电针百会穴经血管加压素改善内淋巴积水豚鼠耳蜗微循环的作用机制

Meniere's disease (MD) refers to aural vertigo in traditional chinese medicine. The primary histopathological character of MD is endolymphatic hydrops (EH). Disorder of cochlear blood flow is closely related to MD. Acupuncture at Baihui has demonstrated significant beneficial effects on MD in clinic, but its mechanics is not very clear. So we hypothesis that cochlear blood flow (CoBF) may be improved after acupuncture at Baihui piont in guinea pigs with EH by adjusting arginine vassopressin (AVP) secretion and recepters expression. The purpose of this project is to analyze the mechanism of electroacupuncture (EA) at baihui to improve CoBF by AVP in the guinea pigs with endolymphatic hydrops that will be administrated desmopressin by intraperitoneal injection. The effect of EA at Baihui point on CoBF in guinea pigs with EH will be observed and compared with that treated by EA at non-acupoints, vasopressin antagonist OPC-31260 oral administered, and combination therapy with EA point and OPC-31260. The attention will be focuse on: (1) Verifying the effect of EA on improving CoBF of guinea pigs with EH. The CoBF will be directly measured by laser doppler flowmetry (LDF). The function and morphology of inner ear is also the evidence of reducing EH. Vestibular and cochlear function changes will be detected by electrophysiology, and the morphological changes of Reissner's membrane will be observed under light microscope along the longitudinal section of cochlea. (2) Comparing the effects of EA at Baihui point and other interventions on AVP secretion in hypothalamus, AVP concentration in plasma and AVP recepter mRNA and protein expression in inner ear. The morphology and distribution of AVP positive cells in guinea pig hypothalamus will be observed systematically with immunohistochemical method. The concentration of AVP in plasma will be detected by enzyme-linked immunosorbent assay (ELISA). The AVP recepter mRNA and protein expression in inner ear will be detected by RT-PCR and Western blotting respectively. (3) Analyzing the relationship among CoBF, AVP positive cells in hypothalamus and AVP recepter expression in cochlea. This study is expected to provide experiment evidences for the mechanism of acupuncture on MD.

梅尼埃病属中医“耳眩晕”范畴，组织病理变化是内淋巴积水，发生与耳蜗血液循环障碍密切相关。针刺治疗梅尼埃病效果显著，但机理不清。临床发现梅尼埃病患者血浆血管加压素浓度增高，我们认为针刺可能通过调节机体血管加压素的分泌及受体表达改善耳蜗微循环，缓解内淋巴积水。因此，本项目以腹腔注射血管加压素建立内淋巴积水豚鼠模型，电针百会穴对其干预，并与电针股部非穴位、血管加压素拮抗剂OPC-31260灌胃和针药联合等比较，采用激光多普勒、电生理检测和组织形态学观察内淋巴积水豚鼠耳蜗血流量、前庭耳蜗功能和形态的改变，验证电针对内淋巴积水豚鼠耳蜗微循环的改善效果；通过单克隆抗体、流式细胞术及分子生物学技术在受体和蛋白水平上研究电针对血管加压素分泌、血浆水平及内耳受体表达的影响，并分析与耳蜗血流量的关系。本项目探讨针刺通过血管加压素途径改善内淋巴积水豚鼠耳蜗微循环的作用机制，为针刺治疗梅尼埃病提供科学的实验依据。

梅尼埃病属中医“耳眩晕”范畴，组织病理变化是耳蜗的内淋巴积水，发生与耳蜗的血流灌注量和水平衡密切相关。临床发现梅尼埃病患者血浆血管加压素（arginine vasopressin, AVP）浓度增高，且针刺“百会”等穴位治疗梅尼埃病效果显著，但其机制不明。本项目通过腹腔注射醋酸去氨加压素建立豚鼠内淋巴积水动物模型，分别采用电针百会穴、电针非穴位、AVP拮抗剂Tolvaptan（OPC-41061）灌胃及电针百会穴联合OPC-41061灌胃等不同方式干预后与模型组及假模型组进行比较，从豚鼠耳蜗血流量、功能和形态的比较，验证了电针百会穴对内淋巴积水豚鼠耳蜗微循环的改善效果；通过ELISA、RT-PCR及Western blotting等分子生物学技术从受体和蛋白水平研究了电针对血浆AVP水平、内耳受体及其蛋白表达的影响，探索机体AVP途径调节耳蜗血流及水平衡的机制。.本研究发现电针百会穴可明显提高内淋巴积水豚鼠百会穴及耳蜗血流灌注量；听觉脑干诱发电位结果显示，模型组听阈明显升高，电针非穴位与模型组相近，无明显改善，而电针百会后，豚鼠听阈与模型组比较虽有明显降低，但仍高于假模型组；药物组与针药联合组的听力改善最为明显。耳蜗电图结果显示电针百会穴可使SP/AP绝对值明显降低，但无药物和针药联合的效果明显。形态学和分子生物学结果显示电针百会穴、药物和针药联合可以改善豚鼠内淋巴积水造成的前庭膜膨隆，豚鼠下丘脑、垂体AVP阳性细胞率明显降低，同时血浆和耳蜗AVP浓度明显下降。我们进一步分析了电针百会穴、药物和针药联合等不同干预对豚鼠耳蜗AVP-V2受体（AVP-V2）和水通道蛋白2(aquerin protein 2, AQP2)的影响，结果表明电针穴位、药物和针药联合均可以上调AVP-V2受体的表达，但使AQP2的mRNA和蛋白水平降低。本项目结果提示AVP参与电针百会穴豚鼠耳蜗血流灌注量和水平衡的调节，其可能的机制是通过抑制下丘脑AVP的分泌并下调耳蜗AQP2蛋白含量，进而水通道关闭，改善耳蜗内淋巴积水。