神经肌接头突触后膜烟碱型乙酰胆碱受体异质化在脓毒症致获得性肌无力综合征中的作用及其机制研究

The acquired weakness syndrome induced by sepsis increase hospital mortality in patients who are critically ill and cause chronic disability in survivors of critical illness, in addition to prolonging mechanical ventilation and hospitalisation. Incidence was 56–80% in those with systemic inflammatory response syndrome (SIRS), and 100% in those with septic shock or severe sepsis and coma. Pathogenesis of acquired weakness syndrome induced by sepsis is not entirely clear. Current clinical treatments could not get satisfied results. Structural changes associated with acquired weakness syndrome include axonal nerve degeneration, muscle myosin loss, and muscle necrosis. Functional changes can cause electrical inexcitability of nerves and muscles with reversible muscle weakness. Neuromuscular junction is an important structural basis of neuromuscular contraction coupling, in which nicotinic acetylcholine (nAChR) plays an important role. It had been found that in previous research heterogeneous nicotinic acetylcholine receptors exepressed in postsynaptic membrane of neuromuscular junction. Expression of nAChR with abnormal function (γ- and α7-nAChR) was up-regulated. While down-regulation of neurogenic factor- neuregulin-1, which regulates the exression of nAChR, had been found in septic rats. This research bases on heterogeneous nicotinic acetylcholine receptors as entry point. The animal model of sepsis will be built by cecal ligation and perforation surgery. The "space-time ralation" of distribution and expression of mRNA and protein of α7- and γ- nAChR on postsynaptic membrane of neuromuscular junction in septic rats will be investigate by western-blot, RT-PCR and immunofluorescence. The whole cell patch clamp is used to assess the effect of heterogeneous nAChR on neuromuscular function. The expression levels of neuregulin-1 protein will be changed by exogenous neuregulin-1 or siRNA given. The object is to clarify the signal pathway which regulates of heterogeneous nicotinic acetylcholine receptors exepressed on postsynaptic membrane of neuromuscular junction in acquired weakness syndrome induced by sepsis. 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，其发生率高达56%~80%。脓毒症获得性肌无力的发病机制尚不完全清楚，目前的临床治疗方法未取得满意疗效。神经肌接头（NMJ）是神经肌肉收缩耦联的重要结构基础，而烟碱型乙酰胆碱（nAChR）在其中发挥了重要作用。本研究前期发现脓毒症导致NMJ突触后nAChR异质化。功能异常的nAChR（γ-和α7-nAChR）表达上调，而参与nAChR表达调控的神经源性neuregulin-1蛋白表达下调。本项目以nAChR异质化为切入点，拟通过免疫荧光等方法观察脓毒症NMJ突触后nAChR异质化的时空变化规律；采用膜片钳研究nAChR异质化对神经肌肉功能的影响；运用siRNA干扰技术，探讨脓毒症致NMJ突触后nAChR异质化的信号通路。本研究将进一步探索脓毒症获得性肌无力发生机制，为寻找治疗靶点提供理论依据。

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（包括骨骼肌能量代谢障碍、氧化应激等）的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，而烟碱型乙酰胆碱（nAChR）在其中发挥了重要作用。本项目以nAChR异质化为切入点，运用不同的模型，分别从蛋白表达，功能和分子水平，应用分子生物学，电生理学等研究方法，阐明了脓毒症导致骨骼肌nAChR异质性对神经肌肉功能的影响及neuregulin-1β对nAChR表达调控的机制。研究表明重症脓毒症和脓毒症休克导致骨骼肌肌颤搐收缩力和复合肌动作电位幅值下降，神经传导速度下降， nAChR发生异质性（ε-nAChR、γ-nAChR和α7-nAChR）、终板膜面积的增加，表明脓毒症引起骨骼肌发生“拟去神经支配”。骨骼肌收缩功能下降与异质性nAChR表达水平呈正相关。α7-nAChR异常表达是导致骨骼肌收缩力下降的主要原因，约发挥60%作用，且异质性nAChR的表达增高导致骨骼肌对强直刺激的衰减加快，易发生疲劳。利用C2C12 myotubes肌管细胞模型证明neuregulin-1β可刺激成熟受体ε-亚基mRNA表达，抑制α7和γ亚基mRNA的表达。通过western-blot检测下游信号分子表达水平，发现Ras、Raf、Erk1/2、GABPα磷酸化水平增强。通过加入特异性分子抑制剂和细胞转染方法进一步证明neuregulin-1β通过Ras-ERK/MAPK信号通路调控异质性nAChR的表达。脓毒症小鼠血清明显抑制成肌细胞的分化，增加细胞凋亡。neuregulin-1β可发挥保护作用，改善脓毒症小鼠血清对C2C12 myotube的损伤作用，其机制主要通过激活PPARγ-NF-κB 通路，减少炎症因子TNF-α，IL-6对C2C12细胞分化的抑制。研究还发现联合应用外源性neuregulin-1β和agrin蛋白可促进C2C12 myotube细胞膜上ε-nAChR表达和聚集。该研究从终板膜nAChR受体异质性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neuregulin-1β联合agrin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。