Effects of cold atmospheric plasma-treated medium on HaCaT and HUVEC cells in vitro

Cold atmospheric plasma (CAP) is an emerging technology that can generate various reactive oxygen and nitrogen species (RONS) at room temperature and shows promising applications for skin wound healing. The effects of plasma-treated medium (PTM) promoting cell proliferation have been verified, but the biological mechanisms involved are not well known. This study aims to assess the proliferation effect and mechanism induced by PTM on human keratinocyte cells (HaCaT) and human umbilical vein endothelial cells (HUVEC) in vitro. The results showed that the concentrations of H2O2 and NO2- inside PTM increased in a time-dependent manner as the atmospheric pressure plasma jet (APPJ) treatment time was prolonged. The biological outcomes were determined by cell counting kit, wound healing assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and western blot analysis. The cell viability and motility assays indicated that appropriate plasma conditions of short treatment time (15s-, 30s- and 45s-PTM) could promote cell proliferation, while long treatment time would inhibit cell proliferation (60s-PTM). With an appropriate time of PTM treatment, the secretion of vascular endothelial growth factor-alpha (VEGF-alpha) and transforming growth factor alpha (TGF-alpha) was promoted and the extracellular signal-regulated kinase/serine-threonine protein kinase pathways were activated, which induced HaCaT and HUVEC cell proliferation eventually. These behaviors of cells were mainly related to the enhancement of intracellular reactive oxygen species levels. These findings established a theoretical foundation for potential clinical applications of PTM in wound healing. With appropriate treatment time of plasma-treated medium (PTM) exposure, the production of intracellular ROS increased compared with the normal control. The intracellular ROS could promote the secretion of vascular endothelial growth factor-alpha (VEGF-alpha) and transforming growth factor alpha (TGF-alpha) via activating the ERK/AKT pathways, and induce HaCaT and HUVEC cells proliferation eventually. image