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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) ›› 2020, Vol. 13 ›› Issue (02) : 198 -203. doi: 10.3877/cma.j.issn.1674-6902.2020.02.015

论著

NLRP3炎症小体在ICU获得性衰弱大鼠呼吸肌和下肢肌中的表达
向朝雪1, 李福祥2,†(), 朱忠立3, 刘畅3, 胡健3, 黎俊雅3, 祝国芸3, 宋羽希3   
  1. 1. 610031　成都，西南交通大学临床医学院
    2. 610031　成都，西南交通大学临床医学院；610083　成都，中国人民解放军西部战区总医院重症医学科
    3. 610083　成都，中国人民解放军西部战区总医院重症医学科
  • 收稿日期:2019-07-10 出版日期:2020-04-25
  • 通信作者: 李福祥

Expression and role of NOD-like receptor thermal-protein domain-related protein 3 inflammasome in skeletal muscles of rats with ICU-acquired weakness

Chaoxue Xiang1, Fuxiang Li2,†(), Zhongli Zhu3, Chang Liu3, Jian Hu3, Junya Li3, Guoyun Zhu3, Yuxi Song3   

  1. 1. Clinical Medical College, Southwest Jiaotong University, Chengdu 610031, China
    2. Clinical Medical College, Southwest Jiaotong University, Chengdu 610031, China; Department of Critical Care Medicine, General Hospital of Western Theater Command, Chengdu 610083, China
    3. Department of Critical Care Medicine, General Hospital of Western Theater Command, Chengdu 610083, China
  • Received:2019-07-10 Published:2020-04-25
  • Corresponding author: Fuxiang Li
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向朝雪, 李福祥, 朱忠立, 刘畅, 胡健, 黎俊雅, 祝国芸, 宋羽希. NLRP3炎症小体在ICU获得性衰弱大鼠呼吸肌和下肢肌中的表达[J]. 中华肺部疾病杂志(电子版), 2020, 13(02): 198-203.

Chaoxue Xiang, Fuxiang Li, Zhongli Zhu, Chang Liu, Jian Hu, Junya Li, Guoyun Zhu, Yuxi Song. Expression and role of NOD-like receptor thermal-protein domain-related protein 3 inflammasome in skeletal muscles of rats with ICU-acquired weakness[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2020, 13(02): 198-203.

目的

探讨重症监护室获得性衰弱(ICU-AW)大鼠吸肌和下肢肌组织中NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎症小体的表达及作用。

方法

将40只健康雄性SD大鼠完全随机分为对照组(sham组)和实验组(脓毒血症组)，采用盲肠结扎穿孔术在实验组大鼠中构建ICU-AW模型，对照组仅行开腹暴露盲肠手术；造模96 h后收集大鼠腓肠肌和膈肌标本，采用HE染色观察病理学变化并计算肌纤维横截面积，采用qRT-PCR和western blot的方法检测大鼠腓肠肌和膈肌中Atrogin-1、MuRF1、NLRP3、Caspase-1、IL-1β和IL-18的表达。

结果

盲肠结扎穿孔术96 h后，大鼠腓肠肌和膈肌纤维排列较对照组疏松，肌纤维横截面积较对照组明显减少(P<0.05)，Atrogin-1、MuRF1基因和蛋白表达量较对照组明显升高(P<0.05)；ICU-AW大鼠腓肠肌和膈肌中NLRP3、Caspase-1、IL-1β和IL-18基因和蛋白表达量较对照组明显升高(P<0.05)。

结论

盲肠结扎穿孔术制备的脓毒血症模型中，吸肌和下肢肌溶解参与ICU-AW的发生，NLRP3炎症小体相关信号通路参与上述过程。

关键词: 重症监护室获得性衰弱, NLRP3炎症小体, 肌肉萎缩, 呼吸肌
Objective

To investigate the expression and role of NOD-like receptor thermal-protein domain-related protein 3 (NLRP3) inflammasome in the skeletal muscles of the rats with Intensive Care Unit-acquired weakness (ICU-AW).

Methods

Forty healthy male Sprague-Dawley rats were randomly divided into a control group (the sham group) and an experimental group (a septic group). The ICU-AW models were established by cecal ligation and puncture, while cecal exposure but no ligation or puncture was carried out in the rats of the control group. The gastrocnemius and diaphragmatic muscle specimens were collected 96 h after modeling. The pathological changes were observed by HE staining and the cross-sectional areas of the muscle fibers were calculated and compared between the two groups. Both gene and protein expressions of atrogin-1, MuRF1, NLRP3, caspase-1, IL-1β and IL-18 in the gastrocnemius and diaphragmatic muscle were detected by qRT-PCR and Western blotting method.

Results

The arrangement of the gastrocnemius and diaphragm muscle fibers was looser 96 h after cecal ligation and puncture compared with that of the controls. And the cross-sectional areas of the muscle fibers from the experimental group were significantly smaller than those of the controls (P<0.05). Both gene and protein expression levels of atrogin-1 and MuRF1 were higher in the muscle samples from the ICU-AW rats than those of the controls (P<0.05). NLRP3 inflammatory body-related molecular studies showed that the expression levels of NLRP3, caspase-1, IL-1β and IL-18 in the gastrocnemius and diaphragmatic muscle of the ICU-AW rats were significantly higher than those of the controls (P<0.05).

Conclusion

Skeletal muscle lysis participates in the occurrence of ICU-AW in the rat models created by cecal ligation and puncture, and NLRP3 inflammasome-related signal pathway participates in the above process.

Key words: Intensive Care Unit-acquired weakness, NLRP3 inflammasome, Muscle atrophy
表1 目的基因qRT-PCR引物序列
名　称 上游 下游
NLRP3 5’-ATTACCCGCCCGAGAAAGG-3’ 5’-TCGCAGCAAAGATCCACACAG-3’
Atrogin-1 5’-CAGCTTCGTGAGCGACCTC -3’ 5’-GGCAGTCGAGAAGTCCAGTC-3’
MuRF1 5’-GTGTGAGGTGCCTACTTGCTC -3’ 5’-GCTCAGTCTTCTGTCCTTGGA -3’
Caspase-1 5’-ACAAGGCACGGGACCTATG -3’ 5’-TCGCAGCAAAGATCCACACAG -3’
IL-1β 5’-GCAACTGTTCCTGAACTCAACT -3’ 5’-ATCTTTTGGGGTCCGTCAACT -3’
IL-18 5’-GACTCTTGCGTCAACTTCAAGG -3’ 5’-CAGGCTGTCTTTTGTCAACGA -3’
图1 盲肠结扎穿孔术后96 h大鼠腓肠肌和膈肌HE染色结果；注：A：对照组　沿腓肠肌纤维纵轴切片；B：对照组　沿腓肠肌纤维纵轴的横截面切片；C：对照组　沿膈肌纤维纵轴切片；D：实验组　腓肠肌纤维纵轴切片；E：实验组　沿腓肠肌纤维纵轴的横截面切片；F：实验组：沿膈肌纤维纵轴切片
表2 对照组和实验组大鼠腓肠肌和膈肌纤维横截面积对比
分　组 例数 腓肠肌(μm2) 膈肌(μm2)
对照组 3 878.74±201.94 573.01±210.46
实验组 3 345.90±127.02 140.40±67.79
  t值   3.869 3.389
  P值   0.018 0.028
表3 对照组和实验组大鼠腓肠肌和膈肌中Atrogin-1和MuRF-1基因表达
组　别 腓肠肌 膈肌
MuRF1 Atrogin-1 MuRF1 Atrogin-1
对照组 0.78±0.12 0.88±0.09 0.63±0.19 0.46±0.27
实验组 3.24±0.20 24.19±3.81 1.22±0.05 61.47±11.76
  t值 10.74 6.12 2.98 5.19
  P值 0.0004 0.0036 0.0408 0.0066
图2 脓毒血症时大鼠腓肠肌、膈肌组织中Atrogin-1和MuRF-1蛋白的表达
表4 脓毒血症时大鼠腓肠肌、膈肌组织中NLRP3及其下游分子的mRNA表达
组　别 腓肠肌 膈肌
NLRP3 Caspase-1 IL-18 IL-1β NLRP3 Caspase-1 IL-18 IL-1β
对照组 0.91±0.16 1.25±0.78 0.72±0.15 0.0003±0.0001 0.59±0.20 0.96±0.23 0.74±0.13 1.17±0.10
实验组 3.16±0.18 18.01±0.45 1.54±0.03 0.0025±0.0006 1.54±0.08 4.16±1.07 5.36±0.87 4.45±0.10
  t值 9.34 18.49 5.36 3.65 4.344 2.90 5.256 23.43
  P值 0.0007 <0.0001 0.0060 0.0218 0.0122 0.0439 0.0063 <0.0001
图3 脓毒血症时大鼠腓肠肌、膈肌组织中NLRP3及其下游分子蛋白表达
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