Supplementary Material for: Alpha-Tocopherol Protects Human Dermal Fibroblasts by Modulating Nitric Oxide Release, Mitochondrial Function, Redox Status, and Inflammation

<b><i>Background:</i></b> The altered balance between oxidants/antioxidants and inflammation, changes in nitric oxide (NO) release, and mitochondrial function have a role in skin aging through fibroblast modulation. Tocopherol is promising in counteracting the abovementioned events, but the effective mechanism of action needs to be clarified. <b><i>Objective:</i></b> The aim of this study was to examine the effects of α-tocopherol on cell viability/proliferation, NO release, mitochondrial function, oxidants/antioxidants, and inflammation in human dermal fibroblasts (HDF) subjected to oxidative stress. <b><i>Methods:</i></b> HDF were treated with H₂O₂ in the presence or absence of 1–10 μM α-tocopherol. Cell viability, reactive oxygen species (ROS), NO release, and mitochondrial membrane potential were measured; glutathione (GSH), superoxide dismutase (SOD)-1 and -2, glutathione peroxidase-1 (GPX-1), inducible NO synthase (iNOS), and Ki-67 were evaluated by RT-PCR and immunofluorescence; cell cycle was analyzed using FACS. Pro- and anti-inflammatory cytokine gene expression was analyzed through qRT-PCR. <b><i>Results:</i></b> α-Tocopherol counteracts H₂O₂, although it remains unclear whether this effect is dose dependent. Improvement of cell viability, mitochondrial membrane potential, Ki-67 expression, and G0/G1 and G2/M phases of the cell cycle was observed. These effects were accompanied by the increase of GSH content and the reduction of SOD-1 and -2, GPX-1, and ROS release. Also, iNOS expression and NO release were inhibited, and pro-inflammatory cytokine gene expression was decreased, confirming the putative role of α-tocopherol against inflammation. <b><i>Conclusion:</i></b> α-Tocopherol exerts protective effects in HDF which underwent oxidative stress by modulating the redox status, inflammation, iNOS-dependent NO release, and mitochondrial function. These observations have a potential role in the prevention and treatment of photoaging-related skin cancers.