Antioxidant Properties of Crocodylus siamensis Blood Components on H2O2-induced Human Skin Fibroblast Cells

Abstract

To elucidate the antioxidant activity of Crocodylus siamensis (C. siamensis) blood components on BJ human skin fibroblasts, hydrogen peroxide (H2O2), a reactive oxygen species (ROS) causing cellular injury associated with the induction of numerous diseases, was selected as the oxidant in this research. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide solution (MTT) assay, C. siamensis blood components were found to exhibit no cytotoxicity on BJ cells. However, the oxidative damage induced by 500 mM H2O2 to BJ cells led to a significant reduction of cell growth. A pronounced protective effect against this damage was observed upon pre-incubation of the cells with 25, 50 and 100 mg/ml of each C. siamensis blood component for 1 h prior to H2O2-exposure. The highest potential to preserve cell viability was found for C. siamensis hemoglobin (cHb) (89.0%). When observed by visible light microscopy, BJ cells exposed to H2O2 displayed diverse morphological alterations, including decreased cell density, cell shrinkage and loss of typical fibroblast appearance. In contrast, BJ cells pre-incubated with C. siamensis blood components remained largely intact after exposure to H2O2. It could further be shown that treatment with C. siamensis blood components did not exhibit any cytotoxic effect on human peripheral blood mononuclear cells (PBMC). To evaluate if the protective effect of cHb against H2O2-induced damage to fibroblast cells is correlated to cellular apoptosis, annexin V-FITC/PI-staining flow cytometry analysis was conducted. The collected data clearly demonstrate that H2O2 induces apoptosis in human skin fibroblast cells, which was evidently decreased by pre-treatment with cHb. It is therefore concluded that C. siamensis blood components possess a great potential to inhibit H2O2-induced oxidative damage in BJ human skin fibroblast cells.