山东大学耳鼻喉眼学报 ›› 2025, Vol. 39 ›› Issue (3): 1-10.doi: 10.6040/j.issn.1673-3770.0.2024.509

• 上海市第六人民医院耳鼻咽喉头颈外科献礼“六院120周年”纪念专题 •    

CAMK4介导胆红素所致听觉中枢神经元氧化应激损伤

柯冰冰1,陈铭1,王洪阳2,李春燕1,殷善开1   

  1. 1.上海交通大学医学院附属第六人民医院 耳鼻咽喉头颈外科/上海交通大学耳鼻咽喉研究所, 上海 200233;
    2.解放军总医院耳鼻咽喉头颈外科医学部 耳鼻咽喉内科/国家耳鼻咽喉疾病临床医学研究中心/教育部聋病重点实验室/聋病防治北京市重点实验室, 北京 100853
  • 发布日期:2025-06-04
  • 通讯作者: 李春燕. E-mail:7250012693@shsmu.edu.cn
  • 基金资助:
    国家自然科学基金优秀青年科学基金(82322020);国家自然科学基金(82071042);国家重点研发计划青年科学家项目(2023YFC2509800)

CAMK4-mediated oxidative stress injury in auditory central neurons induced by bilirubin

KE Bingbing1, CHEN Ming1, WANG Hongyang2, LI Chunyan1, YIN Shankai1   

  1. 1. Shanghai Key Laboratory of Sleep Disordered Breathing, Department of Otolaryngology-Head and Neck Surgery, Otolaryngology Institute of Shanghai JiaoTong University, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200233, China2. Department of Audiology and Vestibular Medicine, College of Otolaryngology Head and Neck Surgery, Chinese PLA General Hospital, Chinese PLA Medical School, National Clinical Research Centerfor Otolaryngologic Diseases, Key Labof Hearing Science, Ministry of Education, Beijing Key Lab of Hearing Impairment for Prevention and Treatment, Beijing 100853, China
  • Published:2025-06-04

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摘要： 目的 探讨钙/钙调蛋白依赖性蛋白激酶4(calcium/calmodulin-dependent protein kinase IV, CAMK4)在胆红素介导的听觉中枢神经毒性中的作用。 方法 通过分子对接和微量热泳动技术(microscale thermophoresis, MST)评估CAMK4与胆红素的结合能力。采用石蜡切片免疫荧光染色法检测CAMK4在耳蜗核中的表达。在体外培养的耳蜗核原代神经元中,分别给予胆红素(25 μmol/L)或胆红素联合CAMK4抑制剂KN-93(1 μmol/L)处理,随后评估神经元的ROS水平、线粒体膜电位变化,进行Annexin V/PI染色及活/死细胞染色。 结果 分子对接分析显示,胆红素与CAMK4的结合能为-9.71 kcal/mol, MST检测得到平衡解离常数值为(1.294 4±1.080 3)μmol/L,提示二者具有较强的结合潜力。在耳蜗核中,CAMK4主要表达于神经元。体外实验结果表明,CAMK4抑制剂KN-93能够显著抑制高浓度胆红素诱导的耳蜗核神经元ROS蓄积、线粒体膜电位下降,以及细胞晚期凋亡和存活率下降。 结论 CAMK4在胆红素介导的听觉中枢氧化应激损伤中发挥重要作用。

关键词: 钙/钙调蛋白依赖性蛋白激酶4, 胆红素, 神经毒性, 氧化应激

Abstract: Objective The present study aims to investigate the role of calcium/calmodulin-dependent protein kinase 4(CAMK4)in bilirubin-mediated auditory central neurotoxicity. Methods The binding ability between CAMK4 and bilirubin was assessed using molecular docking and micro-scale thermophoresis(MST). The expression of CAMK4 in the cochlear nucleus was detected by immunofluorescence staining on paraffin sections. Primary cochlear nucleus neurons were cultured in vitro and treated with bilirubin(25 μmol/L)or bilirubin combined with the CAMK4 inhibitor KN-93(1 μmol/L). The neuronal ROS levels, mitochondrial membrane potential changes, Annexin V/PI staining, and live/dead cell staining were then evaluated. Results The molecular docking analysis revealed that the binding energy between bilirubin and CAMK4 was -9.71 kcal/mol, and the MST analysis gave a dissociation constant of(1.294 4±1.080 3)μmol/L, suggesting a strong binding affinity between the two. In the cochlear nucleus, CAMK4 was predominantly expressed in neurons. In vitro experiments demonstrated that the CAMK4 inhibitor KN-93 significantly suppressed bilirubin-induced ROS accumulation, mitochondrial membrane potential decline, late-stage apoptosis, and reduced cell survival in cochlear nucleus neurons. Conclusion CAMK4 has been demonstrated to play a crucial role in bilirubin-mediated oxidative stress injury in auditory central neurons.

Key words: Calcium/calmodulin-dependent protein kinase IV, bilirubin, neurotoxicity, oxidative stress

中图分类号: 

  • R764.4
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