水杨酸钠诱导大鼠螺旋神经节细胞发生程序性坏死的研究

Mechanism of Salicylate-induced Necroptosis in Rat Spiral Ganglion Neurons

黄巧;尹时华;侯涛;任毅;廖行伟;翟思佳;王晓荣;

1:广西医科大学第二附属医院耳鼻咽喉头颈外科

摘要
目的探讨受体相互作用蛋白激酶-1(receptor-interacting protein kinase 1,RIPK1)/受体相互作用蛋白激酶-3(receptor-interacting protein kinase 3,RIPK3)/混合系列蛋白激酶样结构域(mixed lineage kinase domain-like protein, MLKL)通路介导的程序性坏死在水杨酸钠诱导大鼠螺旋神经节(spiral ganglion neurons, SGN)细胞损伤中的作用,以及程序性坏死特异性抑制剂necrostatin-1(Nec-1)对螺旋神经节细胞损伤的保护作用。方法通过听性脑干反应(auditory brainstem response, ABR)筛选出ABR阈值<40 dB SPL的48只成年健康雄性大鼠,随机分为空白对照组(左耳经圆窗注入外淋巴液,腹腔注入生理盐水)、人工外淋巴液(artificial perilymph, APL)(左耳经圆窗注入含Nec-1人工外淋巴液)组、水杨酸钠(sodium salicylate, SS)组(腹腔注射水杨酸钠350 mg·kg(-1)·d(-1)·d(-1))和Nec-1组(左耳经圆窗注入Nec-1后经腹腔注射水杨酸钠),每组12只。各组连续给药7天后检测ABR,然后,通过HE染色观察螺旋神经节细胞形态变化并计算细胞坏死率;采用实时荧光定量PCR(qRT-PCR)检测各组大鼠螺旋神经节RIPK1、RIPK3、MLKL mRNA表达水平;采用免疫组织化学染色检测各组大鼠螺旋神经节RIPK1、RIPK3、MLKL蛋白表达。结果给药后水杨酸钠组ABR反应阈较对照组及APL组升高,Nec-1组ABR阈值较水杨酸钠组降低;HE染色示与对照组、APL组相比,水杨酸钠组SGN细胞变圆,细胞质肿胀,细胞界限不清;坏死细胞计数(47.8%±2.387%)最高(P<0.0001),Nec-1组SNG中坏死细胞计数(30.6%±1.14%)较水杨酸钠组降低(P<0.0001)。qRT-PCR和免疫组化结果显示,水杨酸钠组SGN中RIPK1、RIPK3和MLKL高表达,Nec-1组SGN中RIPK1、RIPK3和MLKL表达较水杨酸钠组降低。结论在水杨酸钠致大鼠SGN损伤中存在RIPK1/RIPK3/MLKL介导的程序性坏死,Nec-1抑制程序性坏死,对SGN细胞损伤有保护作用。
关键词
螺旋神经节;水杨酸钠;程序性坏死;大鼠;Necrostatin-1
基金项目(Foundation):
广西自然科学基金(2016GXNSFAA380150)
作者
黄巧;尹时华;侯涛;任毅;廖行伟;翟思佳;王晓荣;
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