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Sox2 overexpression alleviates noise-induced hearing loss by inhibiting inflammation-related hair cell apoptosis

Sox2 overexpression alleviates noise-induced hearing loss by inhibiting inflammation-related hair... Background: The transcription factor Sox2 plays important roles in the developmental processes of multiple organs and tissues. However, whether Sox2 can protect mature or terminally differentiated cells against injury is still unknown. Methods: We investigated the roles of Sox2 in cochlear hair cells, which are terminally differentiated cells, using conditional transgenic mice and several hearing loss models. Results: Sox2 overexpression dramatically mitigated the degree of cochlear hair cell loss when exposed to ototoxic drugs. Noise-induced apoptosis of cochlear hair cells and hearing loss were also significantly alleviated by Sox2 overexpression. Notably, noise-induced upregulation of pro-inflammatory factors such as TNF-α and IL6 was inhibited by Sox2 overexpression. Then we used lipopolysaccharide to clarify the effect of Sox2 on cochlear inflammation, and Sox2 overexpression significantly inhibited lipopolysaccharide-induced upregulation of pro-inflammatory factors and alleviated inflammation-related cochlear hair cell death. Conclusions: These results demonstrate a novel protective role of Sox2 in mature and terminally differentiated coch- lear hair cells by inhibiting inflammation. Keywords: Sox2, Cell protection, Hearing loss, Cochlear hair cells, Apoptosis, Inflammatory response, Lipopolysaccharide Background onset of sensorineural hearing loss [1–7]. Because of the In mammals, cochlear mechanosensory hair cells (HCs) inability of HCs to regenerate after injury in the mature convert sound waves into electrical signals that are trans- mammalian cochlea, protecting HCs against injury is mitted to the brain’s auditory centers to produce hearing. extremely important for hearing preservation [8–10]. Aging, noise trauma, ototoxic drugs, and infections can Sox2 (HMG-box transcription factor sex-determining lead to HC loss or degeneration and thus result in the region Y-box  2), a well-studied pluripotent transcrip- tion factor, is critical for the embryonic development of many organs [11–13]. For example, Sox2 serves as a regulator in the process of taste bud sensory cell differ - *Correspondence: wenyan_li2000@126.com; chenyan0528@fudan.edu.cn; niyusu@aliyun.com entiation from endodermal progenitor cells [12], and the Diyan Chen, Gaogan Jia and Yanping Zhang contributed equally to this expression of Sox2 has a strong influence on the branch - work ing process in the respiratory system [13]. Deficiency ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China of Sox2 leads to abnormal cartilage development in the Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 2 of 16 tracheal mesenchyme and to abnormal cellular composi- for genetic identification. The primers used in the geno - tion [14], and Sox2 has been shown to play a significant typing protocols are listed in Additional file 1: Table S2. role in the development of the inner ear [15]. In addition to its essential role in stem cell self-renewal and repro- Auditory brainstem response (ABR) measurement gramming, Sox2 also plays a role in the survival of stem Mice were injected with a solution of Zoletil (70  g/kg) cells and cancer cells [16]. Sox2 directly up-regulates the and dexmedetomidine hydrochloride (1000  mg/kg) expression of Survivin, which inhibits the mitochondria- intraperitoneally to induce anesthesia. The efficacy of dependent apoptotic pathway in neural stem cells [17]. anesthesia was periodically ascertained through foot Although the roles of Sox2 in stem cells and cancer cells pinch response. The body temperature was maintained have been clarified in many studies, so far no studies at 37  °C to avoid any influence on the auditory physi - about the possible role of Sox2 in mature or terminally ology test. ABR measurements were used to assess the differentiated cells have been reported. mouse auditory function with a System 3 ABR work- In the development process of the inner ear, Sox2 is station (Tucker Davis Technology, Alachua, FL, USA). expressed in pro-sensory cells in the embryonic pro- Hearing function at 8, 16, 24, and 32 kHz was evaluated sensory domain. When the terminal differentiation of through an open field speaker, and acoustic thresh - cochlear progenitors is initiated, Sox2 expression is main- olds of sound pressure level were determined using the tained in the supporting cells but gradually decreases in BioSigRP software (Tucker Davis Technology, Alachua, auditory HCs. Once the cochlea becomes fully mature FL, USA). and hearing function has been developed, the expression of Sox2 in HCs becomes undetectable [18, 19]. During Cochlear HC injury models the maturation of the vestibular sensory epithelium, Sox2 To construct the ototoxic drug-induced HC injury expression is maintained in supporting cells and type 2 model in the neonatal stage, cochlear epithelia samples vestibular HCs, but is gradually diminished in type 1 ves- were obtained for explant culture from P2 transgenic tibular HCs. Sox2 is required for the maintenance of HCs mice that had received tamoxifen (Sigma, T5648-5G) during embryonic development of the zebrafish inner ear administration at P 0–1. The tissues were treated with and for HC regeneration in zebrafish embryos after injury 1  mM neomycin (Sigma) for 6  h and then harvested [20]. Our recent study reported that Sox2-negative type after an additional 24 h in the absence of neomycin. 1 vestibular HCs are more vulnerable than Sox2-positive To construct the noise-induced HC injury model, the type 2 vestibular HCs to 3,3’-iminodipropionitrile toxic- hearing function of P30 mice that had received tamox- ity in adult mice [21], suggesting that Sox2 might exert a ifen administration at P21–P22 were examined by ABR positive effect on the survival of sensory HCs. To explore test 24  h before noise exposure. The mice were then the possibility that Sox2 might exert a positive effect on exposed to broadband noise at 116 dB for 2 h. Hearing the survival of auditory HCs, we used conditional Sox2- function was evaluated again by ABR test 7  days after upregulated transgenic mice and several cochlear HC noise exposure, and the mice were killed and cochlear injury models to investigate the possible roles of Sox2 in samples were harvested for morphological analysis. HC damage and survival. To construct the lipopolysaccharide (LPS)-induced HC injury model, mice were given 10  μl LPS (Sigma- Aldrich, L2880) at 1, 2, or 4 mg/kg by tympanum injec- Materials and methods tion and killed 3 days later. Animals and genotyping The Rosa26-CAG-LSL (loxP-stop-loxP)-Sox2 transgenic Immunofluorescence and morphological analysis mice, referred to as Sox2OE mice, were designed by our Immunofluorescence staining was performed as previ - group and Beijing Biocytogen Co., Ltd. The Atoh1CreER ously reported [32]. Rabbit polyclonal anti-Myosin7A and PrestinCreER transgenic mice, which have been (Proteus Biosciences, 1: 1000 dilution, 25-6790), goat described in detail in previous studies [22, 23], were the polyclonal anti-SRY (sex-determining region Y)- kind gifts of Prof. Zuo Jian. For the Atoh1CreER line, box  2 (Sox2) (Santa Cruz Biotechnology, 1:1000 dilu- tamoxifen was injected subcutaneously once daily at tion, sc-17320), mouse monoclonal anti- Parvalbumin 75  mg/kg body weight at postnatal day (P) 0–1. For the (Sigma, 1:1000 dilution, P3088), mouse monoclonal PrestinCreER line, tamoxifen was injected intraperito- anti-Myosin7a (Santa Cruz Biotechnology, 1:500 dilu- neally once daily at 225  mg/kg body weight at P21–22. tion, sc-74516), rabbit mAb anti-TNF-α (Cell Signaling Mice of either sex were used for all experiments. Animals Technology, 1:400 dilution, 11948), and rat monoclo- were bred under specific and opportunistic pathogen- nal anti-IL6 (ThermoFisher Scientific, 1:200 dilution, free conditions, and tail DNA genotyping was performed Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 3 of 16 11-7061-82) were used as primary antibodies, and con- corresponding HRP-conjugated secondary antibodies trol incubations were routinely processed without pri- for 1 h at room temperature. The immunoreactive bands mary antibody treatments. were detected using an ECL kit (Pierce). Propidium iodide (PI) staining was used to detect the apoptotic and necrotic HC nuclei after noise expo- Statistical analysis sure as previously reported . Fixed cochleae were Data were analyzed using GraphPad Prism 8. Stu- permeabilized in 1% Triton X-100 solution for 30  min dent’s 2-tailed t-test was used to analyze the differences and then stained with 5  mg/ml PI dissolved in PBS for between two groups, and one-way ANOVA followed by 1  h at room temperature. After the final washes with the Bonferroni post-test was used for comparisons of PBS (10  min each), the tissue was dissected in PBS by differences among three or four groups. Data are pre - removing the modiolus. sented as the means ± S.E.M, and p < 0.05 was considered TUNEL staining was used to detect apoptotic HC significant. death. Tissues were fixed with 4% PFA for 1  h, treated with 1% Triton X-100 37  °C for 40  s, and incubated in Study approval TUNEL staining solution (Roche) according to the man- This study followed the “Guiding Directive for Humane ufacturer’s instructions. Treatment of Laboratory Animals” reporting guidelines enacted by the Chinese National Ministry of Science and Technology in 2006. All experiments were approved by Confocal imaging and cell counting the Shanghai Medical Experimental Animal Administra- Specimens were examined by confocal fluorescence tive Committee (Permit Number: 2009-0082) and were in microscopy (Leica SP8). To quantify the immunostain- accordance with animal welfare principles. ing-positive cells, nine separate segments along the entire cochlea were selected from the apex to the base. Results Sox2 abundance is correlated with HC tolerance to ototoxic Real‑time PCR drugs in neonatal HCs RNA was extracted from six cochleae of P30 adult mice First, we examined the Sox2 expression pattern in the from each group and was subsequently reverse tran- cochleae of wild type mice. Myosin7a was used as the scribed into cDNA using Superscript III reverse tran- marker of cochlear HCs. The cochlear epithelium is a scriptase (Invitrogen) according to the manufacturer’s delicate tissue containing region-specific HCs, which can instructions. Real-time PCR was performed on an ABI be roughly classified into two categories, namely inner 7500 real-time PCR system (Applied Biosystems) using hair cells (IHCs) and outer hair cells (OHCs), which have ™ ™ the TB Green PrimeScript RT-PCR Kit (Takara). The different functions, gene expression patterns, and mor - Actb gene was used as the endogenous control. Primer phological characteristics [24] (Fig. 1A). Immunofluores - sets are listed in Additional file  1: Table S1. The quantifi - cence results revealed that Sox2 was strongly expressed cation of relative gene expression, compared to Actb, was in all cochlear supporting cells, including inner bor- −ΔΔCT analyzed by the 2 method. der cells, inner phalangeal cells, inner pillar cells, outer pillar cells, Deiters’ cells, and Hensen’s cells (Fig.  1C). +/− Western blotting In the cochleae of adult control (PrestinCreER / −/− Proteins were extracted from isolated cochlear epithe- Sox2OE ) mice, Sox2 was not observed in the coch- lia with lysis buffer containing proteinase inhibitors, lear HCs (Fig.  1C). However, in the cochleae of neonatal separated by SDS-PAGE gel, and transferred onto PVDF mice, Sox2 was also observed in some IHCs and OHCs, membranes (Immobilon-P; Millipore, Bedford, MA, but the Sox2 abundance in HCs decreased from the api- USA). Membranes were blocked in 5% nonfat dried milk cal to the basal turns (Additional file  1: Fig. S1A, note the in TBST (20  mM Tris–HCl, 500  mM NaCl, and 0.1% Sox2 + HCs indicated by arrows in the apical turns). Sim- Tween-20) for 1  h and later incubated with the primary ilar to what was observed previously [25, 26], we found antibodies overnight at 4  °C. The following antibodies that the HC sensitivity to ototoxic drugs increased from were used for Western blot analysis: mouse monoclonal the apical to basal turns because more HCs were lost in anti-beta-Actin (Thermo Fisher Scientific, 1:5000 dilu - the basal turns compared to the apical turns in neonatal tion, MA515739), rabbit mAb anti-TNF-α (Cell Signaling mice (Additional file 1: Fig. S1B). Technology, 1:3000 dilution, 11948), and rat monoclo- nal anti-IL6 (ThermoFisher Scientific, 1:2000 dilution, 11-7061-82). The next day, the blots were washed three times with TBST and subsequently incubated with Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 4 of 16 Fig. 1 The cochlear morphology and hearing function were not affected by Sox2 overexpression. A The cross-section of the cochlear sensory epithelium. The IHCs and OHCs are located on the top layer, and most supporting cells (including BC, IphC, IPC, OPC, DC, and HeC) are located on the under layer. Some HCs can be observed in the top layer. IPC inner pillar cell, OPC outer pillar cell, DC Deiters’ cell, IPhC inner phalangeal cell, BC inner border cell, HeC Hensen’s cell, IS inner sulcus, CC Claudius cell. B Schematic for creating the transgenic mouse models. The Rosa26- CAG-LSL(loxP-stop-loxP)-Sox2 transgenic mice, referred to as the Sox2OE mice, were designed using the CRISPR/Cas9 method. C Images of Sox2 immunofluorescence (whole mount, HC layer) showed that there was no detectable Sox2 expression in the cochlear HCs of the control group, +/– +/– while in the PrestinCreER /Sox2OE group Sox2 expression was clearly observed in OHCs. D Experimental protocol for E–F. Tamoxifen was injected once daily during P21–P22, and the cochlear morphology and hearing function were assessed by immunofluorescence and ABR at P30. +/– +/– E Representative Myosin7a immunofluorescence showed no difference in HC morphology between the PrestinCreER /Sox2OE and control +/– +/– +/– groups. F Quantification of IHCs and OHCs in the PrestinCreER /Sox2OE and control groups. G The ABR thresholds of adult PrestinCreER / +/– Sox2OE and control mice. Data are presented as the mean ± SEM. n = 5 for each group. Scale bar = 20 µm Sox2 overexpression in HCs did not alter HC morphology CAG promoter in a Cre-mediated manner. To exam- or auditory function under physiological conditions ine the effects of Sox2 overexpression (Sox2OE) on To overexpress Sox2 in the cochlear HCs, we gener- cochlear HC survival in the neonatal and adult stages, ated the inducible Rosa26-CAG-LSL-Sox2 mouse line respectively, we used two Cre mouse lines, namely, in which forced expression of Sox2 was driven by the the Atoh1CreER line that targets the HCs including Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 5 of 16 both IHCs and OHCs in the neonatal stage and the turns, respectively. The average numbers of remain - PrestinCreER line that targets OHCs in the adult stage ing HCs in the middle and basal turns from the Atoh1- +/– +/– (Fig.  1B). We generated Pr estinCreER /Sox2OE Sox2OE groups were significantly larger than those from mice in which constitutive expression of Sox2 in control groups (Fig.  2D, p < 0.001). Thus it appears that Prestin cells were obtained by tamoxifen administra- there is a protective effect of Sox2 overexpression against tion, and these are referred to as Prestin-Sox2OE mice. neomycin injury. +/– –/– PrestinCreER /Sox2OE mice served as controls. To evaluate the expression of Sox2 in OHCs and to Sox2 overexpression attenuated noise‑induced HC determine the effect of Sox2OE on HC function under damage and hearing loss in adult mice physiological conditions, Prestin-Sox2OE and control To determine whether the susceptibility of HCs to noise mice were injected intraperitoneally (i.p.) with tamox- exposure changed after Sox2 overexpression, P31 Pres- ifen from P21 to P22, and their hearing function and tin-Sox2OE mice that received tamoxifen at P21–22 were cochlear morphological features were assessed at P30 exposed to 116 dB broadband noise for 2 h (Fig. 3A). The (Fig.  1D). In the control group, no Sox2 staining was hearing thresholds of the mice were determined using observed in OHCs from mature cochleae, while nuclear ABR tests at P38, and the cochlear specimens were har- +/– –/– staining of Sox2 was clearly observed in OHCs of Pres- vested afterwards. PrestinCreER /Sox2OE mice tin-Sox2OE mice, confirming the successful induction served as controls. In the control group, noise expo- of Cre activity and Sox2 overexpression in the Prestin sure led to significant OHC loss in the middle and basal cells (Fig.  1C). Immunofluorescence results showed turns of the cochlea (Fig.  3B–D). However, in the Pres- that the morphological features and quantities of coch- tin-Sox2OE cochlear epithelium, the noise-induced loss lear HCs in the Prestin-Sox2OE group were similar of OHCs was significantly attenuated in the middle and to those of controls (Fig.  1E, F). In addition, the ABR basal turns compared with the control mice (p < 0.01, results showed that the hearing function of adult Pres- Fig. 3B–D), indicating that Sox2-overexpressing cochlear tin-Sox2OE mice was comparable to controls at 8, 16, OHCs were less sensitive to noise-induced injury. 24, and 32  kHz (Fig.  1G). These results suggested that Hearing thresholds before noise exposure (baseline val- Sox2 overexpression in OHCs did not affect the main - ues) showed no significant difference between the Pres - tenance of HC function. tin-Sox2OE and the control groups (data not shown). At 7 days after noise exposure, mice in the control group had significant hearing loss, as demonstrated by the increased Sox2 overexpression protected HCs hearing thresholds at 8, 16, 24, and 32 kHz (Fig. 3E). The against aminoglycoside‑induced injury in the neonatal noise-induced threshold shifts in Prestin-Sox2OE mice stage were significantly lower at all frequencies compared To explore the function of Sox2 in HC survival, we first with those of the control mice (p < 0.05, Fig. 3F), suggest- used an in  vitro aminoglycoside antibiotic injury model. ing that Sox2 overexpression in OHCs protects against +/– +/– We generated Atoh1CreER /Sox2OE mice in which noise-induced hearing loss. constitutive expression of Sox2 in A toh1 HCs was obtained by tamoxifen administration, and these are referred to as the Atoh1-Sox2OE group. Sox2 overexpression protected OHCs by inhibiting The cochlear epithelium from P2 Atoh1-Sox2OE the noise‑induced apoptosis pathway mice that received tamoxifen injections at P 0-1 were To determine the mechanism through which Sox2 over- cultured and treated with 1  mM neomycin for 6  h and expression protects HCs against injury, we examined then allowed to recover without neomycin for an addi- the apoptotic and necrotic changes in HCs after noise +/– tional 24 h prior to harvesting (Fig. 2A). Atoh1CreER / exposure using PI staining based on previous research –/– Sox2OE mice served as controls. Myosin7a immu- showing the morphological changes that occur in OHCs nofluorescence was used to determine the number of after noise exposure [27]. In the control group, noise remaining HCs after neomycin treatment. In the control exposure induced morphological nuclear changes that group, neomycin treatment led to massive HC loss, and included both apoptotic (condensed nuclei) and necrotic the average numbers of remaining M yosin7a HCs were (swollen nuclei) changes in OHCs at 6  h after noise 57.0 ± 0.577, 31.5 ± 2.51, and 16.5 ± 1.52 cells/100  µm in exposure (Fig.  4A, B). However, the number of apop- the apical, middle, and basal turns, respectively (Fig. 2B– totic nuclei was significantly decreased in the Prestin- D). In the Atoh1-Sox2OE group, the average numbers Sox2OE group when compared with the control group of Myosin7a HCs were 56.8 ± 0.601, 54.2 ± 1.08, and (Fig.  4A, B). Meanwhile, no significant differences in the 43.3 ± 2.33 cells/100  µm in the apical, middle, and basal numbers of necrotic nuclei were observed between the Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 6 of 16 Fig. 2 Sox2 overexpression protected HCs from aminoglycoside-induced injury in the neonatal stage A Experimental protocol for C-D. Cochlear +/– +/– epithelia from Atoh1CreER /Sox2OE and control mice that received tamoxifen injection at P0–P1 were dissected and cultured at P2. Cochlear explants were treated with 1 mM neomycin for 6 h and harvested after an additional 24 h. B The scheme for cell counting. C Representative + +/– +/– images of Myosin7a cells in A toh1CreER /Sox2OE and control mice treated with or without neomycin. D Quantification of the cochlear HCs +/– +/– per 100 µm in the Atoh1CreER /Sox2OE and control groups. Data are presented as means ± SEM; n = 6 for each group; ***P < 0.001. Scale bar = 20 µm Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 7 of 16 +/– Fig. 3 Sox2 overexpression alleviated noise-induced hearing loss and HC damage. A Experimental protocol for B–F. Adult P restinCreER / +/– Sox2OE and control mice that received tamoxifen at P 21–22 were exposed to 116 dB broadband noise for 2 h. Cochlear morphology and + +/– +/– hearing function were evaluated 7 days after noise exposure. B Representative images of M yosin7a cells in PrestinCreER /Sox2OE and control +/– +/– mice treated with or without noise exposure. C, D Cochleograms showed noise-induced loss of IHCs and OHCs in the P restinCreER /Sox2OE +/– +/– and control groups. E, F Pure-tone ABR thresholds and threshold shifts in the PrestinCreER /Sox2OE and control group 7 days after the noise exposure. Scale bar = 20 µm. Data are presented as means ± SEM; n = 6 for each group; *P < 0.05, **P < 0.01, ***P < 0.001, indicated the significant +/– +/– +/– –/– difference between the PrestinCreER /Sox2OE with noise treatment and the PrestinCreER /Sox2OE with noise treatment groups Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 8 of 16 Fig. 4 Sox2 overexpression suppressed noise-induced HC apoptosis. A Representative images of PI-stained OHC nuclei in the cochlear epithelium. Both swollen and condensed OHC nuclei were observed in mice treated with noise exposure. Arrowheads point to swollen necrotic nuclei, and arrows point to condensed apoptotic nuclei. B The numbers of swollen or condensed OHC nuclei and missing OHCs in the cochlear epithelium at 6 h after noise exposure. C Representative images of TUNEL staining and Myosin7a immunofluorescence of the cochlear epithelium. TUNEL / + + + Myosin7a OHCs were observed in the mouse cochlea at 6 h after noise exposure. D Quantification of TUNEL /Myosin7a HCs in the cochlear epithelium. Data are presented as means ± SEM.; n = 6. *P < 0.05, **P < 0.01. Scale bar = 20 μm Prestin-Sox2OE and control groups. TUNEL staining (Aif, EndoG, and Bax) and necrosis-related genes (RipK1 was used to further determine the differences in apop - and RipK3) (Fig.  5A, B). In the Prestin-Sox2OE group, totic HCs between the two groups (Fig. 4C). Significantly the expression levels of Aif, Bax, and EndoG were signifi - + + fewer Myosin7a /TUNEL cells were observed in Pres- cantly decreased (Fig.  5A, p < 0.05), while the expression tin-Sox2OE mice compared to control mice (Fig.  4D), levels of RipK1 and RipK3 showed no significant differ - suggesting that Sox2 overexpression suppressed noise- ence when compared to the control group after noise induced apoptosis of OHCs. exposure (Fig.  5B), further confirming that Sox2 overex - We next assessed the relative mRNA expression level pression protected OHCs by inhibiting the noise-induced of apoptosis and necrosis-related genes at 6 h after noise apoptosis pathway. exposure. In the control group, noise exposure induced the increased expression of both apoptosis-related genes Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 9 of 16 Fig. 5 Sox2 overexpression suppressed noise-induced upregulation of inflammatory cytokines. A The mRNA expression levels of apoptosis-related genes (Aif, Bax, EndoG, Casp3, Casp8, and Casp9) were elevated 6 h after the completion of noise exposure. B The mRNA expression levels of necrosis-related genes (Prkaα1, Ripk1, and Ripk3) and pro-inflammatory cytokines (Tnf , Il-1β, and Il-6) were elevated 6 h after the completion of noise exposure. C TNF-α immunofluorescence in HCs after noise exposure. The TNF-α expression level was elevated after traumatic noise exposure in HCs, while it was inhibited in the Sox2 overexpression group. Parvalbumin is used as a marker of cochlear HCs. Data are presented as means ± SEM.; n = 6. *P < 0.05, **P < 0.01. Scale bar = 20 μm Sox2 overexpression alleviated HC damage by inhibiting anti-TNF-α and anti-IL6 antibodies, and we found that inflammation noise-induced TNF-α and IL6 expression in cochlear In consideration of the important role of inflammation HCs was relatively lower in the Prestin-Sox2OE group in HC death, the mRNA expression of pro-inflammation compared to the control group (Figs. 5C, 6). mediators was measured. In the control group, noise To determine the role of Sox2 upregulation in the exposure induced significantly increased expression of inflammatory process, we constructed the lipopolysac - Tnf, Il-1β, and Il-6 at 6  h after noise exposure (Fig.  5B). charide (LPS) challenge model. It has been previously In the Prestin-Sox2OE group, the expression level of reported that inflammation-related mRNA expression these pro-inflammation mediators declined significantly is upregulated in the cochlea within 6  h after LPS chal- to a level comparable to that in the group without noise lenge [23]. To determine the optimal concentration of exposure (Fig.  5B, p < 0.05), indicating that Sox2 over- LPS in inflammation-mediated HC loss, 10 μl LPS at dif - expression dramatically attenuated the noise-induced ferent concentrations (1  mg/kg, 2  mg/kg, and 4  mg/kg) inflammation process. To further evaluate the protein was injected into the tympanic cavity of wild type mice expression level of pro-inflammatory mediators in coch - and the cochleae were dissected for analysis 72  h later. lear epithelia, we performed Western blotting analysis of LPS induced no obvious cochlear HC loss in the 1 mg/kg TNF-α and IL6. These results showed that noise expo - LPS group, mild HC loss in the basal turns in the 2 mg/ sure led to significant increases in TNF-α and IL6 protein kg LPS group, and severe HC loss in the middle and basal levels in the control group, but Sox2 overexpression sig- turns in the 4  mg/kg LPS group. Thus, 4  mg/kg of LPS nificantly inhibited the noise-induced increase of TNF-α was considered the optimal concentration for inducing and IL6 protein (Fig.  6). We also measured the expres- significant HC loss in both the middle and basal turns. sion of TNF-α and IL6 protein in the organ of Corti using Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 10 of 16 Fig. 6 Sox2 overexpression suppressed noise-induced upregulation of IL6 and TNF-α. A IL6 immunofluorescence in cochlear cross-section after noise treatment. The IL6 expression level was elevated after traumatic noise exposure in HCs, while it was inhibited in the Sox2 overexpression group. Myosin7a is used as a marker of cochlear HCs. B Western blot analysis of sensory epithelia shows increased expression levels of TNF-α and IL6 in control mice after noise injury, but it was significantly inhibited in Prestin-Sox2OE mice. β-Actin served as the sample loading control. Data are presented as means ± SEM.; n = 3. *P < 0.05, **P < 0.01. Scale bar = 20 μm (See figure on next page.) Fig. 7 Sox2 upregulation mitigated inflammation-induced HC loss. A Experimental protocol for the LPS-induced HC loss model. B Myosin7a +/– +/– immunofluorescence in cochlear HCs of adult PrestinCreER /Sox2OE mice and control mice with or without LPS treatment. C Cell counting results of cochlear HCs after 4 mg/kg of LPS administration. D TNF-α immunofluorescence in cochlear cross-section after LPS challenge. E The mRNA expression levels of pro-inflammatory cytokines. Sox2 overexpression prevented the LPS-induced increase in pro-inflammatory cytokines. Data are presented as means ± SEM; n = 6; **P < 0.01, and ***P < 0.001. Scale bar = 20 μm Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 11 of 16 Fig. 7 (See legend on previous page.) Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 12 of 16 We compared LPS-induced HC damage between the lower in the Prestin-Sox2OE group, which was a sign Prestin-Sox2OE and control groups. Significantly more that the inflammation reaction was limited (Fig.  7E). cochlear HCs were observed in the middle and basal Western blot analysis also showed very high expression turns in the Prestin-Sox2OE group than in the control levels of TNF-α and IL6 protein in the control mice and group, indicating that Sox2 overexpression largely allevi- limited expression in the Prestin-Sox2OE mice after LPS ated LPS-induced HC damage (Fig. 7A–C). We next eval- treatment (Fig.  8). The immunofluorescence results also uated the mRNA expression level of pro-inflammatory showed that the LPS-induced upregulation of TNF-α cytokines induced by LPS. qPCR results showed that Tnf, and IL6 in HCs was attenuated by Sox2 overexpres- Il-6, and Il-1β increased substantially after LPS treatment sion (Figs.  7D, 8). Collectively, these results showed that in the control group, but their expression was relatively Fig. 8 Sox2 overexpression suppressed LPS-induced upregulation of IL6 and TNF-α. A IL6 immunofluorescence in cochlear cross-section after LPS treatment. Myosin7a is used as a marker of cochlear HCs. (B) Western blot analysis of sensory epithelia shows increased expression levels of TNF-α and IL6 in control mice after LPS treatment, but the LPS-induced increase of the TNF-α and IL6 protein was significantly inhibited in Prestin-Sox2OE mice. β-Actin served as the sample loading control. Data are presented as means ± SEM.; n = 3. *P < 0.05, **P < 0.01. Scale bar = 20 μm Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 13 of 16 Sox2 upregulation inhibited the cochlear inflammatory increasing attention in the past decade [42, 43]. The response and subsequent HC death. impact of noise-induced hearing loss is broad and profound, including the loss of the ability to commu- Discussion nicate with others and subsequent social isolation. Up An estimated 466 million people are living with hear- to now, few medicines approved by the Food and Drug ing loss worldwide. Although the exact mechanisms are Administration have been confirmed to be effective in not fully understood, oxidative stress, inflammation, preventing or treating noise-induced hearing loss in autophagy, apoptosis, and necrosis have been reported clinical trials. Consistent with previous studies [44–46], to be involved in the onset and progression of sensori- our data further confirmed that apoptosis, necrosis, neural hearing loss [27, 28], and various strategies have and inflammation are involved in noise-induced HC been explored to try to protect sensory HCs and hear- death (Figs.  4, 5). Aif and Endo G, which act as apop- ing. Some anti-apoptosis genes, like Bcl2 and Xiap, have totic mediators, translocate into the nucleus during been reported to have protective roles in cochlear HCs HC injury and lead to HC apoptosis [47–49]. Prkaa1 and hearing [29, 30]. Antioxidants such as N-acetyl- (also known as AMPK) and receptor-interacting pro- L-cysteine, coenzyme Q10, and vitamin E have been tein kinase (RipK) have been reported be associated reported to be effective in protecting against ototoxic with necrotic HC death [27, 50]. Although the endog- drugs and noise exposure [31–34]. Some genes, like enous expression of Sox2 is very low in mature coch- Bmi1, G6PD, adenosine A1 receptor, and FoxG1, pro- lear HCs, forced Sox2 overexpression in Prestin OHCs tect HCs by inhibiting oxidative stress or by suppress- has a strong protective effect against noise-induced ing inflammatory pathways that are induced by ototoxic hearing injury (Fig.  3). PI staining results showed that drugs or that are related to aging-related damage [28, Sox2 upregulation decreased the number of HCs that 35–37]. Besides classic anti-apoptosis, anti-necrosis, and underwent apoptosis (Fig.  4), and this was accompa- anti-inflammation factors, some development-related nied by a decrease in noise-induced expression of pro- factors, such as Wnt/beta-catenin and Islet-1, have been apoptosis genes (Aif, EndoG, and Bax) and the number shown to be capable of protecting HCs against injury and of TUNEL HCs (Fig. 5). In contrast, Sox2 overexpres- thus preserving hearing [35, 38]. sion had no obvious effect on the number of HCs that In the cochlea, apical HCs are responsible for sensing underwent necrosis, as demonstrated by the PI staining sound at lower frequencies, and basal HCs are responsi- results and the mRNA expression levels of pro-necrosis ble for sensing sound at higher frequencies. Hearing loss factors (Figs.  4, 5). Together, these results suggest that in patients is generally more severe at higher frequen- Sox2 overexpression protects HCs and hearing against cies than lower frequencies, and our experimental data noise-induced injury by inhibiting HC apoptosis. showed that the sensitivity of HCs to injury increased The inflammatory response activated by extrinsic from the apical to basal turns. The three different treat - attack and infection is also a strictly regulated process, ments—aminoglycosides (Fig.  2), broadband noise and an appropriate inflammatory response can elimi - (Fig.  3), and LPS (Fig.  7)—all led to an apical-to-basal nate the pathogen, repair damaged tissue, and maintain gradient of damage in cochlear HCs, and severe HC loss cell homeostasis [51, 52]. However, dysregulation of the occurred in the basal turn while the mildest HC loss inflammatory response is pivotal in the initiation and occurred in the apical turn. Previous studies showed that progression of multiple diseases, such as age-related dis- exposure to low-decibel broad band noise (8–16  kHz) eases [53] and neurodegeneration diseases [54]. In the results in HC damage mainly in the middle turns, but the auditory system, LPS can penetrate into the labyrinth HC damage extends to the basal turn when the intensity through the round window membrane, where it stimu- of the noise is greater than 100  dB [39–41]. The mecha - lates the production and release of cytokines and medi- nism behind the difference in HC injury sensitivity along ates the initiation and development of inflammatory the cochlear turns remains unclear. In this work, we reactions in the media and labyrinth, which in turn leads found that Sox2 was expressed in the neonatal cochlear to auditory epithelial injury and subsequent sensorineu- HCs, which are less sensitive to injury (Additional file  1: ral hearing loss [55]. In our experiments, LPS adminis- Fig. S1), and Sox2 overexpression alleviated HC loss tration caused a significant increase in the expression of induced by aminoglycosides in the neonatal stage (Fig. 2), pro-inflammatory factors and a significant loss of OHCs, suggesting that deficiency of Sox2 in HCs in the basal but this inflammation-induced HC loss was alleviated by turn might be one of the reasons for their vulnerability to Sox2 overexpression (Fig. 7). Previous studies have shown ototoxic drugs. that many pro-inflammatory cytokines and inflammatory The influence of noise exposure on people’s physi - mediators can lead to cell apoptosis [56–58]. For exam- ological and mental state has been the focus of ple, increased interleukin levels mediate cell apoptosis by Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 14 of 16 Declarations regulating the Bax/Bcl-2 ratio in the cardiovascular sys- tem [56], and the TNF-α signaling pathway manipulates Ethics approval and consent to participate cell apoptosis through the Bax/Bak pathway or through a We performed all animal procedures according to protocols that were approved by the Animal Care and Use Committee of Fudan University and caspase-dependent pathway [57–61]. Our previous study that were consistent with the National Institute of Health’s Guide for the Care showed that inhibition of the TNF-α pathway was an and Use of Laboratory Animals. We made all efforts to minimize the number of effective means of protecting HCs [26]. In this paper, we animals used and to prevent their suffering. found that Sox2 overexpression dramatically suppressed Consent for publication LPS-induced and noise-induced upregulation of inflam - Not applicable. matory cytokines and apoptosis-related genes (Figs.  5, 6, Competing interests 7, 8), indicating that Sox2 overexpression inhibited the The authors declare that they have no competing interests. cascade of inflammatory cytokine release and subsequent inflammation-mediated HC apoptosis and hearing loss. Author details ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China. NHC Key Laboratory of Hearing Conclusions Medicine (Fudan University), Shanghai 200031, China. We report for the first time that Sox2 protects mature Received: 19 August 2021 Accepted: 9 February 2022 and terminally differentiated cells against injury. Sox2 overexpression preserved hearing against noise exposure by inhibiting HC apoptosis, and this was correlated with the decreased expression of pro-inflammatory factors. References These results thus suggest the protective role of Sox2 1. Yamasoba T, Lin FR, Someya S, Kashio A, Sakamoto T, Kondo K. Current overexpression in hearing and sensory HCs and provide concepts in age-related hearing loss: epidemiology and mechanistic novel targets for strategies for the prevention and treat- pathways. Hear Res. 2013;303:30–8. 2. Fridberger A, Flock A, Ulfendahl M, Flock B. Acoustic overstimulation ment of hearing loss. increases outer hair cell Ca2+ concentrations and causes dynamic con- tractions of the hearing organ. Proc Natl Acad Sci U S A. 1998;95:7127–32. 3. Davis A, McMahon CM, Pichora-Fuller KM, Russ S, Lin F, Olusanya BO, Abbreviations Chadha S, Tremblay KL. Aging and hearing health: the life-course Aif: Apoptosis inducing factor; Endo G: Endonuclease G; Prkaa1: Phospho- approach. 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Sox2 overexpression alleviates noise-induced hearing loss by inhibiting inflammation-related hair cell apoptosis

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Abstract

Background: The transcription factor Sox2 plays important roles in the developmental processes of multiple organs and tissues. However, whether Sox2 can protect mature or terminally differentiated cells against injury is still unknown. Methods: We investigated the roles of Sox2 in cochlear hair cells, which are terminally differentiated cells, using conditional transgenic mice and several hearing loss models. Results: Sox2 overexpression dramatically mitigated the degree of cochlear hair cell loss when exposed to ototoxic drugs. Noise-induced apoptosis of cochlear hair cells and hearing loss were also significantly alleviated by Sox2 overexpression. Notably, noise-induced upregulation of pro-inflammatory factors such as TNF-α and IL6 was inhibited by Sox2 overexpression. Then we used lipopolysaccharide to clarify the effect of Sox2 on cochlear inflammation, and Sox2 overexpression significantly inhibited lipopolysaccharide-induced upregulation of pro-inflammatory factors and alleviated inflammation-related cochlear hair cell death. Conclusions: These results demonstrate a novel protective role of Sox2 in mature and terminally differentiated coch- lear hair cells by inhibiting inflammation. Keywords: Sox2, Cell protection, Hearing loss, Cochlear hair cells, Apoptosis, Inflammatory response, Lipopolysaccharide Background onset of sensorineural hearing loss [1–7]. Because of the In mammals, cochlear mechanosensory hair cells (HCs) inability of HCs to regenerate after injury in the mature convert sound waves into electrical signals that are trans- mammalian cochlea, protecting HCs against injury is mitted to the brain’s auditory centers to produce hearing. extremely important for hearing preservation [8–10]. Aging, noise trauma, ototoxic drugs, and infections can Sox2 (HMG-box transcription factor sex-determining lead to HC loss or degeneration and thus result in the region Y-box  2), a well-studied pluripotent transcrip- tion factor, is critical for the embryonic development of many organs [11–13]. For example, Sox2 serves as a regulator in the process of taste bud sensory cell differ - *Correspondence: wenyan_li2000@126.com; chenyan0528@fudan.edu.cn; niyusu@aliyun.com entiation from endodermal progenitor cells [12], and the Diyan Chen, Gaogan Jia and Yanping Zhang contributed equally to this expression of Sox2 has a strong influence on the branch - work ing process in the respiratory system [13]. Deficiency ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China of Sox2 leads to abnormal cartilage development in the Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 2 of 16 tracheal mesenchyme and to abnormal cellular composi- for genetic identification. The primers used in the geno - tion [14], and Sox2 has been shown to play a significant typing protocols are listed in Additional file 1: Table S2. role in the development of the inner ear [15]. In addition to its essential role in stem cell self-renewal and repro- Auditory brainstem response (ABR) measurement gramming, Sox2 also plays a role in the survival of stem Mice were injected with a solution of Zoletil (70  g/kg) cells and cancer cells [16]. Sox2 directly up-regulates the and dexmedetomidine hydrochloride (1000  mg/kg) expression of Survivin, which inhibits the mitochondria- intraperitoneally to induce anesthesia. The efficacy of dependent apoptotic pathway in neural stem cells [17]. anesthesia was periodically ascertained through foot Although the roles of Sox2 in stem cells and cancer cells pinch response. The body temperature was maintained have been clarified in many studies, so far no studies at 37  °C to avoid any influence on the auditory physi - about the possible role of Sox2 in mature or terminally ology test. ABR measurements were used to assess the differentiated cells have been reported. mouse auditory function with a System 3 ABR work- In the development process of the inner ear, Sox2 is station (Tucker Davis Technology, Alachua, FL, USA). expressed in pro-sensory cells in the embryonic pro- Hearing function at 8, 16, 24, and 32 kHz was evaluated sensory domain. When the terminal differentiation of through an open field speaker, and acoustic thresh - cochlear progenitors is initiated, Sox2 expression is main- olds of sound pressure level were determined using the tained in the supporting cells but gradually decreases in BioSigRP software (Tucker Davis Technology, Alachua, auditory HCs. Once the cochlea becomes fully mature FL, USA). and hearing function has been developed, the expression of Sox2 in HCs becomes undetectable [18, 19]. During Cochlear HC injury models the maturation of the vestibular sensory epithelium, Sox2 To construct the ototoxic drug-induced HC injury expression is maintained in supporting cells and type 2 model in the neonatal stage, cochlear epithelia samples vestibular HCs, but is gradually diminished in type 1 ves- were obtained for explant culture from P2 transgenic tibular HCs. Sox2 is required for the maintenance of HCs mice that had received tamoxifen (Sigma, T5648-5G) during embryonic development of the zebrafish inner ear administration at P 0–1. The tissues were treated with and for HC regeneration in zebrafish embryos after injury 1  mM neomycin (Sigma) for 6  h and then harvested [20]. Our recent study reported that Sox2-negative type after an additional 24 h in the absence of neomycin. 1 vestibular HCs are more vulnerable than Sox2-positive To construct the noise-induced HC injury model, the type 2 vestibular HCs to 3,3’-iminodipropionitrile toxic- hearing function of P30 mice that had received tamox- ity in adult mice [21], suggesting that Sox2 might exert a ifen administration at P21–P22 were examined by ABR positive effect on the survival of sensory HCs. To explore test 24  h before noise exposure. The mice were then the possibility that Sox2 might exert a positive effect on exposed to broadband noise at 116 dB for 2 h. Hearing the survival of auditory HCs, we used conditional Sox2- function was evaluated again by ABR test 7  days after upregulated transgenic mice and several cochlear HC noise exposure, and the mice were killed and cochlear injury models to investigate the possible roles of Sox2 in samples were harvested for morphological analysis. HC damage and survival. To construct the lipopolysaccharide (LPS)-induced HC injury model, mice were given 10  μl LPS (Sigma- Aldrich, L2880) at 1, 2, or 4 mg/kg by tympanum injec- Materials and methods tion and killed 3 days later. Animals and genotyping The Rosa26-CAG-LSL (loxP-stop-loxP)-Sox2 transgenic Immunofluorescence and morphological analysis mice, referred to as Sox2OE mice, were designed by our Immunofluorescence staining was performed as previ - group and Beijing Biocytogen Co., Ltd. The Atoh1CreER ously reported [32]. Rabbit polyclonal anti-Myosin7A and PrestinCreER transgenic mice, which have been (Proteus Biosciences, 1: 1000 dilution, 25-6790), goat described in detail in previous studies [22, 23], were the polyclonal anti-SRY (sex-determining region Y)- kind gifts of Prof. Zuo Jian. For the Atoh1CreER line, box  2 (Sox2) (Santa Cruz Biotechnology, 1:1000 dilu- tamoxifen was injected subcutaneously once daily at tion, sc-17320), mouse monoclonal anti- Parvalbumin 75  mg/kg body weight at postnatal day (P) 0–1. For the (Sigma, 1:1000 dilution, P3088), mouse monoclonal PrestinCreER line, tamoxifen was injected intraperito- anti-Myosin7a (Santa Cruz Biotechnology, 1:500 dilu- neally once daily at 225  mg/kg body weight at P21–22. tion, sc-74516), rabbit mAb anti-TNF-α (Cell Signaling Mice of either sex were used for all experiments. Animals Technology, 1:400 dilution, 11948), and rat monoclo- were bred under specific and opportunistic pathogen- nal anti-IL6 (ThermoFisher Scientific, 1:200 dilution, free conditions, and tail DNA genotyping was performed Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 3 of 16 11-7061-82) were used as primary antibodies, and con- corresponding HRP-conjugated secondary antibodies trol incubations were routinely processed without pri- for 1 h at room temperature. The immunoreactive bands mary antibody treatments. were detected using an ECL kit (Pierce). Propidium iodide (PI) staining was used to detect the apoptotic and necrotic HC nuclei after noise expo- Statistical analysis sure as previously reported . Fixed cochleae were Data were analyzed using GraphPad Prism 8. Stu- permeabilized in 1% Triton X-100 solution for 30  min dent’s 2-tailed t-test was used to analyze the differences and then stained with 5  mg/ml PI dissolved in PBS for between two groups, and one-way ANOVA followed by 1  h at room temperature. After the final washes with the Bonferroni post-test was used for comparisons of PBS (10  min each), the tissue was dissected in PBS by differences among three or four groups. Data are pre - removing the modiolus. sented as the means ± S.E.M, and p < 0.05 was considered TUNEL staining was used to detect apoptotic HC significant. death. Tissues were fixed with 4% PFA for 1  h, treated with 1% Triton X-100 37  °C for 40  s, and incubated in Study approval TUNEL staining solution (Roche) according to the man- This study followed the “Guiding Directive for Humane ufacturer’s instructions. Treatment of Laboratory Animals” reporting guidelines enacted by the Chinese National Ministry of Science and Technology in 2006. All experiments were approved by Confocal imaging and cell counting the Shanghai Medical Experimental Animal Administra- Specimens were examined by confocal fluorescence tive Committee (Permit Number: 2009-0082) and were in microscopy (Leica SP8). To quantify the immunostain- accordance with animal welfare principles. ing-positive cells, nine separate segments along the entire cochlea were selected from the apex to the base. Results Sox2 abundance is correlated with HC tolerance to ototoxic Real‑time PCR drugs in neonatal HCs RNA was extracted from six cochleae of P30 adult mice First, we examined the Sox2 expression pattern in the from each group and was subsequently reverse tran- cochleae of wild type mice. Myosin7a was used as the scribed into cDNA using Superscript III reverse tran- marker of cochlear HCs. The cochlear epithelium is a scriptase (Invitrogen) according to the manufacturer’s delicate tissue containing region-specific HCs, which can instructions. Real-time PCR was performed on an ABI be roughly classified into two categories, namely inner 7500 real-time PCR system (Applied Biosystems) using hair cells (IHCs) and outer hair cells (OHCs), which have ™ ™ the TB Green PrimeScript RT-PCR Kit (Takara). The different functions, gene expression patterns, and mor - Actb gene was used as the endogenous control. Primer phological characteristics [24] (Fig. 1A). Immunofluores - sets are listed in Additional file  1: Table S1. The quantifi - cence results revealed that Sox2 was strongly expressed cation of relative gene expression, compared to Actb, was in all cochlear supporting cells, including inner bor- −ΔΔCT analyzed by the 2 method. der cells, inner phalangeal cells, inner pillar cells, outer pillar cells, Deiters’ cells, and Hensen’s cells (Fig.  1C). +/− Western blotting In the cochleae of adult control (PrestinCreER / −/− Proteins were extracted from isolated cochlear epithe- Sox2OE ) mice, Sox2 was not observed in the coch- lia with lysis buffer containing proteinase inhibitors, lear HCs (Fig.  1C). However, in the cochleae of neonatal separated by SDS-PAGE gel, and transferred onto PVDF mice, Sox2 was also observed in some IHCs and OHCs, membranes (Immobilon-P; Millipore, Bedford, MA, but the Sox2 abundance in HCs decreased from the api- USA). Membranes were blocked in 5% nonfat dried milk cal to the basal turns (Additional file  1: Fig. S1A, note the in TBST (20  mM Tris–HCl, 500  mM NaCl, and 0.1% Sox2 + HCs indicated by arrows in the apical turns). Sim- Tween-20) for 1  h and later incubated with the primary ilar to what was observed previously [25, 26], we found antibodies overnight at 4  °C. The following antibodies that the HC sensitivity to ototoxic drugs increased from were used for Western blot analysis: mouse monoclonal the apical to basal turns because more HCs were lost in anti-beta-Actin (Thermo Fisher Scientific, 1:5000 dilu - the basal turns compared to the apical turns in neonatal tion, MA515739), rabbit mAb anti-TNF-α (Cell Signaling mice (Additional file 1: Fig. S1B). Technology, 1:3000 dilution, 11948), and rat monoclo- nal anti-IL6 (ThermoFisher Scientific, 1:2000 dilution, 11-7061-82). The next day, the blots were washed three times with TBST and subsequently incubated with Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 4 of 16 Fig. 1 The cochlear morphology and hearing function were not affected by Sox2 overexpression. A The cross-section of the cochlear sensory epithelium. The IHCs and OHCs are located on the top layer, and most supporting cells (including BC, IphC, IPC, OPC, DC, and HeC) are located on the under layer. Some HCs can be observed in the top layer. IPC inner pillar cell, OPC outer pillar cell, DC Deiters’ cell, IPhC inner phalangeal cell, BC inner border cell, HeC Hensen’s cell, IS inner sulcus, CC Claudius cell. B Schematic for creating the transgenic mouse models. The Rosa26- CAG-LSL(loxP-stop-loxP)-Sox2 transgenic mice, referred to as the Sox2OE mice, were designed using the CRISPR/Cas9 method. C Images of Sox2 immunofluorescence (whole mount, HC layer) showed that there was no detectable Sox2 expression in the cochlear HCs of the control group, +/– +/– while in the PrestinCreER /Sox2OE group Sox2 expression was clearly observed in OHCs. D Experimental protocol for E–F. Tamoxifen was injected once daily during P21–P22, and the cochlear morphology and hearing function were assessed by immunofluorescence and ABR at P30. +/– +/– E Representative Myosin7a immunofluorescence showed no difference in HC morphology between the PrestinCreER /Sox2OE and control +/– +/– +/– groups. F Quantification of IHCs and OHCs in the PrestinCreER /Sox2OE and control groups. G The ABR thresholds of adult PrestinCreER / +/– Sox2OE and control mice. Data are presented as the mean ± SEM. n = 5 for each group. Scale bar = 20 µm Sox2 overexpression in HCs did not alter HC morphology CAG promoter in a Cre-mediated manner. To exam- or auditory function under physiological conditions ine the effects of Sox2 overexpression (Sox2OE) on To overexpress Sox2 in the cochlear HCs, we gener- cochlear HC survival in the neonatal and adult stages, ated the inducible Rosa26-CAG-LSL-Sox2 mouse line respectively, we used two Cre mouse lines, namely, in which forced expression of Sox2 was driven by the the Atoh1CreER line that targets the HCs including Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 5 of 16 both IHCs and OHCs in the neonatal stage and the turns, respectively. The average numbers of remain - PrestinCreER line that targets OHCs in the adult stage ing HCs in the middle and basal turns from the Atoh1- +/– +/– (Fig.  1B). We generated Pr estinCreER /Sox2OE Sox2OE groups were significantly larger than those from mice in which constitutive expression of Sox2 in control groups (Fig.  2D, p < 0.001). Thus it appears that Prestin cells were obtained by tamoxifen administra- there is a protective effect of Sox2 overexpression against tion, and these are referred to as Prestin-Sox2OE mice. neomycin injury. +/– –/– PrestinCreER /Sox2OE mice served as controls. To evaluate the expression of Sox2 in OHCs and to Sox2 overexpression attenuated noise‑induced HC determine the effect of Sox2OE on HC function under damage and hearing loss in adult mice physiological conditions, Prestin-Sox2OE and control To determine whether the susceptibility of HCs to noise mice were injected intraperitoneally (i.p.) with tamox- exposure changed after Sox2 overexpression, P31 Pres- ifen from P21 to P22, and their hearing function and tin-Sox2OE mice that received tamoxifen at P21–22 were cochlear morphological features were assessed at P30 exposed to 116 dB broadband noise for 2 h (Fig. 3A). The (Fig.  1D). In the control group, no Sox2 staining was hearing thresholds of the mice were determined using observed in OHCs from mature cochleae, while nuclear ABR tests at P38, and the cochlear specimens were har- +/– –/– staining of Sox2 was clearly observed in OHCs of Pres- vested afterwards. PrestinCreER /Sox2OE mice tin-Sox2OE mice, confirming the successful induction served as controls. In the control group, noise expo- of Cre activity and Sox2 overexpression in the Prestin sure led to significant OHC loss in the middle and basal cells (Fig.  1C). Immunofluorescence results showed turns of the cochlea (Fig.  3B–D). However, in the Pres- that the morphological features and quantities of coch- tin-Sox2OE cochlear epithelium, the noise-induced loss lear HCs in the Prestin-Sox2OE group were similar of OHCs was significantly attenuated in the middle and to those of controls (Fig.  1E, F). In addition, the ABR basal turns compared with the control mice (p < 0.01, results showed that the hearing function of adult Pres- Fig. 3B–D), indicating that Sox2-overexpressing cochlear tin-Sox2OE mice was comparable to controls at 8, 16, OHCs were less sensitive to noise-induced injury. 24, and 32  kHz (Fig.  1G). These results suggested that Hearing thresholds before noise exposure (baseline val- Sox2 overexpression in OHCs did not affect the main - ues) showed no significant difference between the Pres - tenance of HC function. tin-Sox2OE and the control groups (data not shown). At 7 days after noise exposure, mice in the control group had significant hearing loss, as demonstrated by the increased Sox2 overexpression protected HCs hearing thresholds at 8, 16, 24, and 32 kHz (Fig. 3E). The against aminoglycoside‑induced injury in the neonatal noise-induced threshold shifts in Prestin-Sox2OE mice stage were significantly lower at all frequencies compared To explore the function of Sox2 in HC survival, we first with those of the control mice (p < 0.05, Fig. 3F), suggest- used an in  vitro aminoglycoside antibiotic injury model. ing that Sox2 overexpression in OHCs protects against +/– +/– We generated Atoh1CreER /Sox2OE mice in which noise-induced hearing loss. constitutive expression of Sox2 in A toh1 HCs was obtained by tamoxifen administration, and these are referred to as the Atoh1-Sox2OE group. Sox2 overexpression protected OHCs by inhibiting The cochlear epithelium from P2 Atoh1-Sox2OE the noise‑induced apoptosis pathway mice that received tamoxifen injections at P 0-1 were To determine the mechanism through which Sox2 over- cultured and treated with 1  mM neomycin for 6  h and expression protects HCs against injury, we examined then allowed to recover without neomycin for an addi- the apoptotic and necrotic changes in HCs after noise +/– tional 24 h prior to harvesting (Fig. 2A). Atoh1CreER / exposure using PI staining based on previous research –/– Sox2OE mice served as controls. Myosin7a immu- showing the morphological changes that occur in OHCs nofluorescence was used to determine the number of after noise exposure [27]. In the control group, noise remaining HCs after neomycin treatment. In the control exposure induced morphological nuclear changes that group, neomycin treatment led to massive HC loss, and included both apoptotic (condensed nuclei) and necrotic the average numbers of remaining M yosin7a HCs were (swollen nuclei) changes in OHCs at 6  h after noise 57.0 ± 0.577, 31.5 ± 2.51, and 16.5 ± 1.52 cells/100  µm in exposure (Fig.  4A, B). However, the number of apop- the apical, middle, and basal turns, respectively (Fig. 2B– totic nuclei was significantly decreased in the Prestin- D). In the Atoh1-Sox2OE group, the average numbers Sox2OE group when compared with the control group of Myosin7a HCs were 56.8 ± 0.601, 54.2 ± 1.08, and (Fig.  4A, B). Meanwhile, no significant differences in the 43.3 ± 2.33 cells/100  µm in the apical, middle, and basal numbers of necrotic nuclei were observed between the Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 6 of 16 Fig. 2 Sox2 overexpression protected HCs from aminoglycoside-induced injury in the neonatal stage A Experimental protocol for C-D. Cochlear +/– +/– epithelia from Atoh1CreER /Sox2OE and control mice that received tamoxifen injection at P0–P1 were dissected and cultured at P2. Cochlear explants were treated with 1 mM neomycin for 6 h and harvested after an additional 24 h. B The scheme for cell counting. C Representative + +/– +/– images of Myosin7a cells in A toh1CreER /Sox2OE and control mice treated with or without neomycin. D Quantification of the cochlear HCs +/– +/– per 100 µm in the Atoh1CreER /Sox2OE and control groups. Data are presented as means ± SEM; n = 6 for each group; ***P < 0.001. Scale bar = 20 µm Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 7 of 16 +/– Fig. 3 Sox2 overexpression alleviated noise-induced hearing loss and HC damage. A Experimental protocol for B–F. Adult P restinCreER / +/– Sox2OE and control mice that received tamoxifen at P 21–22 were exposed to 116 dB broadband noise for 2 h. Cochlear morphology and + +/– +/– hearing function were evaluated 7 days after noise exposure. B Representative images of M yosin7a cells in PrestinCreER /Sox2OE and control +/– +/– mice treated with or without noise exposure. C, D Cochleograms showed noise-induced loss of IHCs and OHCs in the P restinCreER /Sox2OE +/– +/– and control groups. E, F Pure-tone ABR thresholds and threshold shifts in the PrestinCreER /Sox2OE and control group 7 days after the noise exposure. Scale bar = 20 µm. Data are presented as means ± SEM; n = 6 for each group; *P < 0.05, **P < 0.01, ***P < 0.001, indicated the significant +/– +/– +/– –/– difference between the PrestinCreER /Sox2OE with noise treatment and the PrestinCreER /Sox2OE with noise treatment groups Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 8 of 16 Fig. 4 Sox2 overexpression suppressed noise-induced HC apoptosis. A Representative images of PI-stained OHC nuclei in the cochlear epithelium. Both swollen and condensed OHC nuclei were observed in mice treated with noise exposure. Arrowheads point to swollen necrotic nuclei, and arrows point to condensed apoptotic nuclei. B The numbers of swollen or condensed OHC nuclei and missing OHCs in the cochlear epithelium at 6 h after noise exposure. C Representative images of TUNEL staining and Myosin7a immunofluorescence of the cochlear epithelium. TUNEL / + + + Myosin7a OHCs were observed in the mouse cochlea at 6 h after noise exposure. D Quantification of TUNEL /Myosin7a HCs in the cochlear epithelium. Data are presented as means ± SEM.; n = 6. *P < 0.05, **P < 0.01. Scale bar = 20 μm Prestin-Sox2OE and control groups. TUNEL staining (Aif, EndoG, and Bax) and necrosis-related genes (RipK1 was used to further determine the differences in apop - and RipK3) (Fig.  5A, B). In the Prestin-Sox2OE group, totic HCs between the two groups (Fig. 4C). Significantly the expression levels of Aif, Bax, and EndoG were signifi - + + fewer Myosin7a /TUNEL cells were observed in Pres- cantly decreased (Fig.  5A, p < 0.05), while the expression tin-Sox2OE mice compared to control mice (Fig.  4D), levels of RipK1 and RipK3 showed no significant differ - suggesting that Sox2 overexpression suppressed noise- ence when compared to the control group after noise induced apoptosis of OHCs. exposure (Fig.  5B), further confirming that Sox2 overex - We next assessed the relative mRNA expression level pression protected OHCs by inhibiting the noise-induced of apoptosis and necrosis-related genes at 6 h after noise apoptosis pathway. exposure. In the control group, noise exposure induced the increased expression of both apoptosis-related genes Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 9 of 16 Fig. 5 Sox2 overexpression suppressed noise-induced upregulation of inflammatory cytokines. A The mRNA expression levels of apoptosis-related genes (Aif, Bax, EndoG, Casp3, Casp8, and Casp9) were elevated 6 h after the completion of noise exposure. B The mRNA expression levels of necrosis-related genes (Prkaα1, Ripk1, and Ripk3) and pro-inflammatory cytokines (Tnf , Il-1β, and Il-6) were elevated 6 h after the completion of noise exposure. C TNF-α immunofluorescence in HCs after noise exposure. The TNF-α expression level was elevated after traumatic noise exposure in HCs, while it was inhibited in the Sox2 overexpression group. Parvalbumin is used as a marker of cochlear HCs. Data are presented as means ± SEM.; n = 6. *P < 0.05, **P < 0.01. Scale bar = 20 μm Sox2 overexpression alleviated HC damage by inhibiting anti-TNF-α and anti-IL6 antibodies, and we found that inflammation noise-induced TNF-α and IL6 expression in cochlear In consideration of the important role of inflammation HCs was relatively lower in the Prestin-Sox2OE group in HC death, the mRNA expression of pro-inflammation compared to the control group (Figs. 5C, 6). mediators was measured. In the control group, noise To determine the role of Sox2 upregulation in the exposure induced significantly increased expression of inflammatory process, we constructed the lipopolysac - Tnf, Il-1β, and Il-6 at 6  h after noise exposure (Fig.  5B). charide (LPS) challenge model. It has been previously In the Prestin-Sox2OE group, the expression level of reported that inflammation-related mRNA expression these pro-inflammation mediators declined significantly is upregulated in the cochlea within 6  h after LPS chal- to a level comparable to that in the group without noise lenge [23]. To determine the optimal concentration of exposure (Fig.  5B, p < 0.05), indicating that Sox2 over- LPS in inflammation-mediated HC loss, 10 μl LPS at dif - expression dramatically attenuated the noise-induced ferent concentrations (1  mg/kg, 2  mg/kg, and 4  mg/kg) inflammation process. To further evaluate the protein was injected into the tympanic cavity of wild type mice expression level of pro-inflammatory mediators in coch - and the cochleae were dissected for analysis 72  h later. lear epithelia, we performed Western blotting analysis of LPS induced no obvious cochlear HC loss in the 1 mg/kg TNF-α and IL6. These results showed that noise expo - LPS group, mild HC loss in the basal turns in the 2 mg/ sure led to significant increases in TNF-α and IL6 protein kg LPS group, and severe HC loss in the middle and basal levels in the control group, but Sox2 overexpression sig- turns in the 4  mg/kg LPS group. Thus, 4  mg/kg of LPS nificantly inhibited the noise-induced increase of TNF-α was considered the optimal concentration for inducing and IL6 protein (Fig.  6). We also measured the expres- significant HC loss in both the middle and basal turns. sion of TNF-α and IL6 protein in the organ of Corti using Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 10 of 16 Fig. 6 Sox2 overexpression suppressed noise-induced upregulation of IL6 and TNF-α. A IL6 immunofluorescence in cochlear cross-section after noise treatment. The IL6 expression level was elevated after traumatic noise exposure in HCs, while it was inhibited in the Sox2 overexpression group. Myosin7a is used as a marker of cochlear HCs. B Western blot analysis of sensory epithelia shows increased expression levels of TNF-α and IL6 in control mice after noise injury, but it was significantly inhibited in Prestin-Sox2OE mice. β-Actin served as the sample loading control. Data are presented as means ± SEM.; n = 3. *P < 0.05, **P < 0.01. Scale bar = 20 μm (See figure on next page.) Fig. 7 Sox2 upregulation mitigated inflammation-induced HC loss. A Experimental protocol for the LPS-induced HC loss model. B Myosin7a +/– +/– immunofluorescence in cochlear HCs of adult PrestinCreER /Sox2OE mice and control mice with or without LPS treatment. C Cell counting results of cochlear HCs after 4 mg/kg of LPS administration. D TNF-α immunofluorescence in cochlear cross-section after LPS challenge. E The mRNA expression levels of pro-inflammatory cytokines. Sox2 overexpression prevented the LPS-induced increase in pro-inflammatory cytokines. Data are presented as means ± SEM; n = 6; **P < 0.01, and ***P < 0.001. Scale bar = 20 μm Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 11 of 16 Fig. 7 (See legend on previous page.) Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 12 of 16 We compared LPS-induced HC damage between the lower in the Prestin-Sox2OE group, which was a sign Prestin-Sox2OE and control groups. Significantly more that the inflammation reaction was limited (Fig.  7E). cochlear HCs were observed in the middle and basal Western blot analysis also showed very high expression turns in the Prestin-Sox2OE group than in the control levels of TNF-α and IL6 protein in the control mice and group, indicating that Sox2 overexpression largely allevi- limited expression in the Prestin-Sox2OE mice after LPS ated LPS-induced HC damage (Fig. 7A–C). We next eval- treatment (Fig.  8). The immunofluorescence results also uated the mRNA expression level of pro-inflammatory showed that the LPS-induced upregulation of TNF-α cytokines induced by LPS. qPCR results showed that Tnf, and IL6 in HCs was attenuated by Sox2 overexpres- Il-6, and Il-1β increased substantially after LPS treatment sion (Figs.  7D, 8). Collectively, these results showed that in the control group, but their expression was relatively Fig. 8 Sox2 overexpression suppressed LPS-induced upregulation of IL6 and TNF-α. A IL6 immunofluorescence in cochlear cross-section after LPS treatment. Myosin7a is used as a marker of cochlear HCs. (B) Western blot analysis of sensory epithelia shows increased expression levels of TNF-α and IL6 in control mice after LPS treatment, but the LPS-induced increase of the TNF-α and IL6 protein was significantly inhibited in Prestin-Sox2OE mice. β-Actin served as the sample loading control. Data are presented as means ± SEM.; n = 3. *P < 0.05, **P < 0.01. Scale bar = 20 μm Chen  et al. Journal of Neuroinflammation (2022) 19:59 Page 13 of 16 Sox2 upregulation inhibited the cochlear inflammatory increasing attention in the past decade [42, 43]. The response and subsequent HC death. impact of noise-induced hearing loss is broad and profound, including the loss of the ability to commu- Discussion nicate with others and subsequent social isolation. Up An estimated 466 million people are living with hear- to now, few medicines approved by the Food and Drug ing loss worldwide. Although the exact mechanisms are Administration have been confirmed to be effective in not fully understood, oxidative stress, inflammation, preventing or treating noise-induced hearing loss in autophagy, apoptosis, and necrosis have been reported clinical trials. Consistent with previous studies [44–46], to be involved in the onset and progression of sensori- our data further confirmed that apoptosis, necrosis, neural hearing loss [27, 28], and various strategies have and inflammation are involved in noise-induced HC been explored to try to protect sensory HCs and hear- death (Figs.  4, 5). Aif and Endo G, which act as apop- ing. Some anti-apoptosis genes, like Bcl2 and Xiap, have totic mediators, translocate into the nucleus during been reported to have protective roles in cochlear HCs HC injury and lead to HC apoptosis [47–49]. Prkaa1 and hearing [29, 30]. Antioxidants such as N-acetyl- (also known as AMPK) and receptor-interacting pro- L-cysteine, coenzyme Q10, and vitamin E have been tein kinase (RipK) have been reported be associated reported to be effective in protecting against ototoxic with necrotic HC death [27, 50]. Although the endog- drugs and noise exposure [31–34]. Some genes, like enous expression of Sox2 is very low in mature coch- Bmi1, G6PD, adenosine A1 receptor, and FoxG1, pro- lear HCs, forced Sox2 overexpression in Prestin OHCs tect HCs by inhibiting oxidative stress or by suppress- has a strong protective effect against noise-induced ing inflammatory pathways that are induced by ototoxic hearing injury (Fig.  3). PI staining results showed that drugs or that are related to aging-related damage [28, Sox2 upregulation decreased the number of HCs that 35–37]. Besides classic anti-apoptosis, anti-necrosis, and underwent apoptosis (Fig.  4), and this was accompa- anti-inflammation factors, some development-related nied by a decrease in noise-induced expression of pro- factors, such as Wnt/beta-catenin and Islet-1, have been apoptosis genes (Aif, EndoG, and Bax) and the number shown to be capable of protecting HCs against injury and of TUNEL HCs (Fig. 5). In contrast, Sox2 overexpres- thus preserving hearing [35, 38]. sion had no obvious effect on the number of HCs that In the cochlea, apical HCs are responsible for sensing underwent necrosis, as demonstrated by the PI staining sound at lower frequencies, and basal HCs are responsi- results and the mRNA expression levels of pro-necrosis ble for sensing sound at higher frequencies. Hearing loss factors (Figs.  4, 5). Together, these results suggest that in patients is generally more severe at higher frequen- Sox2 overexpression protects HCs and hearing against cies than lower frequencies, and our experimental data noise-induced injury by inhibiting HC apoptosis. showed that the sensitivity of HCs to injury increased The inflammatory response activated by extrinsic from the apical to basal turns. The three different treat - attack and infection is also a strictly regulated process, ments—aminoglycosides (Fig.  2), broadband noise and an appropriate inflammatory response can elimi - (Fig.  3), and LPS (Fig.  7)—all led to an apical-to-basal nate the pathogen, repair damaged tissue, and maintain gradient of damage in cochlear HCs, and severe HC loss cell homeostasis [51, 52]. However, dysregulation of the occurred in the basal turn while the mildest HC loss inflammatory response is pivotal in the initiation and occurred in the apical turn. Previous studies showed that progression of multiple diseases, such as age-related dis- exposure to low-decibel broad band noise (8–16  kHz) eases [53] and neurodegeneration diseases [54]. In the results in HC damage mainly in the middle turns, but the auditory system, LPS can penetrate into the labyrinth HC damage extends to the basal turn when the intensity through the round window membrane, where it stimu- of the noise is greater than 100  dB [39–41]. The mecha - lates the production and release of cytokines and medi- nism behind the difference in HC injury sensitivity along ates the initiation and development of inflammatory the cochlear turns remains unclear. In this work, we reactions in the media and labyrinth, which in turn leads found that Sox2 was expressed in the neonatal cochlear to auditory epithelial injury and subsequent sensorineu- HCs, which are less sensitive to injury (Additional file  1: ral hearing loss [55]. In our experiments, LPS adminis- Fig. S1), and Sox2 overexpression alleviated HC loss tration caused a significant increase in the expression of induced by aminoglycosides in the neonatal stage (Fig. 2), pro-inflammatory factors and a significant loss of OHCs, suggesting that deficiency of Sox2 in HCs in the basal but this inflammation-induced HC loss was alleviated by turn might be one of the reasons for their vulnerability to Sox2 overexpression (Fig. 7). Previous studies have shown ototoxic drugs. that many pro-inflammatory cytokines and inflammatory The influence of noise exposure on people’s physi - mediators can lead to cell apoptosis [56–58]. For exam- ological and mental state has been the focus of ple, increased interleukin levels mediate cell apoptosis by Chen et al. Journal of Neuroinflammation (2022) 19:59 Page 14 of 16 Declarations regulating the Bax/Bcl-2 ratio in the cardiovascular sys- tem [56], and the TNF-α signaling pathway manipulates Ethics approval and consent to participate cell apoptosis through the Bax/Bak pathway or through a We performed all animal procedures according to protocols that were approved by the Animal Care and Use Committee of Fudan University and caspase-dependent pathway [57–61]. Our previous study that were consistent with the National Institute of Health’s Guide for the Care showed that inhibition of the TNF-α pathway was an and Use of Laboratory Animals. We made all efforts to minimize the number of effective means of protecting HCs [26]. In this paper, we animals used and to prevent their suffering. found that Sox2 overexpression dramatically suppressed Consent for publication LPS-induced and noise-induced upregulation of inflam - Not applicable. matory cytokines and apoptosis-related genes (Figs.  5, 6, Competing interests 7, 8), indicating that Sox2 overexpression inhibited the The authors declare that they have no competing interests. cascade of inflammatory cytokine release and subsequent inflammation-mediated HC apoptosis and hearing loss. Author details ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China. NHC Key Laboratory of Hearing Conclusions Medicine (Fudan University), Shanghai 200031, China. We report for the first time that Sox2 protects mature Received: 19 August 2021 Accepted: 9 February 2022 and terminally differentiated cells against injury. 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Journal

Journal of Neuroinflammation – Springer Journals

Published: Feb 28, 2022

Keywords: Sox2; Cell protection; Hearing loss; Cochlear hair cells; Apoptosis; Inflammatory response; Lipopolysaccharide

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