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中华肺部疾病杂志(电子版)

中华肺部疾病杂志(电子版) ›› 2022, Vol. 15 ›› Issue (02) : 176 -182. doi: 10.3877/cma.j.issn.1674-6902.2022.02.008

论著

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
赵磊1, 徐朝霞2, 胡健3, 刘畅3, 潘晓佳3, 林正霄3, 冯健3,†(), 李福祥4,†()   
  1. 1. 646000　泸州，西南医科大学临床医学院
    2. 610083　成都，西部战区总医院急诊医学科
    3. 610083　成都，西部战区总医院重症医学科
    4. 646000　泸州，西南医科大学临床医学院；610083　成都，西部战区总医院重症医学科
  • 收稿日期:2022-02-15 出版日期:2022-04-25
  • 通信作者: 冯健, 李福祥
  • 基金资助:
    四川省干部保健科研课题(川干研2022-1303); 西部战区总医院军事医学科研项目(2019LH05)

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

Lei Zhao1, Zhaoxia Xu2, Jian Hu3, Chang Liu3, Xiaojia Pan3, Zhengxiao Lin3, Jian Feng3,†(), Fuxiang Li4,†()   

  1. 1. Clinical College of Southwest Medical University, Luzhou 646000, China
    2. Emergency Department, General Hospital of Western Theater Command, Chengdu 610083, China
    3. Department of Critical Care Medicine, General Hospital of Western Theater Command, Chengdu 610083, China
    4. Clinical College of Southwest Medical University, Luzhou 646000, China; Department of Critical Care Medicine, General Hospital of Western Theater Command, Chengdu 610083, China
  • Received:2022-02-15 Published:2022-04-25
  • Corresponding author: Jian Feng, Fuxiang Li
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引用本文：

赵磊, 徐朝霞, 胡健, 刘畅, 潘晓佳, 林正霄, 冯健, 李福祥. 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[J]. 中华肺部疾病杂志(电子版), 2022, 15(02): 176-182.

Lei Zhao, Zhaoxia Xu, Jian Hu, Chang Liu, Xiaojia Pan, Zhengxiao Lin, Jian Feng, Fuxiang Li. Effect and mechanism of different frequency neuromuscular electrical stimulation on ICU-acquired weakness muscular atrophy in mice[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2022, 15(02): 176-182.

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。

关键词: 急性呼吸窘迫综合征, ICU获得性衰弱, 肌肉萎缩, 神经肌肉电刺激, 腺苷酸活化蛋白激酶
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.

Key words: Acute respiratory distress syndrome, ICU-acquired weakness, Muscular atrophy, Neuromuscular Electrical Stimulation, AMP activated protein kinase
表1 实时荧光定量RT-PCR引物系列
基　因 引物系列(5’- 3’)
Atrogin-1 上游引物：CAGCTTCGTGAGCGACCTC
  下游引物：GGCAGTCGAGAAGTCCAGTC
MuRF-1 上游引物：GTGTGAGGTGCCTACTTGCTC
  下游引物：GCTCAGTCTTCTGTCCTTGGA
GAPDH 上游引物：TTGTGATGGGTGTGAACCACGAGA
  下游引物：CATGAGCCCTTCCACAATGCCAAA
图1 气管内滴注LPS构建ARDS相关性ICU-AW小鼠模型；注：A：小鼠腓肠肌组织病理(HE染色　SP×100)；B：小鼠四肢抓力-时间变化；C：小鼠生存曲线图；D：qRT-PCR检测各组小鼠腓肠肌中Atrogin-1和MuRF-1 mRNA相对表达量；E：Western blot检测各组小鼠腓肠肌组织Atrogin-1和MuRF-1蛋白的表达；F：各组小鼠腓肠肌中Atrogin-1和MuRF-1蛋白相对表达量。注：与C1、2两组比较，*表示P<0.05，#表示P<0.001
图2 AMPK蛋白在LPS诱导的ARDS相关性ICU-AW中的作用；注：A：Western blot检测小鼠腓肠肌p-AMPK和AMPK表达；B：小鼠腓肠肌中Atrogin-1和MuRF-1蛋白相对表达量比较；C：小鼠腓肠肌组织病理(HE染色　SP×100)；D：小鼠四肢抓力-时间变化；E：小鼠腓肠肌中Atrogin-1 、MuRF-1 mRNA相对表达量；F：Western blot检测小鼠腓肠肌Atrogin-1和MuRF-1蛋白表达；G：小鼠腓肠肌Atrogin-1和MuRF-1蛋白相对表达量比较。注：图2B，#表示P<0.01，与C2组比较；图2D、2E和2G，*表示P<0.05，#表示P<0.01，与ICU-AW-V组比较
图3 不同频率NMES刺激对ICU-AW小鼠腓肠肌的影响；注：A：各组小鼠腓肠肌组织病理(HE染色　SP×100)；B：各组小鼠四肢抓力-时间变化；C：各组小鼠腓肠肌中Atrogin-1和MuRF-1 mRNA相对表达量；D：Western blot检测各组小鼠腓肠肌Atrogin-1和MuRF-1蛋白表达情况；E：各组小鼠腓肠肌中Atrogin-1和MuRF-1蛋白相对表达量。注：图3B：a表示P<0.05，ICU-AW-40与ICU-AW组比较；b表示P<0.05，ICU-AW-40与ICU-AW-60组比较；c表示P<0.05，ICU-AW-40与ICU-AW-80组比较；d表示P<0.05，ICU-AW-20与ICU-AW组比较；e表示P<0.05，ICU-AW-60与ICU-AW组比较；图3C和3E：#表示P<0.01和ICU-AW比较，^表示P<0.01和ICU-AW-40比较
图4 AMPK抑制剂对NMES预防小鼠ICU-AW的影响；注：A：两组小鼠腓肠肌组织病理(HE染色　SP×100)；B：两组小鼠四肢抓力-时间曲线变化；C：两组小鼠腓肠肌Atrogin-1和MuRF-1 mRNA相对表达量；D：Western blot检测两组小鼠腓肠肌Atrogin-1和MuRF-1蛋白表达情况；E:两组小鼠腓肠肌中Atrogin-1和MuRF-1蛋白相对表达量。注：*表示P<0.05，#表示P<0.01，与ICU-AW-C组比较
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