复方健耳剂对抗小鼠老年性耳蜗螺旋神经节神经元凋亡效应及机制研究

Effects of healthy ear compound of traditional Chinese medicine against age-induced apoptosis on cochlear spiral ganglion neurons in C57BL/6J mice and research on mechanisms by regulating caspase-3

目的探讨小鼠老年性耳蜗螺旋神经节神经元（SGNs）损害及不同剂量中药复方健耳剂干预作用与可能机制。方法选择刚断奶C57BL／6J小鼠36只，随机分为正常对照组（6只），老年性SGNs凋亡对照组（12只），高剂量中药健耳剂组（12只），低剂量中药健耳剂（6只）。正常对照组小鼠自断奶后每日饮用自来水直到出生后2个月止；老年性SGNs凋亡对照组小鼠自断奶后每日饮用自来水直到出生后7个月止；中药高、低剂量组小鼠自断奶后分别每天饮用3．65g／kg、0．91g／kg中药复方健耳剂直到出生后7个月止。每组6只小鼠在实验终止时立即取出耳蜗用于石蜡包埋切片，进行甲苯胺蓝染色，观察耳蜗SGNs形态学变化。另6只中药高剂量组与6只同龄老年性SGNs凋亡对照组在实验终止时立即取出耳蜗，利用Real-TimePCR技术，检测耳蜗caspase-3表达量，最后进行统计分析。结果2月龄对照组全耳蜗SGNs形态表现健康，密度正常。7月龄老年性SGNs凋亡对照组耳蜗基底部SGNs缺损严重，与2月龄对照组比较差异无统计学意义（P〈0．001）。而中药高剂量组神经元形态表现几乎正常，数量及密度与2月龄正常对照组比较差异无统计学意义（P〉0．05），中药低剂量组神经元数量及密度与其他各组均差异有统计学意义（P〈0．001）。中药高剂量组与同龄老年性SGNs凋亡对照组对caspase-3表达量比较差异有统计学意义（P〈0．01）。各组耳蜗中部以上SGNs基本相同，尚无变化。结论C57BL／6J小鼠伴随着年龄增长出现耳蜗老年性SGNs凋亡由耳蜗底部开始并向顶部发展趋势；中药复方健耳剂具有对抗老年性SGNS凋亡显著效应；药物高剂量疗效优于低剂量；中药作用机制可能与其干预caspase介导细胞程序性死亡径路有关。

Objective To explore the damage of spiral ganglion neurons（SGNs）,the protective effects of different dosages of healthy ear compound（HEC） from traditional Chinese medicine（TCM） against age-induced SGNs degeneration and its possible mechanism in spiral ganglion neurons（SGNs）of C57BL/6J mice. Methods Totally 36 C57BL/6J mice just after ablactation were randomly divided into four groups. Normal control group （n = 6）drank tap water daily from ablactation to 2 months old. Ageing-related SGNs apoptosis control group（n= 12）drank tap water daily from ablactation to 7 months old. High dose TCM group（n= 12）at drank 3.65 g/kg/d of HEC from ablactation to 7 months old. Low dose TCM group（n= 6）drank 0.91 g/kg/d of HEC from ablactation to 7 months old. The animal cochleae were immediately removed at the termination of the experiment. In each group, the cochleae of 6 animals were used for paraffin embedding, slicing and toluidine blue staining to observe neuronal morphological changes. The caspase 3 mRNA expression study was performed by real-time PCR technique in 6 cochleae of High dose TCM group and ageing-related SGNs apoptosis control group. Results The morphological structure of cochlear SGNs represented healthy and normal density in normal control group at 2 months old. In contrast, amount or density of SGNs in cochlear basilar part was significantly damaged and reduced in ageing-related SGNs apoptosis control group at 7 months old（P〈 0. 001）. But the high dose TCM group at 7 months of age was similar to the normal control group at 2 months old in morphological structure, amount or density of SGNs（P〉 0.05）. The low dose TCM group was significantly different from other 3 groups in amount or density of SGNs （P〈0. 001 ）. However, SGNs in the middle part and apical part showed integrity in each group. In addition, the expression level of caspase-3 in the cochlea of high dose TCM group was also obviously different with age-related SGNs apoptosis control group（P〈 0.01） Conclusions Ageing-related damage of SGNs in C57BL/6J mice begins from the base of cochlea and progresses towards the apex. The HEC of TCM could significantly protect SGNs against age-induced apoptosis in SGNs. The efficacy of the high dose TCM is better than that of the low dose TCM. Its SGNs protective mechanisms might be related to involving the caspasemediated cell apoptotic pathway.