Li Jie, Zhu Xiaoyan, Ye Shiming, Dong Qi, Hou Jie, Liu Jing, She Wandong
Department of Otolaryngology-Head and Neck Surgery, Nanjing Drum Tower Hospital, Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210008, China.
Department of Otolaryngology, Nantong Hospital Affiliated to Nanjing University of Chinese Medicine, Nantong 226000, China.
Acta Biochim Biophys Sin (Shanghai). 2024 Nov 1;57(5):727-737. doi: 10.3724/abbs.2024194.
Glucocorticoids (GCs) are commonly used to treat sudden sensorineural hearing loss (SSNHL), although some patients are resistant to this therapeutic approach. Clinical studies have demonstrated the efficacy of tanshinone IIA (TA) in combination with GC for managing various human ailments. However, it remains unclear whether TA can mitigate GC resistance in SSNHL. Our aim is to elucidate the role of NRF2-induced transcriptional regulation of HDAC2 in influencing GC resistance and investigate the involvement of TA-related molecular pathways in GC resistance. Here, HEI-OC1 cells are treated with lipopolysaccharide (LPS) to establish an model for SSNHL. The cells are subsequently treated with dexamethasone (DXE) or DXE + TA. RT-qPCR and western blot analysis are used to measure the mRNA and protein levels of Forkhead box P3 (FOXP3), nuclear factor erythroid 2-related factor 2 (NRF2), and histone deacetylase 2 (HDAC2). Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays are carried out to assess cell proliferation. Flow cytometry analysis is performed to evaluate apoptosis. Mechanistic studies involve chromatin immunoprecipitation (ChIP), luciferase reporter, and DNA pull-down assays. Our results show that treatment with TA + DEX significantly increases proliferation and suppresses apoptosis in LPS-treated HEI-treated OC1 cells. TA upregulates HDAC2 expression by activating NRF2-mediated transcription of HDAC2, with the NRF2-HDAC2 binding site located at bases 419-429 (ATGACACTCCA) in the promoter sequence of . Furthermore, TA upregulates FOXP3 expression to activate NRF2 transcription, with the predicted FOXP3-binding site located at bases 864-870 (GCAAACA) in the promoter sequence of . In summary, these findings suggest that TA enhances the therapeutic effects of GC on the proliferation and apoptosis of HEI OC1 cells by increasing FOXP3/Nrf2 expression. These results indicate that TA may be promising for ameliorating GC resistance in patients with SSNHL.
糖皮质激素（GCs）常用于治疗突发性感音神经性听力损失（SSNHL），尽管一些患者对这种治疗方法有抗性。临床研究已证明丹参酮IIA（TA）与GC联合使用在治疗各种人类疾病方面的有效性。然而，TA是否能减轻SSNHL中的GC抗性仍不清楚。我们的目的是阐明NRF2诱导的HDAC2转录调控在影响GC抗性中的作用，并研究TA相关分子途径在GC抗性中的参与情况。在此，用脂多糖（LPS）处理HEI-OC1细胞以建立SSNHL模型。随后用 dexamethasone（DXE）或 DXE + TA处理细胞。采用RT-qPCR和蛋白质印迹分析来测量叉头框P3（FOXP3）、核因子红细胞2相关因子2（NRF2）和组蛋白去乙酰化酶2（HDAC2）的mRNA和蛋白质水平。进行细胞计数试剂盒-8（CCK-8）和5-乙炔基-2'-脱氧尿苷（EdU）试验以评估细胞增殖。进行流式细胞术分析以评估细胞凋亡。机制研究涉及染色质免疫沉淀（ChIP）、荧光素酶报告基因和DNA下拉试验。我们的结果表明，TA + DEX处理显著增加了LPS处理的HEI处理的OC1细胞的增殖并抑制了细胞凋亡。TA通过激活NRF2介导的HDAC2转录来上调HDAC2表达，NRF2-HDAC2结合位点位于启动子序列的第419-429位碱基（ATGACACTCCA）。此外，TA上调FOXP3表达以激活NRF2转录，预测的FOXP3结合位点位于启动子序列的第864-870位碱基（GCAAACA）。总之，这些发现表明TA通过增加FOXP3/Nrf2表达增强了GC对HEI OC1细胞增殖和凋亡的治疗作用。这些结果表明TA可能有望改善SSNHL患者的GC抗性。
