neuregulin1通过agrin/Lrp4/MuSK信号通路影响面肌和骨骼肌对肌松药敏感性差异的机制研究

The intraoperative facial nerve evoked electromyography (EEMG) is an important method to avoid iatrogenic facial nerve injuries, while muscle relaxant is an essential factor to influence the monitoring. Our research indicated that differences in sensitivities to muscle relaxants existed between facial-nerve-innervated muscles and somatic-nerve-innervated muscles, which might be related to the density of nicotinic acetylcholine receptors (nAChR) and the quantal release of acetylcholine. Facial nerve injury caused resistance to muscle relaxants, which might be due to the changes in the expression and distribution of nAChR as well as the changes in the amount and mode of acetylcholine release. The current research is designed to focus on the signaling pathway for the expression and accumulation of nAChR. To investigate whether neuregulin1 influences the expression of nAChR and muscle contraction by mediating agrin/Lrp4/MuSK signaling pathway, leading to the different sensitivities to muscle relaxants between different muscles. Meanwhile, to investigate the interaction between ectogenic agrin and neuregulin1 on the recovery of the function of injuried facial nerve innervated nueromuscular junctions. The results of the study are of significant importance to determine a proper strategy for rational usage of muscle relaxants in the surgeries requiring EEMG monitoring, as well as to explore the possibility of gene therapy for the facial nerve injuries.

术中面神经诱发肌电位（EEMG）监测是避免医源性面神经损伤的重要手段，而肌松药是影响EEMG监测的关键因素。我们研究发现：面肌和肢体骨骼肌对肌松药敏感性存在差异，其机制包括乙酰胆碱受体（nAChR）密度以及乙酰胆碱（ACh）释放的差异;受损面神经支配的肌肉对肌松药发生了抵抗，其机制与nAChR表达量和表达位置改变及ACh释放量及释放模式改变有关。本项目拟关注nAChR表达和聚集的信号通路，neuregulin1是否通过介导agrin/Lrp4/MuSK信号通路影响了nAChR亚基的表达和肌肉收缩，进而影响不同肌肉对肌松药的敏感性；以及外源性agrin和neuregulin1在发生交互作用后，是否对受损面神经神经肌接头功能恢复有影响。本项目对临床上实施EEMG监测的手术中肌松药的合理使用，探索面神经损伤基因治疗可能性有重要意义。

面肌与肢体骨骼肌对肌松药的敏感性存在差异，这主要和运动终板处乙酰胆碱受体（nAChR）表达量和表达位置有关。既往研究表明Agrin/Lrp4/MuSK和Neuregulin1/ErbB信号通路可以影响nAChR的聚集和合成表达；此外，Neuregulin1（NRG-1）还可以调节肌肉代谢，促进骨骼肌葡萄糖转运体（GLUT）的易位；同时在肌肉再生中也扮演着重要的角色。.我们的研究结果发现，在口轮匝肌中各nAChR亚基的表达量及MuSK的磷酸化水平明显高于腓肠肌；且在口轮匝肌和腓肠肌中Agrin/Lrp4/MuSK信号通路的关键因子的表达无明显差异而Neuregulin1/ErbB信号通路的关键因子的表达水平存在明显差异。接着，我们用AAV敲低Neuregulin1，发现MuSK的磷酸化水平下降，同时伴随nAChR的密度下降，因此，NRG-1促进MuSK的磷酸化从而增加了nAChR的密度。综上，面肌和腓肠肌nAChR表达差异主要是由NRG-1通过调节MuSK的磷酸化水平介导。为了进一步研究NRG-1在肌肉收缩功能中的作用，AAV敲低NRG-1后，我们观察到小鼠腓肠肌肌张力和肌电位明显下降；而给与外源性NRG-1干预后肌张力和肌电位明显升高，且腓肠肌GLUT4易位增加；同时我们也在细胞水平上验证了，外源性NRG-1干预后可促进C2C12细胞的葡萄糖摄取和GLUT4易位。因此，NRG-1 可通过促进GLUT4易位从而改善骨骼肌收缩。骨骼肌占人体的40%的重量，糖尿病导致的肌肉病变在临床中较为常见，因此，我们进一步研究了NRG-1在糖尿病小鼠骨骼肌中的作用。我们发现糖尿病小鼠腓肠肌NRG-1的表达是下降的，给予外源性NRG-1治疗后，糖尿病小鼠的体重和血糖水平有所改善，肌张力和肌电位也明显改善；糖尿病小鼠腓肠肌GLUT4的易位降低，然而给予外源性NRG-1干预后，可明显促进GLUT4的易位；在细胞水平，NRG-1干预可促进棕榈酸钠处理的C2C12细胞的葡萄糖摄取和GLUT4易位。因此，NRG-1可通过促进葡萄糖摄取和GLUT4易位，从而改善糖尿病小鼠骨骼肌收缩功能。.以上研究成果已在J Neuropathol Exp Neurol和Journal of Surgical Reasearch共发表两篇SCI文章，一篇中文核心，另有一篇SCI文章接收待发表。