Pretreatment of HUVECs with SMI

Human umbilical vein endothelial cells (HUVECs) were purchased from iCell (iCell Bioscience Inc, Shang, China). Briefly, HUVECs were cultured in endothelial cell medium (ECM; Sciencell Research Laboratories, CA, USA) with the addition of 5% fetal bovine serum, 1% penicillin/streptomycin, and 1% endothelial cell growth supplement. The cells were maintained at 37°C in a humidified incubator containing 5% CO₂. SMI was diluted to different concentrations with ECM. H₂O₂ was diluted to 0.6 mM with ECM.
In order to explore the toxic effect of SMI on HUVECs, we pretreated HUVECs with SMI (0, 0.5%, 1%, 5%, 10%, 20%, 30%, 50%) for 12 h. Results of cell viability showed that the concentration of SMI should be less than 20%. Then, for selecting the most suitable concentration of intervention of SMI, we pretreated HUVECs with SMI (0, 1%, 2%, 2.5%, 3%, 4%, 5%) for 12 h and then treated with H₂O₂ (0.6 mM) for 2 h to induce an endothelial cell injury model [18 (link), 19 (link)]. According to the test of cell viability, we decided to incubate HUVECs with SMI at the concentrations of 1% and 5% in the further study. Then, HUVECs were pretreated with SMI (1%, 5%) for 12 h and H₂O₂ for 2 h as mentioned above, and the medium and cells were collected for the following test.