nAChR异化重构对脓毒症神经肌肉功能的影响及其机制研究

Acute lung injury (ALI) is a major cause of death in patients with sepsis. Lung injury in sepsis could be aggravated by sepsis-induced neuromuscular dysfunction (NMD), leading to increased mortality. The NMD induced by sepsis increase the hospital mortality in patients who are critically ill and cause chronic disability in survivors of critical illness, let along to prolonging mechanical ventilation duration and hospital stay. Its incidence varied from 56% to 80% in those with systemic inflammatory response syndrome (SIRS), and 100% in those with septic shock or severe sepsis and coma. The pathogenesis of NMD induced by sepsis is not entirely clear. Current clinical treatment could not bring satisfactory outcomes. Structural changes associated with NMD include axonal nerve degeneration, muscle myosin loss, and muscle necrosis. These 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.In previous research,it had been found that heterogeneity existed in exepression of nicotinic acetylcholine receptors in postsynaptic membrane of neuromuscular junction.Expression of nAChRs with abnormal function (γ-nAChR and α7-nAChR) were up-regulated,while down-regulation of neurogenic factor- neuregulin-1, which regulates the exression of nAChR, had been found in septic rats. This research is based on the heterogeneity of nicotinic acetylcholine receptors. The animal model of sepsis will be built by cecal ligation and perforation proceudure. 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 investigated by western-blot, RT-PCR and immunofluorescence. The whole cell patch clamp is used to assess the effect of heterogeneous nAChR on the neuromuscular function.Exogenous neuregulin-1 or siRNA will be given aimed at regulating the levels of neuregulin-1 protein expression.The object is to clarify the signal pathway which regulates the heterogeneity of nicotinic acetylcholine receptors exepressed on postsynaptic membrane of neuromuscular junction in NMD induced by sepsis. This study will reveal the mechanism of pathogenesis of sepsis-induced NMD. It could bring a new breakthrough of prevention and treatment for critically ill patients with NMD.

急性肺损伤是脓毒症患者死亡的重要原因，而脓毒症引起的神经肌肉功能障碍（NMD）进一步加重肺损伤，导致病死率增高。据报道脓毒症NMD发生率高达56%~100%，其机制不明。正常情况下，成熟型烟碱型乙酰胆碱受体（ε-nAChR）仅聚集于终板膜，是神经肌肉兴奋收缩耦联功能的结构基础。本项目前期研究发现脓毒症大鼠的骨骼肌细胞膜出现nAChR异化重构现象：非成熟型nAChR（γ-和α7-nAChR）合成增加，散布于整个细胞膜。同时还发现NRβ-1、agrin参与调控nAChR的合成与分布。因此，本项目以nAChR异化重构为切入点，运用体视学联合免疫荧光、膜片钳等技术，研究脓毒症nAChR异化重构对神经肌肉功能的影响；采用siRNA技术干扰NRβ-1和agrin蛋白表达，探讨脓毒症激活骨骼肌非成熟型nAChR表达的信号转导机制，为临床探索脓毒症NMD发生机制，寻找有效治疗方法提供理论基础。

脓毒症（sepsis）会导致严重的器官功能障碍，并且已成为全球重大的公共卫生问题。脓毒症引起的神经肌肉功能障碍（NMD）发生率高达56%~100%，NMD导致患者病死率增高，但是其机制不明。.本项目以盲肠结扎穿刺术（CLP）建立脓毒症大鼠模型为实验对象，烟碱性乙酰胆受体(nAChR)异化重构为切入点，运用ELISA检测大鼠血清和骨骼肌中炎症因子水平，采用分子生物学技术检测大鼠三种nAchR及其相关调控神经营养因子NRG-1、Agrin的表达水平，辅以神经肌肉电生理技术检测大鼠骨骼肌神经肌肉功能。免疫荧光、细胞膜片钳等技术，检测nAchR异化重构的时空性以及两种异质化受体内向电流的大小。运用药代动力学和药效动力学验证蛋白酶抑制剂乌司他丁改善非去极化激动剂（NMBA）药效作用的有效性以及对nAChR异化重构的作用。.本项目原计划研究内容已基本完成，所取得的结果可充分阐明脓毒症导致神经肌肉功能障碍和nAchR异化重构的关系，并为临床上有效治疗方法提供了理论基础。主要研究结果包括：脓毒症导致骨骼肌对非去极化肌松剂的敏感性下降与两种异质化受体（γ-和α7-nAChR）的表达有关，乌司他丁可抑制脓毒症导致的骨骼肌细胞膜上γ-nAChR和α7-nAChR的表达上调，促进神经肌肉功能的恢复，改善骨骼肌对非去极化肌松剂的敏感性。脓毒症状态下的骨骼肌细胞nAChR产生的通道电流中，成熟型受体（ɛ-nAChR）的内向电流占主要部分，γ-nAChR和α7-nAChR占小部分。验证了NRG-1，agrin/MuSK信号通路在调控脓毒症导致的烟碱型乙酰胆碱受体异化重构的机制。拓展研究发现，脓毒症下自噬不全与肌肉功能障碍密切相关，增强自噬可明显降低全身细菌负荷、减轻全身炎症，雷帕霉素可明显减少α7-nAChR、γ-nAChR的表达量，同时提高神经肌肉功能。给予外源性GDNF后也可以改善脓毒症致骨骼肌功能障碍。.本项目阐明了脓毒症引起的NMD的机制，为临床脓毒症NMD的治疗实践的改进提供新的参考依据，对麻醉在其他学科疾病诊疗中的价值认识和实用探索具有促进意义。为研究治疗脓毒症导致的NMD的干预措施或药物提供了新的靶点。