不同频率电针“百会”、“听宫”穴对梅尼埃病膜迷路积水的作用及机制研究

Meniere’s disease (MD) is a common disease of aural vertigo. A well-documented histopathological substrate of MD is endolymphatic hydrops. Studies have shown that improvement of the endolymphatic hydrops is the key to relieve clinical symptoms. There is considerable clinical and experimental evidence for the belief that endolymphatic hydrops in the inner ear is in part resulted from the mal-regulation of the AVP-V2R-cAMP-ENaC pathway and AQP2. Our previous clinical studies have shown remarkable effects of electroacupuncture (EA) therapy for treatment of MD. Animal experiments have shown that EA can reduce the excess water retention in the endolymph in guinea pigs with endolymphatic hydrops, and the mechanism is likely to be related with down-regulating the expression of AQP2 in cochlea. Despite these benefits of EA, the frequency choice in treatment of MD is lack of uniform standards, and the detailed molecular mechanisms of EA treatment for MD still remain an enigma. Firstly, the behavior, auditory brainstem response (ABR) thresholds, severity of cochlear hydrops, as well as ultrastructure changes of cochlea were investigated to evaluate the effects of different frequency of EA on endolymphatic hydrops. Secondly, the project intends to test the relationship between AVP-V2R-cAMP-ENaC signal pathway and endolymphatic hydrops and AQP2 expression in the inner ear, using technology such as radioimmunoassay, quantitative real-time PCR, Western blot and genetic intervention. To determine whether EA may reduce cochlear hydrops by regulating AVP-V2R-cAMP-ENaC signal pathway. To clarify the regulatory mechanism of EA on ENaC expression associated with the AQP2 expression in cochlea. The project will not only screen out the best frequency parameters, but also clarify the potential mechanism for the treatment of endolymphatic hydrops in MD by EA.

梅尼埃病(MD)是一种常见的耳源性眩晕疾病，其病理基础为膜迷路积水，改善膜迷路积水是缓解MD症状的关键。AVP-V2R-cAMP-ENaC信号通路及AQP2与膜迷路积水的发生具有相关性。课题组发现电针治疗MD疗效确切，且能显著减轻膜迷路积水，下调耳蜗AQP2表达。但电针治疗MD的频率选择尚无统一标准，且治疗机制未明。本课题采用行为学、听力学、耳蜗病理及超微结构观察等方法，进一步明确不同频率电针对膜迷路积水的干预作用。在此基础上采用放免、PCR、免疫印迹、基因干预等技术，验证AVP-V2R-cAMP-ENaC信号通路与膜迷路积水及AQP2表达密切相关，阐明AVP-V2R-cAMP-ENaC信号通路在电针减轻膜迷路积水中的关键作用，研究电针下调耳蜗ENaC表达发挥效应的AQP2相关机制。本项目有助于明确电针改善膜迷路积水的最佳频率，阐明电针治疗膜迷路积水的机制，为临床电针治疗MD提供实验依据。

电针疗法治疗梅尼埃病（MD）有较好的临床疗效，但其效应与机制的研究国内外文献报道较少。本项目采用腹腔注射醋酸去氨加压素（DDAVP）诱导的膜迷路积水模型，明确了不同频率电针“百会”、“听宫”穴对膜迷路积水的实验效应差异，并从血管加压素（AVP）-血管加压素受体2（V2R）-环磷酸腺苷（cAMP）信号通路角度，研究了电针对膜迷路积水的干预机制。我们发现2Hz、15Hz、100Hz、2/15Hz电针是治疗耳蜗膜迷路积水的有效频率，其中100Hz电针疗效优于2Hz、15Hz，2/15Hz电针疗效优于2Hz、15Hz。同时，研究发现，经DDAVP诱导的膜迷路积水豚鼠AVP-V2R-cAMP信号通路及其下游水通道蛋白2（AQP2）、水通道蛋白7（AQP7）、上皮细胞钠通道（ENaC-α）等各个关键因子在血浆及内耳中表达发生改变，电针可逆转上述信号通路中各个因子的表达，减轻膜迷路积水。膜迷路积水模型豚鼠通过基因增强耳蜗V2R及AQP2，使耳蜗内ENaC-α蛋白表达上调后，耳蜗积水程度更明显；通过干扰耳蜗V2R及AQP2，使耳蜗内ENaC-α蛋白表达下调则能逆转DDAVP诱导的模型豚鼠耳蜗积水，100Hz电针治疗能显著下调V2R及AQP2基因增强诱导的耳蜗ENaC-α蛋白上调表达，减轻膜迷路积水。总之，本研究率先发现，膜迷路积水模型中AVP-V2R-cAMP信号通路，可能是电针减轻膜迷路积水的新靶点，100Hz电针，而不是2Hz、15Hz，通过调控AVP-V2R-cAMP信号通路及其下游水通道蛋白、离子通道蛋白，改善耳蜗结构变化，成为减轻膜迷路积水的最佳频率。本项目研究不仅有助于明确电针改善膜迷路积水的最佳频率参数，阐明电针治疗MD膜迷路积水的可能机制，而且还可能为电针治疗MD提供新的思路和干预靶点，对于针灸的研究和推广有一定科学意义和实用价值。

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