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

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。

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

ObjectiveTo investigate the effect and mechanism of neuromuscular electrical stimulation (NMES) at different frequencies in the prevention and treatment of ICU-acquired weakness (ICU-AW). MethodsEighty-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. ResultsWhen 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). ConclusionEarly 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.