黄芩素拮抗顺铂所致的耳蜗毛细胞损伤的体外研究

Baicalein attenuates Cisplatin-induced cochlear hair cell damage: an in vitro study

摘要

目的 探究黄芩素对顺铂所致的耳蜗毛细胞损伤的作用及其潜在分子机制。方法 以小鼠听觉毛细胞系（house ear institute-organ of corti 1，HEI-OC1）及离体培养的小鼠耳蜗组织为模型，采用CCK-8法、细胞显微成像和细胞活死染色评估黄芩素对顺铂所致细胞毒性的影响；通过免疫荧光染色检测耳蜗毛细胞存活率；结合实时荧光定量PCR（real-time quantitative polymerase chain reaction，RT-qPCR）、流式细胞术、蛋白质免疫印迹等技术分析黄芩素拮抗顺铂耳毒性的具体机制。结果 黄芩素可显著抑制顺铂诱导的HEI-OC1细胞死亡（P<0.001），且在离体耳蜗模型中无细胞毒性。与顺铂单药组相比，黄芩素联合治疗组的毛细胞存活率提高（P=0.0005）。黄芩素能有效减少线粒体超氧化物及细胞内总活性氧积累，下调促凋亡蛋白Bax、上调抗凋亡蛋白Bcl-2的表达，并抑制Caspase-3/9的活化。结论 黄芩素通过减轻线粒体氧化应激，拮抗顺铂诱导的耳蜗毛细胞凋亡，为顺铂耳毒性的防护提供了潜在治疗策略。

Abstract

Objective The objective of this study is to investigate the protective effects of baicalein against cisplatin-induced cochlear hair cell damage and to elucidate its underlying molecular mechanisms. Methods Utilizing the mouse auditory hair cell line (house ear institute-organ of corti 1, HEI-OC1) and ex vivo cultured mouse cochlear tissues as models, the impact of baicalin on cisplatin-induced cytotoxicity were assessed via Cell Counting Kit-8 assay, microscopic imaging, and live/dead cell staining. The survival of cochlear hair cells were evaluated through the use of immunofluorescence staining. To this end, mechanistic studies were conducted using real-time quantitative polymerase chain reaction (RT-qPCR), flow cytometry, and Western blot (WB) analysis. Results The finding revealed that baicalein significantly attenuated cisplatin-induced HEI-OC1 cell death (P<0.001) without exhibiting toxicity in the ex vivo cochlear model. In comparison with the cisplatin-only group, the baicalein co-treatment group exhibited a significant increase in hair cell survival (P=0.0005). Mechanistic investigations revealed that baicalein effectively reduced mitochondrial superoxide and total intracellular reactive oxygen species accumulation, Furthermore, baicalein was found to downregulate the pro-apoptotic protein Bax, upregulated the anti-apoptotic protein Bcl-2, and suppressed Caspase-3/9 activation. Conclusion Baicalein has been shown to counteract cisplatin-induced cochlear hair cell apoptosis by attenuating mitochondrial oxidative stress. This finding suggests that balcalein could serve as a promising therapeutic agent for preventing cisplatin-induced ototoxicity.

关键词

黄芩素 / 药物性聋 / 顺铂 / 耳毒性 / 听力损失

Key words

Baicalein / Pharmacologic deafness / Cisplatin / Ototoxicity / Hearing loss

参考文献

[1] Check W. How do things stand with cisplatin? [J]. JAMA, 1978, 240(23): 2521-2525

[2] Lee J, Fernandez K, Cunningham LL. Hear and now: ongoing clinical trials to prevent drug-induced hearing loss [J]. Annu Rev Pharmacol Toxicol, 2024, 64: 211-230. DOI: 10.1146/annurev-pharmtox-033123-114106

[3] Dhillon S. Sodium thiosulfate: pediatric first approval [J]. Paediatr Drugs, 2023, 25(2): 239-244. DOI: 10.1007/s40272-022-00550-x

[4] Freyer DR, Chen L, Krailo MD, et al. Effects of sodium thiosulfate versus observation on development of cisplatin-induced hearing loss in children with cancer (ACCL0431): a multicentre, randomised, controlled, open-label, phase 3 trial[J]. Lancet Oncol, 2017, 18(1): 63-74. DOI: 10.1016/S1470-2045(16)30625-8

[5] Freyer DR, Brock PR, Chang KW, et al. Prevention of cisplatin-induced ototoxicity in children and adolescents with cancer: a clinical practice guideline [J]. Lancet Child Adolesc Health, 2020, 4(2): 141-150. DOI: 10.1016/S2352-4642(19)30336-0

[6] Lye J, Delaney DS, Leith FK, et al. Recent therapeutic progress and future perspectives for the treatment of hearing loss [J]. Biomedicines, 2023, 11(12): 3347. DOI: 10.3390/biomedicines11123347

[7] Nan BY, Zhao ZR, Jiang KL, et al. Astaxanthin attenuates cisplatin ototoxicity in vitro and protects against cisplatin-induced hearing loss in vivo [J]. Acta Pharm Sin B, 2022, 12(1): 167-181. DOI: 10.1016/j.apsb.2021.07.002

[8] Lin YH, Liang R, Xie KR, et al. Puerarin inhibits cisplatin-induced ototoxicity in mice through regulation of TRPV1-dependent calcium overload [J]. Biochem Pharmacol, 2024, 220: 115962. DOI: 10.1016/j.bcp.2023.115962

[9] Hsieh CY, Lin JN, Kang TY, et al. Otoprotective effects of fucoidan reduce cisplatin-induced ototoxicity in mouse cochlear UB/OC-2 cells [J]. Int J Mol Sci, 2023, 24(4): 3561. DOI: 10.3390/ijms24043561

[10] 高鑫, 尹柏坤, 左军. 黄芩提取物及其有效成分的药理作用研究进展 [J]. 中医药学报, 2025, 53(3): 112-117. DOI: 10.19664/j.cnki.1002-2392.250063

[11] 于文静, 杨苗, 贺春香, 等. 黄芩苷通过TLR4/MyD88/NF-κB通路抑制链脲佐菌素诱导的阿尔茨海默病大鼠模型神经炎症反应 [J]. 中国药理学通报, 2023, 39(1): 83-89. DOI: 10.12360/CPB202204021

[12] 张倩, 李惠香, 刘攀, 等. 黄芩素的体外抗炎及抗氧化活性研究 [J]. 烟台大学学报(自然科学与工程版), 2018, 31(3): 232-238. DOI: 10.13951/j.cnki.37-1213/n.2018.03.008

[13] Guo MY, Zhang NS, Li DP, et al. Baicalin plays an anti-inflammatory role through reducing nuclear factor-κB and p38 phosphorylation in S. aureus-induced mastitis [J]. Int Immunopharmacol, 2013, 16(2): 125-130. DOI: 10.1016/j.intimp.2013.03.006

[14] Wang CZ, Li XL, Wang QF, et al. Selective fraction of Scutellaria baicalensis and its chemopreventive effects on MCF-7 human breast cancer cells [J]. Phytomedicine, 2010, 17(1): 63-68. DOI: 10.1016/j.phymed.2009.07.003

[15] Chattaraj A, Syed MP, Low CA, et al. Cisplatin-induced ototoxicity: a concise review of the burden, prevention, and interception strategies [J]. JCO Oncol Pract, 2023, 19(5): 278-283. DOI: 10.1200/OP.22.00710

[16] Wang XL, Zhou YY, Wang DL, et al. Cisplatin-induced ototoxicity: From signaling network to therapeutic targets [J]. Biomed Pharmacother, 2023, 157: 114045. DOI: 10.1016/j.biopha.2022.114045

[17] 王岩, 韩锋产. 顺铂耳毒性的研究进展 [J]. 中华耳科学杂志, 2023, 21(3): 401-404. DOI: 10.3969/j.issn.1672-2922.2023.03.021

[18] Xiang L, Liu Y, Liu JL. Regarding the effect of cisplatin-based chemotherapy on hearing loss in children with cancer [J]. Am J Otolaryngol, 2024, 45(4): 104367. DOI: 10.1016/j.amjoto.2024.104367

[19] Yu DH, Gu JY, Chen YM, et al. Current strategies to combat cisplatin-induced ototoxicity [J]. Front Pharmacol, 2020, 11: 999. DOI: 10.3389/fphar.2020.00999

[20] Tang Q, Wang XR, Jin H, et al. Cisplatin-induced ototoxicity: Updates on molecular mechanisms and otoprotective strategies [J]. Eur J Pharm Biopharm, 2021, 163: 60-71. DOI: 10.1016/j.ejpb.2021.03.008

[21] Moyer A, Tanaka K, Cheng EH. Apoptosis in cancer biology and therapy [J]. Annu Rev Pathol, 2025, 20(1): 303-328. DOI: 10.1146/annurev-pathmechdis-051222-115023

[22] Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity relationships of flavonoids and phenolic acids [J]. Free Radic Biol Med, 1996, 20(7): 933-956. DOI: 10.1016/0891-5849(95)02227-9

[23] Kuwar OK, Kalia N. Anti-inflammatory and antioxidant effects of baicalein: targeting Nrf2, and NF-B in neurodegenerative disease [J]. Inflammopharmacology, 2025, 33(3): 1303-1310. DOI: 10.1007/s10787-025-01698-x

[24] Bock FJ, Tait SWG. Mitochondria as multifaceted regulators of cell death [J]. Nat Rev Mol Cell Biol, 2020, 21(2): 85-100. DOI: 10.1038/s41580-019-0173-8

[25] Abate M, Festa A, Falco M, et al. Mitochondria as playmakers of apoptosis, autophagy and senescence [J]. Semin Cell Dev Biol, 2020, 98: 139-153. DOI: 10.1016/j.semcdb.2019.05.022

[26] Chen QS, Zheng AC, Xu XL, et al. Nrf3-mediated mitochondrial superoxide promotes cardiomyocyte apoptosis and impairs cardiac functions by suppressing Pitx2 [J]. Circulation, 2025, 151(14): 1024-1046. DOI: 10.1161/CIRCULATIONAHA.124.070286

基金资助

国家自然科学基金(82192862)

国家自然科学基金(82571318)

江苏省重点研发计划项目(BE2023653)

江苏省医学重点学科/实验室(ZDXK202243)