不同频率神经肌肉电刺激对ARDS相关性ICU-AW小鼠肌肉萎缩防治及临床意义

Effect and mechanism of different frequency neuromuscular electrical stimulation on ICU-acquired weakness muscular atrophy in mice

目的不同频率下神经肌肉电刺激(neuromuscular electrical stimulation,NMES)在ARDS相关性ICU获得性衰弱(ICU-acquired weakness,ICU-AW)中的作用及机制。方法健康雄性C57BL/6小鼠88只随机分为11组,每组8只。分为:空白对照组(C1)、气管内注入无菌水组(C2)、ICU-AW模型组(ICU-AW)、ICU-AW+AMPK激动剂A-769662组(ICU-AW-A)、ICU-AW+A-769662溶剂对照组(ICU-AW-V)、NMES 20 Hz组(ICU-AW-20)、NMES 40 Hz组(ICU-AW-40)、NMES 60 Hz组(ICU-AW-60)、NMES 80 Hz组(ICU-AW-80)、ICU-AW-40+AMPK抑制剂Compound C组(ICU-AW-40-C)、ICU-AW-40+Compound C溶剂对照组(ICU-AW-40-V)。检测小鼠四肢抓力和存活状态,7 d后收集小鼠肺组织和腓肠肌标本,采用HE染色观察肺和肌肉病理学变化,采用western blot以及qRT-PCR的方法检测小鼠腓肠肌中Atrogin-1、MuRF-1 mRNA和蛋白表达。结果与C1、C2组相比,ICU-AW小鼠出现肺损伤及腓肠肌萎缩,四肢抓力及存活率显著降低(P<0.05),腓肠肌组织中MuRF-1、Atrogin-1基因及蛋白表达显著降低(P值分别为<0.001和<0.05);和C2组相比,ICU-AW组p-AMPK蛋白水平显著降低(P<0.01);与ICU-AW-V组相比,ICU-AW-A组小鼠腓肠肌肌肉萎缩改善,四肢抓力显著提高,Atrogin-1和MuRF-1蛋白及基因表达量显著降低(P<0.01);相比于ICU-AW组,ICU-AW-20组、ICU-AW-60组、ICU-AW-40组四肢抓力均显著提升(P<0.05),其中,ICU-AW-40提升最为显著(P<0.05);ICU-AW-40组Atrogin-1和MuRF-1蛋白及基因表达量也较其它四组显著降低(P<0.01);而AMPK抑制剂Compound C干预后能够显著逆转NMES 40 Hz方案的ICU-AW肌无力的保护作用(P<0.05,ICU-AW-40-V vs.ICU-AW-40-C)。结论早期应用40 Hz NMES能够显著改善ARDS相关性ICU-AW小鼠肌肉萎缩无力,其保护机制可能是通过激活AMPK发挥作用。

Objective To investigate the effect and mechanism of neuromuscular electrical stimulation(NMES)at different frequencies in the prevention and treatment of ICU-acquired weakness(ICU-AW).Methods Eighty-eight healthy male C57BL/6 mice were randomly divided into 11 groups with 8 mice in each group.The specific groups were as follows:blank control group(C1),tracheotomy+aseptic water infusion control group(C2),ICU-AW model group(ICU-AW),NMES 20 Hz group(ICU-AW-20),NMES 40Hz group(ICU-AW-40),NMES 60 Hz group(ICU-AW-60),NMES 80 Hz group(ICU-AW-80),ICU model group+AMPK agonist group(ICU-AW-A),ICU model group+AMPK agonist control group(ICU-AW-V),ICU model group+AMPK inhibitor group(ICU-AW-40-C),and ICU model group+AMPK inhibitor control group(ICU-AW-40-C).The limbs holding power and survival status of the mice were detected after operation.After 7 days,mice lung tissue and gastrocnemius specimens were collected,and pathological changes were observed by HE staining,and Western blot and qRT-PCR were applied to detect Atrogin-1 MuRF-1 expression in mice gastrocnemius.Results When compared with C1 and C2,lung injury and gastrocnemius muscle atrophy were apparently noted,and the limbs holding power significantly reduced in ICU-AW(P<0.05).The mRNA and protein expressions of Atrogin-1 and MuRF-1 were significantly decreased in ICU-AW when compared to C1 and C2(P<0.01 and P<0.05,respectively).There was a significant reduction of p-AMPK level in ICU-AW when compared with that in C2(P<0.01).The mice in ICU-AW-A treated with A-769662,an agonist of AMPK,the gastrocnemius muscle atrophy improved,and the grasping power was significantly elevated and the level of mRNA and protein expressions of Atrogin-1 and MuRF-1 were significantly reduced(P<0.01,vs.ICU-AW-C).After NMSE treatment,the grasping power in ICU-AW-20,ICU-AW-40,and ICU-AW-60 was improved when compared with that in ICU-AW(P<0.05),the gastrocnemius muscle atrophy was improved,and Atrogin-1 and MuRF-1 protein and gene expression were significantly decreased(P<0.05).The muscle weakness in ICU-AW-40 alleviated most dramatically than any other groups(P<0.05,vs.ICU-AW-20 or ICU-AW-60).The mice was treated compound C or Compared with the control group,the grasping ability and gastrocnemius muscle atrophy were improved in ICU-AW-A group,and the protein and gene expression levels of Atrogin-1 and MuRF-1 were significantly decreased in ICU-AW-A group(P<0.01).Compound C,an AMPK inhibitor,significantly reversed the protective effect of NMES 40 Hz on ICU-AW(P<0.05,ICU-AW-40-C vs.ICU-AW-40-V).Conclusion Early intervention with 40 Hz NMES can significantly improve ARDS related ICU-AW skeletal muscle atrophy in mice,and the protective mechanism may be through the regulation of AMPK.