摘要
目的研究水杨酸钠(sodium salicylate, SS)诱导豚鼠耳蜗螺旋神经节细胞(spiral ganglion neuron, SGN)凋亡及凋亡的机制。
方法40只成年花色雄性黑目豚鼠随机分为4组,每组10只。A组:空白对照组;B组:人工外淋巴液(artificial perilymph, APL)组(左耳经圆窗龛注入APL后未给SS);C组:SS(400 mg·kg~(-1)·d~(-1))+APL组(左耳经圆窗龛注入APL后经腹腔SS给药);D组:SS(400 mg·kg~(-1)·d~(-1))+zDEVD-FMK组(左耳经圆窗龛注入zDEVD-FMK后经腹腔SS给药)。所有动物均于给药前后分别进行听性脑干反应(auditory brainstem response, ABR)阈值测试。10 % SS(溶于APL配制而成)均采用腹腔注射给药方式,400 mg·kg~(-1)·d~(-1),并于连续给药10天后即时处死动物取出耳蜗,采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling, TUNEL)检测耳蜗SGN凋亡细胞,免疫组织化学染色方法检测SGN内激活型caspase-3蛋白表达情况,透射电镜(transmission electron microscopy, TEM)观察SGN超微结构变化。
结果ABR测试结果显示A、B两组动物听阈均无明显变化,C组动物给药后听阈提高,与A、B两组比较有显著性差异(p<0.01,p<0.01),D组动物听阈较C组降低(p<0.05);TUNEL标记显示A、B两组动物耳蜗中均未见SGN凋亡细胞,亦无激活型caspase-3蛋白表达,而C组则出现较多TUNEL阳性着色SGN,并伴有激活型caspase-3表达的增高,D组较C组TUNEL阳性着色SGN减少,激活型caspase-3表达亦降低,A、B两组与C、D两组比较差异均有显著统计学意义(p<0.01,p<0.01),而C、D两组比较亦有显著差别(p<0.01);透射电镜观察结果显示A、B两组SGN超微结构正常,C组SGN伴有明显细胞凋亡形态学特征,D组SGN超微结构未见明显凋亡征象。
结论①SS可诱导豚鼠耳蜗SGN发生凋亡;②SGN凋亡与caspase-3激活有关;③zDEVD-FMK可在一定程度上拮抗caspase-3的激活,并阻断其引起的SGN凋亡。
Objective: In the present study, in vivo experiments are conducted to test efficacy of caspase-3 inhibitor zDEVD-FMK for protecting spiral ganglion neuron (SGN) from salicylate-induced apoptosis in the guinea pig cochlea.
Methods: Forty adult guinea pigs were divided randomly into four experimental groups, each for ten, they were: Group A, served as blank control without any disposal; Group B, APL only, The animals were disposed with APL through RWN by the means of cochlear micro-injection, the left ear was implemented with an operation; Group C, APL plus salicylate. The animals were treated with APL through RWN before drug injection, with a does of 400 mg·kg~(-1)·d~(-1) i.p., for ten consecutive days; Group D, zDEVD-FMK plus sodium salicylate. The animals were treated with caspase-3 inhibitor zDEVD-FMK through RWN before drug exposure in the same way described in the Group C. All animals received ABR thresholds measurement before drug administration and after last injection; they were then sacrificed for cochleae followed by staining. Programmed cell death (PCD) executioner was evaluated with immunohistochemistry detection of activated caspase-3. Apoptosis was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Transmission electron microscopy (TEM) was also used to evaluate morphological change in the auditory neuron.
Results: Animals disposed with salicylate appeared a high elevation in response of ABR thresholds testing (p<0.01 vs. Group A and B). By contrast, for those received zDEVD-FMK in advance, their hearing improved (p<0.05 vs. Group C). Salicylate strengthened immune reaction in the fields of SGNs, which was in accordance with apoptotic auditory effectors emergence (p<0.01 vs. Group A and B). On the contrary, topical inhibitor delivery alleviated pigmentation of nuclei (p<0.01 vs. Group C), keeping pace with antigen expression decrease, but a few immunostaining could be still detected (p<0.01 vs. Group C). With TEM to SGNs, no ultrastructural change occurred in the Group A and B, while neurons obtained from Group C demonstrated obvious apoptotic changes, characterized with chromatin condensation and nucleus margination. Treatment with zDEVD-FMK prevented the salicylate-damaged SGNs by apoptosis in a highly significant manner.
Conclusion: These findings suggest that long-term administration of high-dose salicylate can activate caspase-3 pathway to induce SGN apoptosis.
引文
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