Protective mechanisms of melatonin against hydrogen-peroxide-induced toxicity in human bone-marrow-derived mesenchymal stem cells

Abstract

Many obstacles compromise the efficacy of bone marrow mesenchymal stem cells (BM-MSCs) by inducing apoptosis in the grafted BM-MSCs. The current study investigates the effect of melatonin on important mediators involved in survival of BM-MSCs in hydrogen peroxide (H2O2) apoptosis model. In brief, BM-MSCs were isolated, treated with melatonin, and then exposed to H2O2. Their viability was assessed by MTT assay and apoptotic fractions were evaluated through Annexin V, Hoechst staining, and ADP/ATP ratio. Oxidative stress biomarkers including ROS, total antioxidant power (TAP), superoxide dismutase (SOD) and catalase (CAT) activity, glutathione (GSH), thiol molecules, and lipid peroxidation (LPO) levels were determined. Secretion of inflammatory cytokines (TNF-α and IL-6) were measured by ELISA assay. The protein expression of caspase-3, Bax, and Bcl-2, was also evaluated by Western blotting. Melatonin pretreatment significantly increased viability and decreased apoptotic fraction of H2O2-exposed BM-MSCs. Melatonin also decreased ROS generation, as well as increasing the activity of SOD and CAT enzymes and GSH content. Secretion of inflammatory cytokines in H2O2-exposed cells was also reduced by melatonin. Expression of caspase-3 and Bax proteins in H2O2-exposed cells was diminished by melatonin pretreatment. The findings suggest that melatonin may be an effective protective agent against H2O2-induced oxidative stress and apoptosis in MSC.