Li Lihua, Xu Kai, Bai Xue, Wang Zhi, Tian Xiaoyan, Chen Xubo
Department of Otorhinolaryngology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Exp Ther Med. 2023 Jan 9;25(2):94. doi: 10.3892/etm.2023.11793. eCollection 2023 Feb.
Age-related hearing loss (ARHL) is the most common cause of hearing loss in the elderly. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme involved in several types of human disease. The present study aimed to investigate the effect of UCHL1 on a hydrogen peroxide (HO)-induced ARHL model in cochlear hair cells and uncover its underlying mechanism. Reverse transcription-quantitative (RT-q)PCR and western blot analysis were used to assess UCHL1 expression in HEI-OC1 cells exposed to HO. Following UCHL1 overexpression in HO-induced HEI-OC1 cells, cell activity was assessed by Cell Counting Kit-8 assay. The content of oxidative stress-associated markers including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and reactive oxygen species (ROS ) was measured using corresponding commercial kits. Cell apoptosis was evaluated by TUNEL assay and western blot analysis. Cell senescence was assessed by senescence-associated β-galactosidase staining and western blot analysis. RT-qPCR and western blot analysis were applied to measure mRNA and protein expression levels, respectively, of specificity protein 1 (Sp1) in HO-treated HEI-OC1 cells. In addition, the association between UCHL1 and Sp1 was verified by luciferase reporter and chromatin immunoprecipitation (ChIP) assay. The mRNA and protein expression levels of UCHL1 were also determined in Sp1-overexpressing cells by RT-qPCR and western blot analysis, respectively. Following Sp1 overexpression in UCHL1-overexpressing HO-treated HEI-OC1 cells, cell activity, oxidative stress, apoptosis and senescence were assessed. Finally, the expression levels of NF-κB signaling-related proteins p-NF-κB p65 and NF-κB p65 were detected using western blot analysis. The results showed that UCHL1 was downregulated in HO-treated HEI-OC1 cells. In addition, UCHL1 overexpression enhanced cell viability and promoted oxidative damage, apoptosis and senescence in HO-induced HEI-OC1 cells. Furthermore, Sp1 was upregulated in HO-treated HEI-OC1 cells. Additionally, luciferase reporter and ChIP assays demonstrated that Sp1 interacted with the UCHL1 promoter to inhibit UCHL1 transcription. Sp1 overexpression reversed the effect of UCHL1 overexpression on cell viability, oxidative stress, apoptosis, senescence and activation of the NF-κB signaling pathway in HOexposed HEI-OC1 cells. Collectively, the results suggested that UCHL1 transcriptional suppression by Sp1 protected cochlear hair cells from HO-triggered senescence and oxidative damage.
年龄相关性听力损失（ARHL）是老年人听力损失的最常见原因。泛素羧基末端水解酶L1（UCHL1）是一种去泛素化酶，与多种人类疾病有关。本研究旨在探讨UCHL1对过氧化氢（H₂O₂）诱导的耳蜗毛细胞ARHL模型的影响，并揭示其潜在机制。采用逆转录定量（RT-q）PCR和蛋白质免疫印迹分析评估暴露于H₂O₂的HEI-OC1细胞中UCHL1的表达。在H₂O₂诱导的HEI-OC1细胞中过表达UCHL1后，通过细胞计数试剂盒-8法评估细胞活性。使用相应的商业试剂盒测量氧化应激相关标志物的含量，包括超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和活性氧（ROS）。通过TUNEL法和蛋白质免疫印迹分析评估细胞凋亡。通过衰老相关β-半乳糖苷酶染色和蛋白质免疫印迹分析评估细胞衰老。分别应用RT-qPCR和蛋白质免疫印迹分析测量H₂O₂处理的HEI-OC1细胞中特异性蛋白1（Sp1）的mRNA和蛋白质表达水平。此外，通过荧光素酶报告基因和染色质免疫沉淀（ChIP）试验验证UCHL1与Sp1之间的关联。还分别通过RT-qPCR和蛋白质免疫印迹分析测定Sp1过表达细胞中UCHL1的mRNA和蛋白质表达水平。在过表达UCHL1的H₂O₂处理的HEI-OC1细胞中过表达Sp1后，评估细胞活性、氧化应激、凋亡和衰老。最后，使用蛋白质免疫印迹分析检测NF-κB信号通路相关蛋白p-NF-κB p65和NF-κB p65的表达水平。结果表明，在H₂O₂处理的HEI-OC1细胞中UCHL1表达下调。此外，UCHL1过表达增强了H₂O₂诱导的HEI-OC1细胞的活力，并促进了氧化损伤、凋亡和衰老。此外，在H₂O₂处理的HEI-OC1细胞中Sp1表达上调。此外，荧光素酶报告基因和ChIP试验表明，Sp1与UCHL1启动子相互作用以抑制UCHL1转录。Sp1过表达逆转了UCHL1过表达对H₂O₂暴露的HEI-OC1细胞的细胞活力、氧化应激、凋亡、衰老和NF-κB信号通路激活的影响。总体而言，结果表明Sp1对UCHL1的转录抑制可保护耳蜗毛细胞免受H₂O₂触发的衰老和氧化损伤。
