore the effects and mechanisms of Erlong Zuoci Wan（ELZC）on age-related hearing loss through regulation of autophagy and apoptosis via miR-34a/SIRT1/p53 pathway.Methods:1.Ex vivo:Organ of Corti explants were divided into 5 goups in random:a normal control group,a H₂O₂group and 3 H₂O₂+ELZC groups（5μg/m L,10μg/m L and50μg/m L）.Tissues were immunolabeled with phalloidin-TRTIC and a mouse monoclonal antibody against neurofilament to label the stereocilia and nerve fibers,respectively.For cochleograms,the numbers of the missing or surviving inner hair cells（IHC）and outer hair cells（OHC）were counted in continuous 0.24 mm segments from the apex to the base of the cochlea under a fluorescence microscope.Fluorescence immunohistochemical technique was used to observe the protein expression in hair cells.Apoptosis was examined by TUNEL method.Western blot was used to evaluate the protein expression in cochlear organotypic cultures.2.In vivo:The animals were randomly divided into 3 groups:the control group,ARHL group and ELZC group.Auditory brainstem responses（ABR）were used to detect the changes of hearing thresholds in C57BL/6J mice.HE staining was used to evaluate the morphologic changes.Fluorescence immunohistochemical technique was used to detect the protein expression in the spiral ganglion neurons in mice.TUNEL method was used to test apoptosis.3.In vitro:MiR-34a mimic,miR-34a mimic negative control,and miR-34a inhibitor were transfected with HEI-OC1 cells by adding 10,20,and 50μg/m L of ELZC Real-time q PCR technique was used to test the expression of miR-34a.Cell vibility was examined by CCK-8 method.Western blot was used to observe the protein expression,as well as the effects of both autophagy inhibitor CQ and caspase 3 inhibitor Ac-DEVD-CHO.Results:1.Ex vivo:A 4h exposure to H₂O₂ led to a significant loss of HCs and SGNs and more TUNEL-positive cells.The protein levels of LC3-Ⅱand p62 were increased after H₂O₂exposure.In addition,the protein expression of SIRT1 was decreased and p53 and Ac-p53 expression were increased in H₂O₂group.Moreover,H₂O₂-exposed cochleae displayed significantly increased levels of miR-34a.Compared with H₂O₂ group,hair cell damage and disarray were alleviated in ELZC group,accompanied by the improvement of nerve fiber thinning and fragmentation.Besides,the SGN damage,shrinkage,and condensation were attenuated after ELZC treatment,paralleled by a marked recovery of autophagy flux and deduction of apoptosis,which were associated with a significant improvement in the SIRT1 expression and reduction of p53acetylation,respectively.2.In vivo:ARHL group showed higher hearing thresholds loss of OHC and SGNs compared with control group.The protein levels of LC3-Ⅱand p62 were increased and more TUNEL-positive cells were found in ARHL group.In addition,the expression of SIRT1 was decreased and the expression of p53 was increased in ARHL group compared with control group.ELZC lessened the hearing loss of ARHL mice.HE staining results showed that ELZC treatment remarkably attenuated the decreased SGN numbers and morphology at the basal cochlear turn compared to ARHL group.Further experiments revealed that the age-related apoptosis was attenuated and autophagy induction was promoted,which were associated with increment of SIRT1 expression and p53 inactivation.3.In vitro:The effects of ELZC on autophagy and apoptosis induced by miR-34a were observed,and the crosstalk between autophagy and apoptosis in the protection effects of ELZC was further explored.Western blot results showed that inhibition of autophagy flux in HEI-OC1 cells enhanced miR-34a overexpression-induced apoptosis.However,there was no effect on autophagy flux when apoptosis was inhibited.These results showed that autophagy occurred before apoptosis.Moreover,ELZC significantly reduced miR-34a-induced HEI-OC1 cell death by restoring autophagy flux and down-regulation of apoptosis-related protein expression via SIRT1/p53 pathway.Conclusion:1.ARHL mice exhibited higher hearing thresholds than control group.ARHL mice and H₂O₂ damaged cochlear cultures showed that the number of OHCs and SGNs decreased significantly,the protein expression levels of LC3-II and p62 increased and the number of TUNEL-positive cells increased.These suggested that autophagy flux impaired and apoptosis was promoted.In addition,the expression of SIRT1 was decreased and the expression of p53 protein was increased.Moreover,the expression of miR-34a in cochlea basement membrane was significantly increased by H₂O₂.2.The overexpression of miR-34a could inhibit the SIRT1/p53 pathway in HEI-OC1 cells,resulting in autophagy flux impairment and apoptosis promotion.Moreover,it was observed that autophagy occurred before apoptosis in this experimental system.3.ELZC has protective effects against ARHL through regulating autophagy and inhibiting apoptosis,which might be related to modulating of miR-34a/SIRT1/p53pathway.