| Part 1 HDAC2 and Efficacy of Glucocorticoids on Refractory SSNHLObjectives: Glucocorticoids(GC)was used to treat SSNHL via intratympanic perfusion(IP)for many years because of its permeability through round window membrane,and had clinical effects in some patients.However,some other cases didn't response to GCs and showed GCs resistance.Recent studies demonstrated that glucocorticoid resistance was closely associated with the dysfunction of histone deacetylase 2(HDAC2).Whether it happened in SSNHL was still unclear.The aim of this study was to observe the effect of reducing HDAC2 protein on intratympanic methylprednisolone perfusion(IMP)in refractory SSNHL.Methods: A total of 34 refractory SSNHL patients,who underwent a 10-day IMP treatment after failed systemic GCs treatment from Jan 2013 to Jul 2015,were collected in the present study.The Pure-tone average(PTA)data of all patients before and the next day after IMP,as well as every 2 to 4 weeks within a 3-month follow-up period were checked.And twenty milliliters peripheral blood was drawn from the 34 patients before and after IMP treatment in order to extract peripheral mononuclear cells(PBMC).According to whether the gain of PTA(0.25-8 k Hz)? 15 d B at 3 months after SSNHL onset,the patients were divided into IMP-sensitive(IMPS)group(PTA gain ? 15 d B)and IMP-insensitive(IMPI)group(PTA gain < 15 d B).Enzyme-linked immunosorbent assay(ELISA)and Western blot were used to detect the level of H3,H4 acetylation and HDAC2 in the two groups to investigate the influence of HDAC2 protein changes to the patients of refractory SSNHL who underwent IMP.Results: According to standards we set,15/34 patients got PTA gain(0.25-8 k Hz)?15 d B after IMP treatment and the total efficacy was 44.11%.The low-frequency PTA gain(0.25,0.5,1 k Hz)was 17.94±20.11 db,which is higher than in high-frequency(2,4,8 k Hz 5.65±13.17 db P< 0.01).HDAC2 protein level in SSNHL patients was lower and H3,H4 acetylation was higher compared to normal reference.And both ELISA and Western blot showed reduction of HDAC2 protein level,while increase in H3,H4 acetylation in IMPS group,while no obvious changes in IMPI group.Conclusions: Most refractory SSNHL patients who failed systemic treatments could improve their hearing level by IMP.The effect was related to the frequency of hearing loss.IMP could improve HDAC2 protein level,reduce H3 and H4 acetylation in IMPS group.This may be one of the possible mechanism of IMP treatment in refractory SSNHL.Part 2 Mechanism of HDAC2 Mediated the Apoptosis of Cochlea Hair CellsObjective: In previous studies,we had found that the down regulation of histone deacetylase 2(HDAC2)could reduce the effect of intratympanic methylprednisolone perfusion(IMP)on refractory sudden sensorineural hearing loss(SSNHL).However,the mechanism about it had not been elucidated.Recent studies showed that cochlear cell apoptosis was associated with SSNHL.Here,we investigated the potential mechanism of HDAC2 mediated the apoptosis of cochlea hair cells through guinea pig model induced by lipopolysaccharides(LPS)and cochlear cell line(HEI-OC1)model in vitro.Subjects and Methods: As inflammation and hypoxia are common reasons for acute hearing loss,we made an acute hearing loss model of guinea pig model by LPS infusion and developed a hypoxia HEI-OC1 cells model under 1% hypoxygen concentrations.We firstly used immunohistochemical method to observe the cell apoptosis in the cochleae of guinea pig model.Then we examined the RNA expression via q RT-PCR and the protein level via western blot of the HDAC2,Bcl-2,Bcl-x L,cleaved-caspase-3,cleaved-caspase-9 and cleaved-PARP in cochlear tissue isolated from LPS-infused guinea pigs and the HEI-OC1 cells cultured in hypoxia condition to analyze the relationship between HDAC2 and the apoptosis in acute hearing loss.By Venn diagram of online analyses,we found that mi R-204-5p and mi R-211-5p might be involved in the HDAC2-mediated Bcl-2 regulation.Using transfected with si-HDAC2,si-HDAC2 and mi R-204-5p inhibitor in HEI-OC1 cells to observe the effect of mi R-204-5p and mi R-211-5p in the HDAC2-mediated Bcl-2 regulation.Subsequently,we analyzed the regions of Mir204,the host gene of mi R-204-5p and found that multiple Sp1 binding sites in the promoter regions of Mir 204,indicating that Sp1 might be a key transcription factor of mi R-204-5p.Assenssment of the activities of the Mir 204 promoter by p GL3 basic firefly luciferase reporter was performed to reveal whether SP1 participated in HDAC2 down-regulation on mir-204-5p with the HEI-OC1 cells transfected with si-RNA,si-HDAC2,si-HDAC2 and si-SP1 and the HEI-OC1 cells mutated Sp1-binding sites.Using co-immunoprecipitation analyses and Chromatin Immunoprecipitation(CHIP)to analyze whether HDAC2 could combine with Sp1 and have regulation on Sp1.Finally,we observed the cell viability and apoptosis rate in HEI-OC1 cells co-transfected with si-HDAC2 and mi R-204-5p inhibitor or Bcl-2 expression vector to further clarify the effect of HDAC2/Sp1/mi R-204-5p/Bcl-2 axis on HEI-OC1 cells.Results: More severe hearing loss and cell apoptosis were observed in the guinea pigs with cochlear LPS infusion compared to AP-infused controls.The protein levels of HDAC2,Bcl-2,Bcl-xl were lower and the protein level of cleaved-caspas3,cleaved-caspas9,cleaved-PARP were higher in the cells derived from LPS-treated guinea pigs.The m RNA of HDAC2,Bcl-2,Bcl-xl were lower and the m RNA of Bcl-2 and Bcl-xl were positively correlated with HDAC2 in the cells derived from LPS-treated guinea pigs.The same results were also observed in HEI-OC1 cells cultured in hypoxia condition compared to controls.These hinted that HDAC2 was associated with the apoptosis of cochlear hair cells.The Venn diagram of online analysis tools identified overlapping of mi RNAs,mi R-204-5p and mi R-211-5p,might be involved in the HDAC2-mediated Bcl-2 regulation.However,only mi R-204-5p increased in HEI-OC1 cells transfected with HDAC2 si RNA(si-HDAC2).Besides,mi R-204-5p was also significantly increased in cochlear cells derived from LPS-treated guinea pigs.Adding mi R-204-5p inhibitor could reverse the down-regulation of Bcl-2 protein and RNA inducion by si-HDAC2 transfection,indicating mi R-204-5p was involved in HDAC2-mediated Bcl-2 regulation.Multiple Sp1 binding sites existed in the promoter regions of Mir 204.Mutated Sp1-binding sites could decrease the activity of Mir-204 promoter induced by transfecting with si-HDAC2.Co-transfected with si-Sp1 could be partially reversed the up-regulation of mi R-204-5p induced by transfecting si-HADC2.The two results mentioned above indicated SP1 was invovled in HDAC2-mediated mi R-204-5p.Subsequently,we confirmed the interaction between HDAC2 and Sp1 protein with co-immunoprecipitation analyses and demonstrated the P1 and P2 were the co-localization of HDAC2 and Sp1 binding sites via Ch IP assays.Furthermore,the increased H3 acetylation level at Sp1-binding sites after transfected with si-HDAC2 was observed,which reduced the activity of Mir 204 promoters and negatively regulated mi R-204-5p expression in HEI-OC1 cells.Finally,we found the decreased cell viability and the increased apoptosis of HEI-OC1 cells transfected with si-HDAC2 could be partially reversed by co-transfection with the mi R-204-5p inhibitor or a Bcl-2 expression vector.Conclusion: HDAC2 mediated the apoptosis of cochlea hair cells in SSNHL via HDAC2/SP1/mi R-204-5p/Bcl-2 regulatory axis. |
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