水杨酸钠增强氧化应激介导耳蜗螺旋神经节神经元损伤作用的实验观察  

Experimental observation on the effect of sodium salicylate enhancing oxidative stress-mediated injury of cochlear spiral ganglion neurons

作　　者：祝小婷 黄佳琳 林晓宇 苏纪平[1] ZHU Xiao-ting;HUANG Jia-lin;LIN Xiao-yu(Department of Otolaryngology Head and Neck Surgery,the First Affiliated Hospital of Guangxi Medical University,Guangxi Key Laboratory of Early Prevention and Treatment for Regional High Frequency Tumor,Nanning 530021,China)

机构地区：[1]广西医科大学第一附属医院耳鼻咽喉头颈外科,广西区域性高发肿瘤早期防治研究重点实验室,南宁530021

出　　处：《中国临床新医学》2023年第5期447-451,共5页CHINESE JOURNAL OF NEW CLINICAL MEDICINE

基　　金：国家自然科学基金地区科学基金项目(编号:82260221);广西自然科学基金面上项目(编号:2023GXNSFAA026288)。

摘　　要：目的观察水杨酸钠(SS)作用大鼠耳蜗螺旋神经节神经元(SGN)后,SGN中氧化应激水平的变化。方法将6只SD大鼠随机分为对照组和SS组,每组3只。通过耳蜗器官培养48 h后,用5 mM SS处理48 h,对照组不予处理,采用免疫荧光染色技术定位耳蜗器官中活性氧自由基(ROS)的主要生成位置。将15只SD大鼠随机分成5组,即对照组、SS组、SS+N-乙酰-L-半胱氨酸(NAC)组、阳性对照过氧化氢(H_(2)O_(2))组、H_(2)O_(2)+NAC组,每组3只。通过急性分离SGN,原代培养48 h后分别用5 mM SS、5 mM SS联合100μM NAC、300μM H_(2)O_(2)、300μM H_(2)O_(2)联合100μM NAC处理48 h,对照组不予处理。采用荧光染色法检测并量化各组大鼠SGN中ROS荧光探针DCFH-DA的平均荧光强度;采用CCK8法检测SGN细胞存活率。结果在耳蜗器官培养中,SS作用后,免疫荧光染色显示ROS荧光增强,并且主要在SGN中表达,而在其他细胞中荧光强度无明显变化。为进一步量化荧光强度,在原代培养SGN中加入5 mM SS处理后,荧光染色法显示ROS平均荧光强度较对照组升高(P<0.001),与H 2O 2组结果一致(P<0.0001)。在加入ROS抑制剂NAC之后,ROS平均荧光强度较SS组下降(P<0.01)。CCK8法结果显示,SS作用之后细胞存活率较对照组下降41.34%(P<0.01);在加入NAC之后,细胞存活率较SS组上升36.05%(P<0.01),与对照组比较差异无统计学意义(P>0.05)。H_(2)O_(2)组细胞存活率较对照组下降52.31%(P<0.001),在加入NAC之后,细胞存活率较H_(2)O_(2)组上升34.73%(P<0.01)。结论SS增强SGN的氧化应激,并且导致了SGN损伤。氧化应激抑制剂NAC可以降低SGN的氧化应激,并且对SS致SGN损伤具有保护作用。Objective To observe the changes of oxidative stress level in spiral ganglion neurons(SGN)after the action of sodium salicylate(SS)on SGN of rat cochlea.Methods Six Sprague-Dawley(SD)rats were divided into control group and SS group,with 3 rats in each group.The cochlear organs were treated with 5 mM SS for 48 hours after they were incubated for 48 hours.The control group was left untreated,and the main locations of reactive oxygen species(ROS)production in the cochlear organs were localized by immunofluorescence staining technique.Fifteen SD rats were divided into five groups,namely,control group,SS group,SS+N-acetyl-L-cysteine(NAC)group,positive control hydrogen peroxide(H_(2)O_(2))group,and H_(2)O_(2)+NAC group,with 3 rats in each group.The SGN were isolated by acute isolation and treated with 5 mM SS,5 mM SS combined with 100μM NAC,300μM H_(2)O_(2),300μM H_(2)O_(2)combined with 100μM NAC for 48 hours after 48 hours of primary culture,respectively,and the control group was left untreated.The mean fluorescence intensity of ROS fluorescent probe DCFH-DA in SGN of rats in each group was detected and quantified by fluorescent staining method;the CCK8 method was used to detect the survival rate of SGN cells.Results In the cochlear organ culture,after SS treatment,immunofluorescence staining showed that ROS fluorescence was enhanced and mainly expressed in SGN,while the fluorescence intensities in the other cells were not significantly changed.To further quantify the fluorescence intensity,in primary culture SGN,after adding 5 mM SS treatment,the fluorescence staining method showed that the mean fluorescence intensity of ROS increased compared with that of the control group(P<0.001),which was consistent with the result of the H_(2)O_(2)group(P<0.0001).After the addition of the ROS inhibitor NAC,the mean fluorescence intensity of ROS decreased compared with that of the SS group(P<0.01).The results of the CCK8 method showed that the cell survival rate decreased by 41.34%after the action of SS compared to the contr

关 键 词：水杨酸钠 耳蜗螺旋神经节神经元 氧化应激 免疫荧光 耳蜗培养 

分 类 号：R764.4[医药卫生—耳鼻咽喉科]