Burn Serum Stimulated Mitochondrial Fission Was Decreased with IL-6 Antibody Treatment
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INTRODUCTION: Burn patients suffer muscle mass loss associated with hyperinflammation and hypercatabolism. We previously observed that burn serum resulted in cell death with elevated mitochondrial fragmentation in C2C12 myoblast. IL-6 as the key cytokine response to thermal injury has been showed to increase mitochondrial fragmentation. We thus posit that inhibition of IL-6 expression in burn serum will alleviate mitochondrial fragmentation. The aim of this study is to investigate the neutralization effect of IL-6 antibody in burn serum stimulated myoblasts. METHODS: Murine myoblasts C2C12 cells were cultured with recombinant IL-6 protein from 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, and 100 ng/ml. Cells were labeled with MitoTracker Green dye and live cell images were captured with Confocal microscopy. Next, C2C12 cells were exposed to medium containing 1) 10% serum from control rat, 2) 10% serum from burn rat, and 3) 10% serum from burn rat and 0.5 ug/ml of IL-6 antibody. All cells were labeled as the first experiment and live cell images were recorded. The caspase 3 activity was further examined from cell protein lysate. RESULTS: The 1 ng/ml dose of r-IL6 showed a fourfold increase in mitochondrial volume (μm3) at 24 hours post challenge. The intensity signal of Mitotracker was significantly increased in the 1 ng/ml IL-6 dose group at 48 hours. The 1 ng/ml dose of r-IL6 showed an increase in mitochondrial fragmentation. In cells cultured with 10% serum from control rats, mitochondrial morphology maintained the elongated linear shape during the 48 hours. In cells cultured with 10% serum from burned rats, a reduction in mitochondrial sizes was significant. In cells cultured with 10% serum and IL-6 antibody treatment this effect was reversed. Further, the intensity signals increased in response to the burn serum challenge. However, with addition of IL-6 antibody treatment the intensity signals decreased. In addition, burn serum increased the total volume of mitochondria about 1.4 fold, while with IL-6 antibody treatment the total volume was decreased. Consistently, a significant decrease in the expression of caspase 3 in the IL-6 antibody treatment group was observed. CONCLUSION: IL-6 stimulates an increase in mitochondria fragmentation in myoblasts, while IL-6 antibody treatment decreases mitochondrial fragmentation and cell death in burn serum stimulated myoblasts. This project has shown that targeting cytokine levels may be an effective treatment strategy in the management of burn patients.