P2Y2受体调控疑核运动神经元自噬及凋亡的作用

Injury to recurrent laryngeal nerve induces ambiguous nucleus motoneurons autophagy and apoptosis in the CNS, which damages the ability of nerve regeneration and functional recovery of laryngeal muscles. The protective role of P2Y2 receptors is increasingly attractive in CNS. Our previous study showed that the recurrent laryngeal nerve lesion induces in vivo P2Y2 receptor upregulation in ambiguous nucleus motoneurons. And P2Y2 agonist PSB1114 in vitro could inhibit autophagy activation and apoptosis in primary motoneurons, while P2Y2 antagonist Suramin stimulus opposite effect. In this event, increasing of intracellular calcium ions transient [Ca2+]i and upregulation of ERK phosphorylation are simultaneously detected. However, Ca2+ antagonist could abolish the effect of P2Y2 receptor inhibiting autophagy and apoptosis of motoneurons. Thus, we hypothesize that activation of P2Y2 receptors maintains the vitality of ambiguous nucleus motoneurons via ERK and/or Ca2+-dependent signal pathways, which may inhibit autophagy and apoptosis of motoneurons following recurrent laryngeal nerve injury. For this purpose, recurrent laryngeal nerve injury of rats model and primary motoneuron culture will be utilized to classify the role of P2Y2 signaling in inhibition of motoneuron autophagy and apoptosis. P2Y2 agonist and antagonist combined with constituted activation and inactivation of signaling components will be applied in this investigation both in vivo and in vitro to classify its underlying mechanism. The present study aims at exploring the role of P2Y2 signaling networks in regulating motoneuron autophagy and apoptosis, and are expected to provide a novel and potential therapeutic medicine for peripheral nerve regeneration in the clinic.

喉返神经损伤诱导疑核运动神经元自噬及凋亡损害了神经再生能力，是导致长期声带麻痹功能恢复不佳的重要原因。嘌呤受体P2Y2的神经保护作用日益受到重视，申请人已证实，损伤诱导疑核运动神经元P2Y2表达上调，特异性激活P2Y2可抑制原代运动神经元自噬和凋亡，并伴有Ca2+升高和pERK上调；而Ca2+阻断剂可抵消P2Y2激活的效应。我们提出假设：喉返神经损伤后，疑核运动神经元P2Y2受体的激活通过调控依赖Ca2+相关信号通路，抑制运动神经元的自噬及凋亡。本项目拟采用药物干预和组成型激活/失活技术，离体研究P2Y2调控运动神经元自噬及凋亡的分子机制；在体研究明确P2Y2调控疑核运动神经元自噬及凋亡的作用。从分子、细胞和组织水平，系统地阐明P2Y2参与调控疑核运动神经元自噬及凋亡的作用及信号转导机制。旨在为保护疑核运动神经元的存活，促进喉返神经再生和喉功能的恢复，寻找有潜力的药物作用靶点提供理论依据。

喉返神经损伤诱导疑核运动神经元自噬及凋亡损害了神经再生能力，是导致长期声带麻痹功能恢复不佳的重要原因，本研究也证实喉返神经损伤时程延长可诱导疑核运动神经元发生不可逆的凋亡，导致喉返神经自发性再生程度不佳及喉功能难以恢复正常。本研究建立喉返神经损伤诱导的神经核自噬及凋亡模型，研究发现生理条件下疑核运动神经元P2Y2受体表达水平低，喉返神经损伤后诱导疑核运动神经元P2Y2表达继续上调，两周达最高，HE 染色观察到神经核运动神经元水肿，核分裂，尼氏体减少，tunel检测到运动神经元凋亡，ELISA检测到FAS表达上调，wb检测到LC3Ⅱ/ LC3Ⅰ先降低后升高，mTOR表达先升高后降低，我们构建了P2Y2 knock out 小鼠，采用ko小鼠及野生型小鼠建立面神经损伤诱导的面神经核损伤及自噬的模型，得到相似的结果，ko小鼠自噬及凋亡的指标表达高于野生型小鼠，结果显示抑制P2Y2受体促进神经元的自噬及凋亡，采用原代培养的运动神经元，离体研究P2Y2调控运动神经元自噬及凋亡的分子机制；以lps刺激，血清饥饿等处理神经元诱导细胞自噬及凋亡，ELISA检测细胞凋亡指标FAS，P2Y2 受体激动剂2-ThioUTP tetrasodium 处理下调FAS表达，而P2Y2 受体拮抗剂AR-C118925则表现为促细胞凋亡的作用。cck8检测细胞存活率发现P2Y2 ko小鼠细胞存活率低于野生型小鼠，提示P2Y2受体表现为促细胞凋亡的作用。从分子、细胞和组织水平，系统地阐明P2Y2参与调控疑核运动神经元自噬及凋亡的作用及信号转导机制。为保护疑核运动神经元的存活，促进喉返神经再生和喉功能的恢复，提供了有潜力的药物作用靶点提供理论依据。