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Attenuation of cellular toxicity by calpain inhibitor induced by bacterial endotoxin: a mechanistic study using muscle precursor cells as a model system

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Abstract

This investigation was under taken to explore probable mechanisms and signal pathways involved in cytotoxicity induced by bacterial endotoxin lipopolysaccharide (LPS). Herein, we selected muscle precursor C2C12 myoblasts as representative cells to test effect of calpain inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) on LPS induced inflammation and apoptosis. In order to rule out the toxicity of endotoxin, mouse myoblasts were exposed to various concentrations of LPS and viability of cells and morphology were assessed using CCK-8 assay and simple microscopy respectively. Apoptotic cell death was examined by fluorescence microscope at regular time intervals. Additionally, LPS induced apoptosis in C2C12 cells were determined by mRNA expression of µ-calpain, caspase-3 and tumor necrosis factor alpha (TNF-α) and were quantified by qRT-PCR. Our results point out that LPS stimulation produced dose dependent toxicity in muscle precursor cells. Pre-treatment with a calpain inhibitor can significantly attenuate LPS-induced inflammation/apoptosis. Results of present research determined that mRNA expression of aforesaid genes was amplified (p < 0.05) in LPS stimulated C2C12 cells, whereas a noticeable drop off in mRNA expression of these genes was observed when pre-exposed with calpain inhibitor PD150606. Our study has outlined the current understanding regarding the connection between µ-calpain and caspase-3 in skeletal muscle wasting and as a result provides suitable choice for designing promising chemotherapeutic system for muscle illness and atrophy.

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Acknowledgments

This work was supported by a grant from the Rural Development Administration, Republic of Korea (No. PJ010170) and the Next Generation Biogreen 21(No. PJ011101). This research was partly supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Project no. 2014R1A1A2007175). Dr Touseef Amna acknowledges the research Grant from NRF.

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    Authors and Affiliations

    1. Department of Animal Science and Biotechnology, Chonbuk National University, Chonju, 561-756, Republic of Korea

      Ke Shang, Junfeng Zhang, Touseef Amna, Jieun Yang & Inho Hwang

    2. Animal Disease and Public Security Academician Workstation of Henan Province/The Key Lab of Animal Disease and Public Health, Henan University of Science and Technology, Luoyang, 471003, China

      Ke Shang, Junfeng Zhang, Xiangchao Cheng & Chunjie Zhang

    3. Departmnet of Biology, Faculty of Science, Albaha University, P.O. Box 1988, Albaha, Kingdom of Saudi Arabia

      Touseef Amna

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    Correspondence to Touseef Amna or Inho Hwang.

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    Ke Shang, Junfeng Zhang and Touseef Amna have contributed equally to this work.

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    Shang, K., Zhang, J., Amna, T. et al. Attenuation of cellular toxicity by calpain inhibitor induced by bacterial endotoxin: a mechanistic study using muscle precursor cells as a model system. Mol Biol Rep 42, 1281–1288 (2015). https://doi.org/10.1007/s11033-015-3869-7

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