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Abstract：
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 (H2O2)-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 H2O2. Following UCHL1 overexpression in H2O2-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 H2O2-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 H2O2-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 H2O2-treated HEI-OC1 cells. In addition, UCHL1 overexpression enhanced cell viability and promoted oxidative damage, apoptosis and senescence in H2O2-induced HEI-OC1 cells. Furthermore, Sp1 was upregulated in H2O2-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 H2O2-exposed HEI-OC1 cells. Collectively, the results suggested that UCHL1 transcriptional suppression by Sp1 protected cochlear hair cells from H2O2-triggered senescence and oxidative damage.
摘要：
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，与几种类型的人类疾病有关。本研究旨在研究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。逆转录-定量(RT-q)PCR和蛋白质印迹分析用于评估暴露于H2O2的HEI-OC1细胞中的UCHL1表达。在H2O2诱导的HEI-OC1细胞中UCHL1过表达后，通过细胞计数试剂盒-8测定评估细胞活性。氧化应激相关标志物的含量，包括超氧化物歧化酶（SOD），使用相应的商业试剂盒测量谷胱甘肽过氧化物酶（GSH-Px）和活性氧（ROS）。通过TUNEL测定和蛋白质印迹分析评估细胞凋亡。通过衰老相关的β-半乳糖苷酶染色和蛋白质印迹分析评估细胞衰老。应用RT-qPCR和Westernblot分析检测mRNA和蛋白表达水平,分别,H2O2处理的HEI-OC1细胞中的特异性蛋白1（Sp1）。此外,UCHL1和Sp1之间的关联通过荧光素酶报告基因和染色质免疫沉淀(ChIP)分析得到验证.通过RT-qPCR和westernblot分析在Sp1过表达细胞中也测定了UCHL1的mRNA和蛋白表达水平,分别。在UCHL1过表达H2O2处理的HEI-OC1细胞中Sp1过表达后，细胞活性,氧化应激，评估细胞凋亡和衰老。最后,采用蛋白质印迹法检测NF-κB信号相关蛋白p-NF-κBp65和NF-κBp65的表达水平。结果显示UCHL1在H2O2处理的HEI-OC1细胞中下调。此外,UCHL1过表达增强细胞活力并促进氧化损伤，H2O2诱导的HEI-OC1细胞凋亡和衰老。此外，Sp1在H2O2处理的HEI-OC1细胞中上调。此外,荧光素酶报告基因和ChIP分析证明Sp1与UCHL1启动子相互作用以抑制UCHL1转录。Sp1过表达逆转了UCHL1过表达对细胞活力的影响，氧化应激，凋亡，H2O2暴露的HEI-OC1细胞中NF-κB信号通路的衰老和激活。总的来说，结果表明，Sp1抑制UCHL1转录保护耳蜗毛细胞免受H2O2引发的衰老和氧化损伤。
