Basic Research Of Hair Cells Protection By Inhibition Of H3K9me2in Mice

Backgrouds:As one of the most common sensory defects in human, sensorineural hearing loss (SNHL) happen to more than half of individuals aged between50years and80years and more than30000children annually. SNHL is mainly associated with permanent hair cell damage in the inner ears, as hair cells can’t regenerate theirs elves spontaneously in mammalian. Protection of hair cells from apoptosis and hair cell regeneration provides two processes to prevent and control SNHL. However, efficient regeneration of functional hair cells is yet challenging. Active protection of cochlear hair cells is thus of critical importance for SNHL management.Ototoxic action of aminoglycosides is one of the major causative factors for permanent SNHL. In previous studies, it was found that the responses of hair cells to ototoxic challenge were complex and potentially involved the change of the mitochondrial membrane potential as part of the earliest response to neomycin in hair cells. Besides aminoglycosides, hair cells are susceptible to other vulnerabilities, such as cisplatin, noise, and so on. Therefore, we propose it deserves special attention on reduction of hair cells’ susceptibility to injury and the mitochondrial protection during hair cells injury.Epigenetic modifications play an important role in the regulation of many chromosomal functions and are closely linked to certain biological events such as transcriptional regulation, cell survival, differentiation, and death. Much work in recent years has shown that the dimethylated histone H3lysine9(H3K9me2) has a key role in embryogenesis and carcinogenesis. Moreover, previous research suggested that the expression of H3K9me2was followed by the stage of cells in zebrafish. However, it is unclear whether the same or different results show up after reversal of H3K9me2in hair cells’ responses to stress.Objectives:To describe H3K9me2regional distribution in organ of Corti and to observe H3K9me2expression changes in different condition. To explore hair cells protecion by inhibition of H3K9me2methyltransferase G9a/GLP in vitro and in vivo. To further understand the molecular mechanisms underlying the otoprotection of inhibition of H3K9me2in the organ of Corti. Methods:This design included2parts.1. In vitro studies:We use neonatal cochlea explant culture to invesatigate otoprotection of inhibition of H3K9me2and it possible mechanisms. Firstly, we tested the expression of H3K9me2by immune fluorescent chemical technology and/or western blot. Then we quantified the survival hair cells and apoptotic bodies, which were also confirmed by examination of TUNEL, PI and LC3, in the four groups. Finally, we tested the change of the stability in mitochondrial membrane potential (MMP) and the expression of cleaved caspase-3using immune fluorescent chemical technology and/or western blot technology.2. In vivo studies:We adopt self-controlled study. The left ears were included as experimental group, the right ears as control group. We observed the stereociliary bundles of the hair cells using scanning electron microscopy (SEM) technology. We quantified the survival hair cells using immune fluorescent chemical technology. Further, we tested hearing status using brainstem response (ABR).Results:1. In vitro studies:The expression of H3K9me2presented regional distribution in organ of Corti and consistently increased transiently in diverse hair cells injury models, such as cisplatin, copper, neomycin and UV damage. Meanwhile, it was easily decreased after administration of BIX-01294, an inbitor of H3K9me2. Easily from these data in vitro, we could conclude the survival hair cells in pre-treat group were more than neo groups, presenting striking difference (P<0.01). By the same token, apoptotic bodies in pre-treat group were more in evidence than others in the middle and basal segments with statistical meaning (P<0.01). Furthermore, GTTR or FM1-43FX can efficiently entered hair cells in BIX-01294preconditioning Corti organ. To further understand the underlying mechanisms, we found that three styles of hair cell death, apoptosis/type1cell death, autophagy/type2cell death and necrosis/type3cell death, were detected morphologically. And it was notable that Corti organs with BIX-01294preconditioning showed higher levels of visualized TMRM fluorescence than neomycin-alone treated tissue, while lower expression of cleaved caspase-3in BIX-01294pre-treat samples.2. In vivo studies:From SEM examination, the stereocilia fusions mainly in apical segment were observed in neomycin-exposed cochleas, while stereocilia bundle loss medially and basally, with a massive loss of outer hair cells preceding inner hair cells loss. Contralateral cochleae pretreated with BIX-01294appeared slightly less damaged, with more survival stereocilia bundle in basal segment. From immunofluorescent histochemical staining, the location of the "transitional" zone in BIX-01294pre-treated ears was found to be more close to the basal border with middle segment. Within this region, we observed some remaining nuclei that appeared apoptotic, with marginated and condensed chromatin as demonstrated by Hoechst33342staining. In addition, the amount of survived hair cells in BIX-01294pre-treated ears was significantly more than neomycin-alone ears. From the functional standpoint, neomycin-alone treatment caused serious ABR threshold shift, while BIX-01294precondition ameliorated the threshold shift of hearing. Quantitative analysis of ABR test showed the amelioration was greater or equal to15dB in8000Hz and16000Hz (P<0.01).Conclusions:A crew of different pattern of H3K9me2in outer hair cells and inner hair cells indicated that H3K9methylation was associated with the differentiation or fate of cells. Meanwhile, increased tendency of H3K9me2showed that H3K9me2was involed in the physiologic and pathologic process in hair cells. Results obtained from neonatal Corti explants in vitro and adult Corti organs in vivo demonstrated that the downregulation of H3K9me2prevented ototoxic and ongoing hair cells death and permanent hearing loss significantly. We proposed that the regulation of H3K9me2was followed by the change of hair cells’ susceptibility to stress. Further, reduced susceptibility of hair cells to stress by downregulation of H3K9me2is of potential therapeutic value clinically for hearing protection of Corti organ morphologically and functionally.