nAChR异位聚集分布在脓毒症相关获得性肌无力发生发展中的作用及机制研究

The sepsis-related acquired weakness syndrome is an import factor to increase hospital mortality in septic patients and causes chronic disability in survivors of severe sepsis, in addition to prolonging mechanical ventilation and hospitalization. Pathogenesis of acquired weakness syndrome induced by sepsis is not entirely clear. Current clinical treatments could not get good long-term effect. The endplate is an important structural basis of neuromuscular function, in which nicotinic acetylcholine (nAChR) plays an important role. In our previous research, perijunctional nAChR diffusion and ectopic clustering had been found in septic postsynaptic membrane of neuromuscular junction, which induced postsynaptic nAChR density decreased. That’s one of the important reason for neuromuscular dysfunction. It has been known the agrin, which is released from motor fiber terminal, mediates nAChR trafficking and clustering in membrane. We found sepsis inhibited phosphorylation of PI3K/Akt, which was downstream of agrin, and expression of perijunctioncal microtubule decreased. That indicated there might be a relationship of trafficking abnormality and clustering dysfunction with neuromuscular dysfunction. This research bases on trafficking abnormality and clustering dysfunction of nAChR as entry point. The movement of postsynaptic receptors of neuromuscular junction in sepsis will be recorded using immunofluorescence combining with single partial tracking technique. Intracellular microelectrode will test the endplate potential in nAChR distribution abnormality. Using electroporation transfection in vivo technique, the signal pathway and regulatory molecule will be identified in trafficking abnormality and clustering dysfunction of nAChR induced by sepsis. And specific therapeutic methods would be investigated in this project. This study will reveal the mechanism of pathogenesis of sepsis-induced acquired weakness syndrome. It’s advantage to find a new breakthrough of prevention and treatment for critical patients with acquired weakness syndrome.

脓毒症相关获得性肌无力是导致脓毒症患者死亡率增高的重要原因，而机制尚不清楚。我们前期研究发现脓毒症导致骨骼肌乙酰胆碱受体（nAChR）发生异位聚集和围接头区离散，引起终板膜nAChR密度下降，从而导致神经肌肉功能障碍。已知神经来源的聚集蛋白agrin介导nAChR转运和膜内定位过程，而我们实验发现脓毒症抑制agrin下游PI3K/Akt磷酸化，引起胞内微管表达下降，提示nAChR胞内转运和膜内定位障碍可能参与nAChR异位聚集分布。本项目以nAChR异位聚集分布为切入点，拟通过免疫荧光结合单颗粒示踪等方法观察脓毒症骨骼肌nAChR的运动变化规律；采用胞内微电极研究nAChR异常聚集分布对终板膜电位的影响；运用病毒转染、电穿孔质粒转染等方法分别探讨脓毒症影响nAChR聚集分布的胞内转运和跨膜定位两大过程的信号调控机制。本研究将进一步探索脓毒症相关获得性肌无力发病机制，为寻找治疗靶点提供思路。

脓毒症导致的获得性肌无力加重肺部感染，延长机械通气时间，是导致患者死亡率增高的危险因素。脓毒症获得性肌无力的发病机制尚不完全清楚。基于已有发病机制，包括改善氧化应激等治疗措施并未取得良好的临床疗效。神经肌接头是神经肌肉收缩耦联的重要结构基础，而nAChR的表达和分布在其中发挥了重要作用。本项目以突触后nAChR异位聚集分布为切入点，运用不同模型，分别从蛋白表达，功能和分子水平，应用多种研究方法，系统地研究了脓毒症导致NMJ突触后 nAChR 异常聚集分布的过程及其分子调控机制。研究发现脓毒症骨骼肌nAChR在终板膜区在时空上的分布变化与脓毒症严重程度有关。NMJ发生突触结构改变，nAChR发生“围接头区离散”和“异位聚集”，伴随NMJ突触传递功能障碍。脓毒症通过抑制神经源性聚集蛋白 agrin介导的Agrin-PI3K/Akt-microtubule/CLASP2胞内转运信号通路引起 nAChR 在膜内转运障碍，可通过增强自噬-溶酶体途径，增强骨骼肌细胞内nAChR的降解，从而破坏nAChR内循环转运。同时，脓毒症抑制agrin-Laminin/DGC-Rapsyn信号通路破坏围终板区nAChR“补偿池”作用，导致nAChR向终板膜聚集的膜定位障碍。脓毒症通过抑制胞内[Ca2+]i释放调节nAChR的膜内定位过程。通过外源性给予agrin处理以及外周神经电刺激治疗，可有效缓解终板膜nAChR的异常聚集程度以及NMJ的形态改变，骨骼肌功能障碍可得到有效改善。该研究从终板膜nAChR受体异位聚集分布的角度进一步阐明了脓毒症导致获得性肌无力的机制，并提示agrin联合神经电刺激在改善脓毒症导致获得性肌无力治疗方面的临床应用价值。

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