膜联蛋白A2-S100A10对大鼠背根神经节TRPV4通道的调控作用及机制

DRG neurons are primary afferent sensory neurons and responsible for the peripheral noxious stimuli transduction as sensory cells with aggregation of transducer receptors and/or ion channels. Our previous studies suggested that TRPV4 contributes to mechanical allodynia after CCD. In CCD model, we found an increased expression and sensibilized function of TRPV4 channel, with an increased expression of annexin A2. Interaction of TRPV4 with annexin A2 exists,but they did not associated directly together in a complex. TRPV4 belongs to TRP channel families, and is a Ca2+ permeable mechanosensitive cation channel which could be activated by many stimuli. TRPV4 is a mechanoreceptor in the DRG neurons and plays an important role in neuropathic pain. Annexin A2 is a Ca2+-dependent membrane-binding protein, associated with endosomal membrane dynamics and vesicular trafficking, and participates in fibrinolysis, blood clotting and angiogenesis. The S100A10-annexin A2 complex could bind to TRPV5 and TRPV6 and regulate the channel activities. The formation of a heterotetrameric complex between annexin A2 and S100A10 plays an important role in regulating the cellular biochemical properties. We supposed annexin A2-S100A10 complex assosciated with TRPV4 and regulated the channel functions and expressions. Thus, this interaction played a part in the process of neuropathic pain. In this study, we aimed to investigate if there exists an interaction of TRPV4 with annexin A2-S100A10 invivo and in vitro. We will conduct laser confocal scanning, small interfering RNA, western blot and coimmunoprecipitation to investigate the regulation of annexin A2-S100A10 complex on TRPV4 channel. This will facilitate the study of mechanisms of neuropathic pain.

背根神经节（DRG）细胞作为初级神经元，参与外周伤害性刺激传导，在神经病理性疼痛的外周机制中起重要作用，其胞膜上有多种离子通道，包括香草素相关性瞬时感受器电位离子通道4(TRPV4)。我们研究发现，在大鼠背根神经节持续压迫（CCD）模型中，受压侧DRG上的TRPV4通道表达明显增高，功能增强，膜联蛋白A2的表达明显增高。对TRPV4及膜联蛋白A2的研究提示，膜联蛋白A2参与TRPV4的调控，但不是通过直接相连，而是通过某种中间介质。膜联蛋白A2常与 S100A10形成复合体，参与多种离子通道功能的调节。由此，我们推测，膜联蛋白A2通过与S100A10形成复合体，参与TPPV4通道表达及功能调控，从而参与神经病理性疼痛的发生。本课题拟通过激光共聚焦、RNA干扰、免疫印迹等方法，研究膜联蛋白A2-S100A10复合体对TRPV4通道的调控及机制，揭示其在神经病理性疼痛发生机制中的作用。

背根神经节（DRG）细胞作为初级神经元，参与外周伤害性刺激传导，在神经病理性疼痛的外 周机制中起重要作用，其胞膜上有多种离子通道，包括香草素相关性瞬时感受器电位离子通道 4(TRPV4)。多种中间介质参与了TRPV4的调控，从而参与了神经病理性疼痛的发生。我们在前期研究的基础上，应用动物模型、分子生物学、免疫组织化学、基因工程学等手段对膜联蛋白A2-S100A10复合体对TRPV4的影响进行的研究。应用免疫沉淀方法检测TRPV4、膜联蛋白A2、S100A10三者相连接，确定了三者存在相关关系；免疫组织化学检测正常与CCD大鼠DRG中TRPV4分别与膜联蛋白A2、S100A10的共分布情况；然后Western blot研究提示大鼠CCD模型中TRPV4、膜联蛋白A2、S100A10三者的表达增加，检测到鞘内注射Forskolin（FSK）后膜联蛋白A2、S100A10、TRPV4的表达无明显变化；最后通过鞘内注射siRNA干扰膜联蛋白A2的合成，从而减少膜联蛋白A2-S100A10复合体的形成，Western blot检测、Realtime PCR检测DRG中TRPV4的表达降低，且对大鼠的行为学检测显示干扰膜联蛋白A2后CCD模型大鼠的痛阈较CCD组增加，进一步确定了膜联蛋白A2-S100A10复合体对TRPV4表达的影响；最后通过合成TRPV4、膜联蛋白A2、S100A10过表达载体，转染HEK293T细胞，观察三者在细胞内的共分布情况，进一步验证膜联蛋白A2-S100A10复合体与TRPV4的相关关系。结果提示膜联蛋白A2-S100A10复合体参与TRPV4通道表达及功能的调控，从而参与了神经病理性疼痛的发生。本研究进一步揭示了神经病理性疼痛的发生机制，以期为临床上神经病理性疼痛的治疗和镇痛药物的开发提供新的靶点。