调节内耳ANP/NPR-A/cGMP/PKG信号通路对运动病的影响及其机制研究

Literature and our study have suggested that motion sickness, similar to Meniere's disease, will be induced by an elevated level of arginine vasopressin (AVP) in the blood, which in turn activates V2R/cAMP/PKA signaling pathway, elevates the expression and membran translocation of AQP2 in the inner ear, increases the production of endolymph and eventually, similar to endolymphatic hydrops, results in many symptoms such as vertigo, nausea and vomiting. Atrial natriuretic peptide (ANP) could antagonize the effects of AVP in the body. ANP and its receptors are also distributed in the inner ear, and similar to its effects in the kidney, ANP is supposed to influence the endolymph and ion balances and thus to be involved in maintaining the auditory and vestibular senses. However, related studies are much less, and the effects of ANP signaling pathway in the development of motion sickness are unclear. Our preliminary results revealed that motion sickness-evoking rotatory stimulatus in rat elevated the expression of ANP receptor NPR-A in the inner ear, and ANP reduced the expression and phosphorylation of AQP2 induced by AVP in cultured epithelial cells from the endolymphatic sac of rat. Therefore, present study will focus on ANP signaling pathway, and investigate the relationship between the individual differences of motion sickness susceptibility and the basal and responsive expressions of this pathway signaling molecules in the inner ear; and at the same time, using pharmacological methods and gene regulating techniques to activate or inhibit ANP signaling pathway we intend to observe the inhibitory effect of ANP on motion sickness and to identify ANP/NPR-A/cGMP/PKG signaling pathway-related mechanisms, especially the influences of ANP on the downstream signaling molecules such as AQP2 and ENaC, so as to uncover the pathophysiological mechanisms of motion sickness and then, to find new targets aiming at this signaling pathway for the development of new drugs against motion sickness.

现有研究提示运动病与梅尼埃病相似，血液AVP水平↑，通过V2R/cAMP/PKA信号系统，使内耳AQP2表达及上膜↑，内淋巴液生成↑，类似膜迷路积水，诱发眩晕、恶心、呕吐等症状。ANP在体内能拮抗AVP作用，内耳有ANP及其受体分布，可能与在肾脏作用类似，影响AQP2和ENaC等靶蛋白活动，调节内淋巴液及离子平衡，参与维持内耳听觉与前庭感觉功能，但相关研究较少，在运动病中作用不明。初步研究显示，旋转刺激使大鼠内耳ANP受体NPR-A表达↑，在培养的内淋巴囊上皮细胞ANP抑制AVP诱导AQP2表达及磷酸化。本项目从内耳ANP信号通路入手，探讨运动病敏感性与该通路在内耳活动的联系，并通过药理学与基因调控方法影响ANP信号通路，探讨ANP对运动病抑制作用及NPR-A/cGMP/PKG通路相关机制，尤其对靶蛋白AQP2与ENaC的影响，帮助阐明运动病机制，以便以此通路为靶点，研究开发抗运动病新药。

本研究发现，腹腔注射与中耳微注射心房钠尿肽（ANP）对旋转刺激及AVP诱导的大鼠运动病有抑制作用，NPR-A拮抗剂可抑制ANP的作用，提示ANP抑制运动病发生通过内耳受体NPR-A介导；ANP通过NPR-A激动内耳ANP信号通路，抑制AQP-2表达与磷酸化，使其在膜上分布减少；ANP还抑制内淋巴囊上皮细胞ENaC、NKCC1表达，促进NKCC2表达，降低胞内Na+和胞外K+浓度，拮抗由AVP引起的胞内Ca2+升高，从而可抑制内淋巴液生成，促进重吸收，降低前庭敏感性。腹腔注射ANP，旋转刺激使大鼠内耳表达上调的基因有41个被ANP下调（下调一半以上7个，3个涉及前庭感觉调控）；旋转刺激使表达下调的基因有25个被ANP上调（上调2倍以上7个，4个涉及前庭感觉调控）。运动病不敏感大鼠内耳ANP水平高，ANP受体与AQP2在内耳主要结构左右侧分布有所差异，可能对运动病敏感性个体差异有影响。前庭功能锻炼后大鼠内耳ANP/NPR-A信号通路有适应性改变，旋转刺激后PKG反应性上调更明显。上述结果为利用前庭功能锻炼习服抗运动病及靶向内耳ANP/NPR-A信号通路研发抗运动病新药提供了理论依据。