骨化三醇通过调节 ATF3/DUSP1 信号通路缓解噪声性听力损失。

Calcitriol alleviates noise-induced hearing loss by regulating the ATF3/DUSP1 signalling pathway.

机构信息

Department of Otolaryngology Head and Neck Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi'an, China.

Department of Otorhinolaryngology-Head and Neck Surgery, Zhongnan Hospital of Wuhan University, Wuhan, China.

出版信息

Ecotoxicol Environ Saf. 2024 Oct 1;284:116906. doi: 10.1016/j.ecoenv.2024.116906. Epub 2024 Aug 24.

DOI:10.1016/j.ecoenv.2024.116906

PMID:39182283

Abstract

BACKGROUND

Calcitriol (Cal) is the most active metabolite of vitamin D and has antioxidant and anti-inflammatory properties. The aim of this study was to investigate the role of Cal in noise-induced hearing loss (NIHL) to further elucidate the mechanism of noise-induced oxidative stress in the mouse cochlea.

METHODS

C57BL/6 J mice were given six intraperitoneal injections of Cal (500 ng/kg/d). After 14 days of noise exposure, auditory brainstem response (ABR) thresholds, and the cochlear outer hair cell loss rate were analysed to evaluate auditory function. Real-time fluorescence quantitative PCR, immunofluorescence and western blotting were performed in vitro after the treatment of cochlear explants with 100 µM tert-butyl hydroperoxide (TBHP) for 2.5 h and HEI-OC1 cells with 250 µM TBHP for 1.5 h.

RESULTS

In vivo experiments confirmed that Cal pretreatment mitigated NIHL and outer hair cell death. The in vitro results demonstrated that Cal significantly reduced TBHP-induced cochlear auditory nerve fibre degradation and spiral ganglion neuron damage. Moreover, treatment with Cal inhibited the expression of oxidative stress-related factors (3-NT and 4-HNE) and DNA damage-related factors (γ-H2A.X) and attenuated TBHP-induced apoptosis in cochlear explants and HEI-OC1 cells. A total of 1479 upregulated genes and 1443 downregulated genes were screened in cochlear tissue 1 h after noise exposure. The level of transcription factor 3 (ATF3) was significantly elevated in HEI-OC1 cells after TBHP stimulation. Gene Transcription Regulation Database （GTRD）and Cistrome database analyses revealed that the downstream target gene of ATF3 is dual specificity phosphatase 1 (DUSP1). Cistrome DB Toolkit database results showed that the transcription factor of DUSP1 was ATF3. In addition, the ChIP-PCR results indicated that ATF3 might be a direct transcription factor of DUSP1.

CONCLUSION

The results of our study suggest that Cal attenuates NIHL and inhibits noise-induced apoptosis by regulating the ATF3/DUSP1 signalling pathway.

摘要

背景

钙三醇（Cal）是维生素 D 的最活跃代谢物，具有抗氧化和抗炎特性。本研究旨在探讨 Cal 在噪声性听力损失（NIHL）中的作用，以进一步阐明小鼠耳蜗中噪声诱导氧化应激的机制。

方法

C57BL/6J 小鼠给予六次腹腔注射 Cal（500ng/kg/d）。在噪声暴露 14 天后，分析听觉脑干反应（ABR）阈值和耳蜗外毛细胞损失率，以评估听觉功能。体外培养耳蜗组织 2.5 小时后用 100μM 叔丁基过氧化物（TBHP）处理，HEI-OC1 细胞 1.5 小时后用 250μM TBHP 处理，实时荧光定量 PCR、免疫荧光和 Western blot 检测。

结果

体内实验证实 Cal 预处理减轻了 NIHL 和外毛细胞死亡。体外实验结果表明，Cal 显著减少了 TBHP 诱导的耳蜗听神经纤维降解和螺旋神经节神经元损伤。此外，Cal 处理抑制了氧化应激相关因子（3-NT 和 4-HNE）和 DNA 损伤相关因子（γ-H2A.X）的表达，减轻了 TBHP 诱导的耳蜗组织和 HEI-OC1 细胞凋亡。噪声暴露后 1 小时，耳蜗组织共筛选出 1479 个上调基因和 1443 个下调基因。TBHP 刺激后，HEI-OC1 细胞中转录因子 3（ATF3）的水平显著升高。基因转录调控数据库（GTRD）和 Cistrome 数据库分析显示，ATF3 的下游靶基因是双特异性磷酸酶 1（DUSP1）。Cistrome DB Toolkit 数据库结果显示，DUSP1 的转录因子是 ATF3。此外，ChIP-PCR 结果表明，ATF3 可能是 DUSP1 的直接转录因子。