Neferine, a bisbenzylisoquinoline alkaloid, offers protection against cobalt chloride-mediated hypoxia-induced oxidative stress in muscle cells

BACKGROUND Neferine, a bisbenzylisoquinoline alkaloid, isolated from Nelumbo nucifera has a wide range of biological activities. Cobalt chloride (CoCl2) was known to mimic hypoxic condition. In the present study, we assessed the cytoprotective effect of neferine against CoCl2-induced oxidative stress in muscle cells. METHODS Rhabdomyosarcoma cells were exposed to different concentrations of CoCl2, and the IC50 value was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Lactate dehydrogenase and NO assays were performed in order to determine the cytotoxic effect of CoCl2. Reactive oxygen species generation and cellular antioxidant status were determined for evaluating oxidative stress. For analyzing the effect of neferine on CoCl2-induced apoptosis, propidium iodide staining was performed. RESULTS The results of the present study indicate that CoCl2 induces cell death in a dose-dependent manner. Neferine pretreatment at 700 nM concentration offers better cytoprotection in the cells exposed to CoCl2. Lactate dehydrogenase and NO release in the culture medium were restored after neferine pretreatment. CoCl2 triggers time-dependent reactive oxygen species generation in muscle cells. Further, results of propidium iodide staining, mitochondrial membrane potential, and intracellular calcium accumulation confirm that neferine offers protection against CoCl2-induced hypoxic injury. Depleted activities of antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase due to CoCl2 exposure were also reinstated in the group that received neferine pretreatment. CONCLUSION Our study suggests that neferine from N. nucifera offers protection to muscle cells by counteracting the oxidative stress induced by CoCl2.