JNK信号通道在强噪声诱导豚鼠耳蜗细胞凋亡的作用

Study of the effect of JNK signal transduction pathway in intense noise-induced apoptosis in cochlea of guinea pig

目的:探讨强噪声对豚鼠耳蜗细胞死亡的机制及磷酸化c-Jun氨基末端激酶(JNK)信号通道在强噪声诱导耳蜗细胞凋亡的作用。方法:将实验组豚鼠暴露在4kHz窄带噪声120dBSPL噪声环境中4h,噪声刺激停止后1d、4d、14d组及对照组(每组各8只)在处死前测ABR。取每组4只豚鼠耳蜗作石蜡切片,另外4只豚鼠提取耳蜗总蛋白。脱氧核糖核甘酸末端转移酶介导的缺口末端标记技术(TUNEL)检测耳蜗凋亡细胞,免疫组织化学及WesternBlot方法检测JNK信号途径蛋白质P-JNK、P-c-Jun的表达。结果:实验组耳蜗Corti器毛细胞、血管纹和螺旋神经节细胞存在TUNEL阳性细胞,以1d组最多,逐渐减少,14d组最少,而对照组未见阳性细胞。免疫组织化学观察到实验组P-JNK、P-c-Jun有免疫反应阳性,定位于细胞核,对照组未见阳性细胞。Western Blot检测P-JNK、P-c-Jun含量在噪声刺激后迅速增高并快速活化,1d、4d达到高峰,随后逐渐下降,但在14d仍然维持较高水平。结论:强噪声可以通过诱导凋亡造成耳蜗细胞损伤,同时P-JNK标志着JNK信号途径的激活,提示JNK信号通道可能也是介导强噪声诱导豚鼠耳蜗细胞凋亡信号通道之一。

Objective：To investigate the mechanism of intense noise-induced cochlea cells death in guinea pig,and the effect of JNK signal transduction pathway in the procedure of cochlea cells apoptosis by intense noise-induced.Method：Thirty-two guinea pigs were randomly divided into 4 groups. The guinea pigs in the experiment groups were exposed to 4 kHz narrow band noise at 120 dB SPL for 4 h. After the noise expose for 1,4,14 days of the experiment guinea pigs,ABR of the guinea pigs on experiment and control groups were tested before put them to death.Four guinea pig＇s cochleas of every group were taken to paraffin section,and the rest was extracted the total cochlear＇s protein.Apoptosis was tested by terminal deoxynucleotidyl Transferase（TdT）-mediated deoxyuridine triphosphate （d-UTP） nick and labeling method （TUNEL）. The phosphorylation of JNK and c-Jun were tested by immunohistochemistry and western blot methods.Result：Tunel-Positve cells in the Corti＇s,SGC and SV of experiment groups,and there have significant differences compared with the control group（P〈0.01）and Tunel-Positve cells are most in 1 d experiment group. The positive cells of P-JNK and P-c-Jun could be dectected in guinea pig＇s cochleas after noise exposed ,but no positive cells were found in the control.Protein levels of P-JNK,and P-c-Jun were risen up and activated quickly after noise exposed,and achieved peak in 1 d,4 d,and then fallen-offs ,but still maintained higher levels within 14 d.Conclusion：Intense noise causes cochlea cell lesion by inducing apoptosis to result in and JNK signal transduction pathway plays an important role in the procedure of apoptosis.