基于内耳水通道蛋白-1的表达研究电针对耳性眩晕豚鼠模型膜迷路积水的效应及机制

Aural vertigo of Meniere's disease is one of the highest incidences of diseases. Our acupuncture clinic studies have shown remarkable effect of electro-acupuncture therapy for treatment of Meniere's disease, but until now its effect and mechanis have not seen in animal experiment study on literature reported at home and abroad. This project proposed will establish classic endolymphatic hydrops vertigo model in guinea-pig with intraperitoneal injection of aldosterone，and use behaviors and auditory brainstem reflex as success criteria of the model. Through behavioral and related ion concentration in the serum, electronystagmogram , and auditory brainstem reflex, the experiment effect after treatment in different therapy groups will be observed. At the same time, through to the inner ear Reissner membrane morphology observation and cochlear image analysis, changes of endolymphatic hydrops after treatment in different therapy groups will be objective judgment. On these bases, through light microscope observation of the cochlear AQP-1 and quantitative detection of its area, can verify expressed of the AQP-1 in the inner ear is closely related to endolymphatic hydrops，clarify the effect mechanism of electroacupuncture for improving endolymphatic hydrops in the inner ear. This reseach can determine the curative effect in the good and bad in the commonly used acupuncture therapies; clear and definite the treatment result of aural vertigo is through changes of endolymphatic hydrops intervention; and clarify its regulatory mechanism from the way of the AQP-1.

耳源性眩晕中梅尼埃病是发病率相当高的一种耳病，我们前期的针灸临床研究表明电针等针灸疗法治疗梅尼埃病有明显临床疗效，但其效应及机制的实验研究至今未见国内外文献报道。本课题拟采用经典的腹腔注射醛固酮建立符合耳性眩晕膜迷路积水表现的豚鼠模型，并以行为学和听性脑干反射作为评价模型成功的标准。通过行为学、血清相关离子浓度、眼震电图和听性脑干反射检测，观察电针和其它不同疗法组治疗后的实验效应，同时通过对内耳Reissner膜形态观察和耳蜗图像分析，客观判断不同疗法组治疗后内耳膜迷路积水这一效应途径的动态变化。在此基础上通过对耳蜗水通道蛋白-1（AQP-1）的光镜观察和面积量化检测，验证AQP-1在内耳的表达与膜迷路积水密切相关，从而阐明电针改善内耳膜迷路积水的效应机制。本研究能确定电针在常用针灸疗法中的优效作用；明确电针治疗耳性眩晕主要是通过对膜迷路积水的干预；并从AQP-1的角度阐明其调节机制。

针灸疗法治疗梅尼埃病有较好的临床疗效，但其效应与机制的研究至今未见国内外文献报道。本项目首先采用醛固酮诱导的梅尼埃病膜迷路积水模型，明确了不同针灸疗法对梅尼埃病的实验效应差异，并从水通道蛋白1(aquaporin1，AQP1)的角度，研究了电针对膜迷路积水的干预机制。我们发现，针刺、艾灸、电针刺激“百会”、“听宫”穴均能减轻豚鼠膜迷路积水，并且改善耳蜗听功能。电针在减轻豚鼠膜迷路积水及改善听力方面表现出的效应最好，优于艾灸、针刺；艾灸优于针刺。同时，研究发现，醛固酮诱导的膜迷路积水模型豚鼠耳蜗螺旋韧带处AQP1表达下调，K+、Ca2+、Na+、Cl–浓度发生改变，电针可使膜迷路积水模型AQP1表达水平上调，并对血清离子浓度具有良性调整作用，提示电针对膜迷路积水的改善可能与上调耳蜗AQP1的表达有关，并受离子浓度变化的影响。此外，我们还从与梅尼埃病膜迷路积水发生密切相关的AVP-V2R-cAMP信号轴及其下游的水通道蛋白与离子通道蛋白角度进一步研究了电针对膜迷路积水的干预机制。采用腹腔注射AVP的方法成功复制了膜迷路积水模型，发现经AVP处理的膜迷路积水动物前庭细胞密度下降，血浆AVP浓度升高，耳蜗V2R表达下调，耳蜗cAMP、AQP2表达上调，Na+-K+-ATPase α1亚基表达下调，电针干预能使前庭细胞密度增加，血浆AVP、耳蜗V2R、cAMP、AQP2及Na+-K+-ATPase α1亚基表达发生逆转，提示AVP-V2R-cAMP信号轴及其下游的水通道蛋白与离子通道蛋白参与电针对膜迷路积水的干预作用。同时，我们还发现，常规针刺可使AVP诱导的膜迷路积水模型动物耳蜗AQP2表达下调，艾灸可使血浆AVP、耳蜗AQP2及AQP7表达下调，从而实现干预膜迷路积水效应。总之，本研究发现，膜迷路积水模型中内耳水通道蛋白及离子通道蛋白表达改变，可能是针灸减轻积水，改善梅尼埃病临床症状的靶点。本项目研究不仅有助于明确电针在常用针灸疗法中的优效作用，明确针灸治疗内耳眩晕主要是通过对膜迷路积水的干预，并从内耳水通道蛋白及离子通道蛋白角度阐明了针灸对膜迷路积水的调节机制。