NLRP3炎症小体激活对水杨酸钠诱导大鼠耳蜗螺旋神经节细胞损伤的作用  

Activation of NLRP3-related Inflammasome in Rat Cochlear Spiral Ganglion Neurons by Sodium Salicylate

作　　者：翟思佳 尹时华[1] 侯涛[1] 任毅[1] 廖行伟 黄巧 李念燊 Zhai Sijia;Yin Shihua;Hou Tao;Ren Yi;Liao Xingwei;Huang Qiao;Li Nianshen(Department of Otolaryngology Head and Neck Surgery,the Second Affiliated Hospital of Guangxi Medical University,Nanning,530007,China)

机构地区：[1]广西医科大学第二附属医院耳鼻咽喉头颈外科,南宁530007

出　　处：《听力学及言语疾病杂志》2021年第3期327-333,共7页Journal of Audiology and Speech Pathology

基　　金：广西自然科学基金(2016GXNSFAA380150)。

摘　　要：目的通过研究NLRP3炎症小体在大鼠耳蜗中的激活,探讨水杨酸钠诱导螺旋神经节神经元(SGN)损伤的关键分子机制。方法64只SD大鼠随机分为4组对照组(腹腔注射等量生理盐水)、人工外淋巴液(artifieial perilymph fluid,APL)组(左耳圆窗注入APL后,腹腔注射生理盐水)、水杨酸钠(sodium salicylate,SS)组(左耳经圆窗注入APL后,腹腔注射10%水杨酸钠)、水杨酸钠+NLRP3拮抗剂(SS+MCC950)组(左耳圆窗注入溶于APL的NLRP3抑制剂MCC950后,腹腔注射水杨酸钠),每组16只;各组连续用药7 d。通过听性脑干反应(ABR)、畸变产物耳声发射(DPOAE)检测动物造模前后的听觉损害程度;HE染色观察耳蜗SGN的病理生理改变;采用实时荧光定量PCR对各组大鼠耳蜗组织中炎症复合体相关及炎性相关因子的表达水平进行检测;最后采用免疫组化方法评估水杨酸钠诱导耳蜗炎症复合体及相关炎性因子的蛋白表达水平及其在耳蜗中的定位。结果造模后SS组大鼠ABR反应阈明显升高,DPDAE幅值下降,造模成功;造模后SS组的SGN中形态异常细胞计数(39.33±8.618个)明显高于对照组(16.83±6.555个)、APL组(13.83±3.312个)和SS+MCC950组(23±6.573个)(P<0.001),可见SS+MCC950组SGN损伤较SS组明显减轻(P<0.01)。PCR检测结果示SS组SGN区域中的NLRP3、IL-1β和caspase-1的转录水平(ΔCT值分别为5.41±0.622、4.035±0.26、5.831±0.059)较对照组(分别为7.438±0.483、6.811±0.05、8.547±0.157)、APL组(分别为7.254±0.848、6.76±0.401、8.363±0.375)显著增加,而SS+MCC950组(分别为6.378±0.479、5.602±0.157、7.271±0.296)经过NLRP3阻滞剂预处理明显减弱了这一作用,其中ΔCT值越大,转录水平越低。免疫组化检测示对照组NLRP3、IL-1β和Caspase-1的蛋白表达分别为0.117±0.003、0.132±0.028、0.127±0.021,APL组分别为0.114±0.009、0.137±0.024、0.137±0.011,SS组分别为0.192±0.043、0.215±0.026、0.205±0.009,SS+MCC950组分别为0.136±0.015、0.176±0.00Objective To study the activation of inflammasome and related pathways in the cochlea of rats and the key molecular mechanisms of sodium salicylate-induced SGN injury.Methods In this study,64 SD rats were used as the research object,and they were randomly divided into 4 groups control group(16)intraperitoneal injection of equal volume of saline;artificial perilymph fluid(APL)group(16)after injecting APL into the round window niche of the left ear,intraperitoneal injection of normal saline;sodium salicylate(SS)group(16)after injecting APL through the round window of the left ear,intraperitoneally inject 10%sodium salicylate;Sodium salicylate+NLRP3 antagonist(SS+MCC950)group(16)after injection of NLRP3 inhibitor MCC950 dissolved in APL through the left ear round window niche,sodium salicylate was injected intraperitoneally.Continuous administration 7d.We used electrophysiological methods such as ABR,DPOAE to detect the animals before and after modeling to confirm the degree of hearing damage;morphological methods such as HE staining were used to observe the pathophysiology of the cochlea SGN;rt-qPCR was used to detect the expression level of inflammasome related inflammatory factors of the cochlea tissue of each group of rats;finally,the expression level of sodium salicylate-induced NLRP3 inflammasome and related inflammatory factor protein in SGN were evaluated by immunohistochemistry.Results After modeling,the SGN in the SS group was significantly damaged compared with the control group and the APL group(P<0.001),and the SGN damage pretreated by MCC950 was significantly improved compared with the SS group(P<0.01).At the same time,PCR found that the transcription levels of NLRP3,caspase-1 and IL-1βin the SNG region of the SS group(△CT 5.41±0.622,4.035±0.26,5.831±0.059)were higher than those in the control group(△CT 7.438±0.483,6.811±0.05,8.547±0.157)and APL group(△CT 7.254±0.848,6.76±0.401,8.363±0.375)significantly,while SS+MCC950 group after NLRP3 blocker pretreatment significantly weakened this e

关 键 词：水杨酸钠 螺旋神经节神经元 NLRP3炎症小体 半胱胺酸天冬氨酸蛋白酶-1 白介素-1Β 

分 类 号：R764.5[医药卫生—耳鼻咽喉科]