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Effect of burn injury on apoptosis and expression of apoptosis-related genes/proteins in skeletal muscles of rats

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Abstract

The purpose of this study was to investigate the occurrence and possible mechanisms of apoptosis in skeletal muscles after burn injury. After a 40% body surface area burn to rats, TA muscles were examined for apoptosis at varying times by TEM, TUNEL and cell death ELISA assay. Thermal injury was found to induce apoptosis in skeletal muscle on the first day and maximal apoptosis appeared 4 days post-injury. Apoptotic ligands in serum assessed by ELISA revealed rapidly increase of TNF-α and subsequent increase of sFasL to sFas ratio after burn injury. It implied TNF-α induced apoptosis in early stage and FasL induced apoptosis in later stage after burn injury. Apoptosis-related genes/proteins in skeletal muscles examined by real-time PCR array and Western blotting showed pro-apoptotic genes/proteins, including Tnfrsf1a, Tnfrsf1b and Tnfsf6 in TNF ligand and receptor family, Bax and Bid in Bcl-2 family, caspase-3 and caspase-6 in caspase family, Dapk1, FADD and Cidea in death and CIDE domain family, Apaf-1 in CARD family, and Gadd45a were up-regulated, while anti-apoptotic gene Bnip1 was down-regulated compared with that of time-matched controls. In addition, increment of caspase-3, caspase-8 and caspase-9 activity provided further evidence for their role in apoptosis in skeletal muscle. Significant increase in expression in pro-apoptotic genes/proteins and activity of caspases suggested that death receptor-mediated signaling pathways and other apoptotic related pathways participated in apoptosis in skeletal muscle after burn injury. However, it was found that some anti-apoptotic genes such as Bcl2l1, Mcl-1, Nol-3, Il-10 and Prok2 were also up-regulated, which might imply the co-existence of protective response of the body after burns. In conclusion, the data suggest that apoptosis and pro-apoptotic signaling are enhanced in muscles of burned rats. To further elucidate the underlying apoptotic mechanisms mediating the atrophic response is important in establishing potential therapeutic interventions that could prevent and/or reduce skeletal muscle wasting and preserve its physiological function.

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Abbreviations

TA:

Tibialis anterior

EDL:

Extensor digital longus

SOL:

Soleus

TNF-α:

Tumor necrosis factor-α

sFasL:

Soluble Fas ligand

sFas:

Soluble Fas

Cyt c :

Cytochrome c

Apaf-1:

Apoptosis-protease activating factor-1

ELISA:

Enzyme linked immunosorbent assay

LM:

Light microscopy

TEM:

Transmission electron microscopy

TUNEL:

Transferase–mediated dUTP nick end labeling

PBS:

Phosphate buffered saline

HRP:

Horseradish peroxidase

AEC:

3-Amino-9-ethyl-carbazole

SA:

Specific activity

CIDE:

Cell death-inducing DNA fragmentation factors

CARD:

Caspase recruitment domain

FADD:

Fas-associated death domain

HKG:

Housekeeping genes

JNK:

Jun N-terminal kinase

MAPK:

Mitogen-activated protein kinase

Gadd45:

Growth-arrest and DNA damage inducible 45

Dapk1:

Death-associated protein kinase 1

ARC:

Apoptosis repressor with caspase recruitment domain

FLIP:

FADD-like IL-1-converting enzyme-like inhibitory protein

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Acknowledgments

We thank Prof. Fusheng Wang (Infectious Diseases Institute of PLA) and Dr. Jiangyang Lu (Pathological Department, First Hospital Affiliated to General Hospital of PLA) for technological supports of histological assay. This work was supported by Grants from the National Natural Science Foundation of China (Project No. 30571919 and No. 7072077).

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Authors and Affiliations

  1. Department of Burns and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA (Formerly 304th Hospital of PLA), 51# Fucheng Road, Beijing, 100037, China

    Hongjie Duan, Jiake Chai, Zhiyong Sheng, Yongming Yao, Huinan Yin, Liming Liang, Chuanan Shen & Jing Lin

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  1. Hongjie Duan
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Correspondence to Jiake Chai.

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Duan, H., Chai, J., Sheng, Z. et al. Effect of burn injury on apoptosis and expression of apoptosis-related genes/proteins in skeletal muscles of rats. Apoptosis 14, 52–65 (2009). https://doi.org/10.1007/s10495-008-0277-7

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