Huvecs

In order to examine the molecular mechanisms underlying the impact of monophasic pulsed current ES in vitro, the primary HUVECs (<8 passages, ScienCell, CA, USA) were cultured in endothelial cell medium (ECM, ScienCell, CA, USA) with 5% fetal bovine serum, 1% endothelial cell growth supplement (ECGS, ScienCell, CA, USA), and 1% penicillin/streptomycin solution (P/S, ScienCell, CA, USA) at 37 °C in a humidified atmosphere of 5% CO₂. The medium was changed every 2 days, and cells were passaged when cells reached 80% confluence. To mimic a high-glucose and high-fat environment, HUVECs were incubated in a medium supplemented with 25 mM d-glucose (Sigma-Aldrich, MO, USA) and 0.2–0.4 mM BSA-conjugated palmitate (Sigma-Aldrich, MO, USA) for 24h. HUVECs cultured in ECM medium containing 5.5 mM d-glucose, 19.5 mM l-glucose (Sigma-Aldrich, MO, USA), and 0.4 mM BSA were used as controls. For electrical stimulation, HUVECs in the experimental group were exposed to electrical stimulation environment using a C-Dish (Ion-Optics Co., MA, USA) and an isolated pulse stimulator (A-M systems, USA). The stimulating carbon electrodes were placed in the wells and submerged in the media.

HUVECs (Sigma-Aldrich; Merck KGaA) were maintained in endothelial growth medium (Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂. HUVECs were pretreated with Mtp (0.1, 1, 10, 100 or 1,000 µM; dissolved in deionized water; purity >98%; Chengdu Herbpurify Co., Ltd.) for 24 h at 37°C. H₂O₂ has been widely used to induce an oxidative environment in vascular endothelial cell models in vitro (16 (link)). To induce cell senescence, HUVECs were treated with 100 µM H₂O₂ for 12 h at 37°C. HUVECs were incubated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich; Merck KGaA) for 12 h at 37°C to activate NF-κB, as previously described (17 (link)).