骨骼肌不同亚型nAChR在大鼠脓毒症相关神经肌肉功能障碍中的作用:神经电生理法

Role of different nicotinic acetylcholine receptor subtypes in skeletal muscles in sepsis-related nervemuscle dysfunction in septic rats:neuroelectrophysiology

目的 采用神经电生理法评价骨骼肌不同亚型烟碱型乙酰胆碱受体(nAChR)在大鼠脓毒症相关神经肌肉功能障碍中的作用.方法 选择SPF级成年雄性SD大鼠,体重200~220 g,2~3月龄,采用盲肠结扎穿孔法制备脓毒症模型.脓毒症建模成功后第3天取右侧坐骨神经-胫前肌12份为脓毒症组(S组),另取正常大鼠坐骨神经-胫前肌12份为对照组(C组).采用细胞内微电极记录法监测神经肌接头终板电位(EPP).取6份坐骨神经-胫前肌采用终浓度为1μmol∕L的成熟型nAChR(ε-nAChR)特异性拮抗剂waglerin-1、终浓度为250 nmol∕L的胎儿型nAChR(γ-nAChR)特性拮抗剂αA-OIVA及终浓度为6 nmol∕L的神经型nAChR(α7-nAChR)特异性拮抗剂MLA孵育.于孵育15 min时记录EPP幅值,计算EPP幅值的差值(ΔEPP)和EPP变化率(ΔEPP%).另取6份坐骨神经-胫前肌,分别采用终浓度为1μmol∕L waglerin-1+250 nmol∕LαA-OIVA的Kreb混合液、终浓度为1μmol∕L waglerin-1+6 nmol∕L MLA的Kreb混合液、终浓度为250 nmol∕LαA-OIVA+6 nmol∕L MLA的Kreb混合液孵育15 min,记录EPP幅值.不同拮抗剂处理之间使用不含拮抗剂的Kreb液洗脱15 min.结果 与C组比较,S组拮抗ε-nAChR后ΔEPP和ΔEPP%降低,拮抗γ-nAChR后ΔEPP%降低,拮抗α7-nAChR后ΔEPP和ΔEPP%升高,拮抗ε-nAChR+γ-nAChR和拮抗ε-nAChR+α7-nAChR后EPP幅值升高,拮抗γ-nAChR+α7-nAChR后EPP幅值降低(P<0.05).与拮抗γ-nAChR比较,S组拮抗α7-nAChR后ΔEPP和 ΔEPP%明显增高(P<0.05).与拮抗 ε-nAChR+γ-nAChR比较,拮抗 ε-nAChR+α7-nAChR后EPP幅值降低(P<0.05).结论 骨骼肌终板膜 ε-nAChR功能减弱和 α7-nAChR功能增强在脓毒症相关神经肌肉功能障碍中占主导作用,且两者的作用相当.

Objective To evaluate the role of different nicotinic acetylcholine receptor ( nAChR) subtypes in skeletal muscles in sepsis-related nerve-muscle dysfunction in septic rats by neuroelectrophysiol-ogy. Methods SPF adult male Sprague-Dawley rats, weighing 200-220 g, aged 2-3 months, were stud-ied. Sepsis was induced by cecal ligation and puncture in chloral hydrate-anesthetized rats. Twelve portions of sciatic nerve-tibialis anterior muscles taken at day 3 after successful establishment of the sepsis modelserved as group S. Another 12 portions of sciatic nerve-tibialis anterior muscles of normal rats served as group C. The endplate potential ( EPP ) was monitored by intracellular microelectrode recording. Six por-tions of nerve-muscle samples were incubated with the adult nAChR (ε-nAChR ) specific antagonist wa-glerin-1 at a concentration of 1 μmol∕L, the fetal nAChR (γ-nAChR) specific antagonist αA-OIVA at a concentration of 250 nmol∕L and neuronal nAChR (α7-nAChR) antagonist methyllycaconitine ( MLA) at a final concentration of 6 nmol∕L. The amplitude of EPP was recorded at 15 min of incubation, and the differ-ence in the amplitude of EPP (ΔEPP) and changing rate of EPP (ΔEPP%) were calculated. Another 6 portions of samples were incubated for 15 min with the Kreb mixture of waglerin-1 and 250 nmol∕LαA-OIVA at a final concentration of 1 μmol∕L, the Kreb mixture of waglerin-1 and 6 nmol∕L MLA at a final concen-tration of 1 μmol∕L, and the Kreb mixture of αA-OIVA and 6 nmol∕L MLA at a final concentration of 250 nmol∕L, and then the amplitude of EPP was recorded. Washout was performed with antagonist free Kreb so-lution for 15 min between treatments with different blockers. Results Compared with group C, ΔEPP andΔEPP% were significantly decreased after antagonizing ε-nAChR, ΔEPP% was decreased after antagoni-zing γ-nAChR, ΔEPP and ΔEPP% were increased after antagonizing α7-nAChR, the amplitude of EPP was increased after antagonizingε-nAChR+γ-nAChR andε-nAChR+α7-nAChR, and the amplitude of EPP was decreased after antagonizing γ-nAChR+α7-nAChR (P<0. 05). ΔEPP and ΔEPP% were significantly higher after antagonizing α7-nAChR than after antagonizingγ-nAChR in group S ( P<0. 05) . The EPP was significantly lower after antagonizingε-nAChR+α7-nAChR than after antagonizingε-nAChR+γ-nAChR ( P<0. 05) . Conclusion Weakened function of ε-nAChR and enhanced function of α7-nAChR in the mem-brane of the end-plate in skeletal muscle are the main reasons for sepsis-related nerve-muscle dysfunction in septic rats, and the role is comparable between them.