复方健耳剂对抗老年性耳蜗神经细胞凋亡超微结构观察及上调NeuN和BDNF定位表达作用

Ultrastructural age-related effects of a compound healthy ear agent on apoptosis in cochlear neurocytes and brain-derived neurotrophic factor and neuron-specific nuclear antigen expression

目的利用透射电镜观察中药复方健耳剂干预小鼠老年性耳蜗外毛细胞(outer hair cell,OHC)、螺旋神经节神经元(spiral ganglion neurons,SGN)凋亡作用;共聚焦显微镜,结合形态学多重免疫荧光技术,观察复方健耳剂对耳蜗SGN的特异性核抗原蛋白(Neu N)、脑源性神经营养因子(BDNF)定位表达影响作用,探讨其作用机制。方法选择1月龄C57BL/6J小鼠22只并随机分为两组,其中11只小鼠每日饮用自来水直到7个月作为老年性耳蜗退变对照组(简称7月龄对照组);11只小鼠每日饮用复方健耳剂1.83 g/(kg·d)直到7个月作为中药干预组(简称7月龄中药组)。所有动物期满取出耳蜗,制备石蜡包埋切片,在透射电镜下,重点选择观察7月龄对照组耳蜗底回存留尚未解体的OHC以及对应部位的SGN超微结构变化,同时与7月龄中药组相同部位的OHC和SGN进行对照。利用激光共聚焦显微镜,结合形态学多重免疫荧光技术,观察Neu N与BDNF在SGN定位表达,并进行统计分析。结果 7月龄对照组耳蜗底回存留尚未解体的OHC以及对应部位的SGN,胞体萎缩,胞核不整,核染色质聚集成团、边集,纹理结构模糊,电子密度高,尤其是SGN大小形态不一,数量明显减少,甚至核染色质溶解,形成空泡,或解体缺如等,呈现严重凋亡状况。而7月龄中药组耳蜗底回OHC或SGN凋亡状况不明显,细胞形态结构较完整,数量较多,核染色质分布较均匀,纹理结构清晰,电子密度低,仅个别胞核溶解,形成空泡。与7月龄对照组比较,7月龄中药组耳蜗底回SGN中的Neu N与BDNF定位表达密度较高,数量较多(P<0.05)。结论耳蜗OHC或SGN细胞核超微结构改变、Neu N表达减少是老年性耳蜗神经细胞凋亡的重要特征;同期SGN凋亡早于OHC;中药复方健耳剂具有明显对抗OHC或SGN超微结构改变,其对SGN中的Neu N表达明显增多,提示中药具有明显保护SGN作用,与透射电镜观察一致,其作用机制与促进BDNF表达,从而发挥系列生物学效应有关。

Objective To examine the effect of a compound healthy ear agent(CHEA)from traditional Chinese medicine(TCM)on i)age-related apoptosis of cochlear hair cells(OHC)and spiral ganglion neurons(SGN)using transmission electron microscopy(TEM),and on ii)localized expression of brain-derived neurotrophic factor(BDNF)and neuron-specific nuclear antigen(NeuN)to investigate mechanisms of CHEA actions.Methods Twenty-two C57BL/6J mice were randomly divided into two groups at 1 month of age.Eleven animals were provided tap water daily until 7 months of age;the age-related cochlear degeneration control group(7-month-old control group).Another 11 animals drank CHEA 1.83 g/(kg·d)until 7 months of age;the TCM-treated group(7-month-old TCM group).Cochlea of euthanized animals were removed and processed into paraffin-embedded slices.Using TEM,ultrastructural changes were examined in the remaining and undisintegrated OHC and SGN associated with OHC at the basal region of cochlea from 7-month-control and 7-month-old TCM groups.Localized expression of BDNF and NeuN in SGN was observed by LCM combined with a morphological multi-overlap immunofluorescence technique,and examined using statistical analyses.Results In the 7-month-old control group,remaining and undisintegrated OHC and SGN associated with OHC in cochlea basal regions exhibited atrophied cytostomes,irregular nuclei and chromatin structure,texture structure presented vague,and high electron density;in particular,SGN were different in size and shape,significantly decreased in number,and exhibited nuclei chromatin that had disintegrated or dissolved to form vacuoles,representing the presence of apoptosis.However,the 7-month-old TCM group had no obvious apoptosis in OHC or SGN of basal cochlear regions,which exhibited more complete morphological structures and nuclei with a uniform chromatin distribution,texture structure presented clear,a lower electron density,and only individual nuclei had dissolved to form vacuoles.The localized expression levels of BDNF and NeuN in the basal cochlear region were higher(P<0.05)in the 7-month-old TCM versus the 7-month-control group.Conclusions Ultrastructural changes of nuclei and decreased expression of NeuN in cochlear OHC or SGN were important apoptotic features in aging cochlear neurocytes.Apoptosis in SGN was earlier than in OHC.The TCM reduced the ultrastructural changes in OHC or SGN,and increased NeuN expression in SGN,which suggests that TCM had a protective effect upon SGN,consistent with TEM observations.The mechanism of action may involve promotion of BDNF expression,which exerts a series of biological effects.