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Diazoxide protects L6 skeletal myoblasts from H2O2-induced apoptosis via the phosphatidylinositol-3 kinase/Akt pathway

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Abstract

Objectives and design

Transplanted cell survival might greatly improve the therapeutic efficacy of cell therapy. Diazoxide (DZ), a highly selective mitochondrial ATP-sensitive potassium channel opener, is known to suppress cell apoptosis and protect cells in oxidative stressed ischemic environment. We explored the mechanisms involved in DZ pre-treatment-induced anti-apoptotic effect on L6 skeletal myoblast (SKM).

Materials and methods

L6 SKMs were divided into control group, H2O2 group, DZ + H2O2 group and DZ + LY + H2O2 group. Treatments of 400 μmol/L H2O2 for 24 h alone, or after 200 μmol/L DZ pre-treatment for 30 min, or after DZ and 50 μmol/L LY294002 co-administration for 30 min were performed. The cell apoptosis rates were assessed by flow cytometric analysis. The changes of mitochondrial membrane potential were determined by JC-1 mitochondrial staining. The activation of phosphatidylinositol-3 kinase (PI3K)/Akt, caspase-9 and caspase-3 was detected by western blot.

Results

Compared with the H2O2 group, DZ pre-treatment protected cells from H2O2-induced damage, increased Akt phosphorylation, prevented mitochondrial membrane depolarization as well as the activation of caspase-9 and caspase-3 and decreased the cell apoptosis rate. However, the DZ-induced cytoprotective and anti-apoptosis effects were partly inhibited by co-administration of a PI3K inhibitor, LY294002.

Conclusions

These data suggest that DZ pre-treatment contributes to protection of L6 SKMs against apoptosis at least partly by activating the PI3K/Akt pathway and subsequently inhibiting the mitochondrial-mediated caspase-dependent apoptotic signalling pathway.

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Abbreviations

SKM:

Skeletal myoblast

DZ:

Diazoxide

PI3K:

Phosphatidylinositol-3 kinase

mitoKATP channel:

Mitochondrial ATP-sensitive potassium channels

ROS:

Reactive oxygen species

MTT:

Methyl thiazolyl tetrazolium

Annexin V-FITC/PI:

Annexin V-fluorescein isothiocyanate/propidium iodide

IOD:

Integral optical density

JC-1:

5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide

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Acknowledgments

This research was supported by the Nature Science Foundation of Hebei Province, No. H2015206440.

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Authors and Affiliations

  1. Department of Histology and Embryology, Basic Medical College, Hebei Medical University, Zhongshan East Road 361, Shijiazhuang City, 050017, Hebei Province, People’s Republic of China

    Wei Chen, Guoyu Xue, Lisi Zhang, Lei Zhang & Suxia Shao

  2. Department of Endocrinology, The 3rd Hospital of Hebei Medical University, Ziqiang Road 139, Shijiazhuang City, Hebei Province, People’s Republic of China

    Yan Liu

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  1. Wei Chen
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Correspondence to Suxia Shao.

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Responsible Editor: Yoshiya Tanaka.

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Chen, W., Liu, Y., Xue, G. et al. Diazoxide protects L6 skeletal myoblasts from H2O2-induced apoptosis via the phosphatidylinositol-3 kinase/Akt pathway. Inflamm. Res. 65, 53–60 (2016). https://doi.org/10.1007/s00011-015-0890-1

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