Non-thermal Dielectric-barrier Discharge Plasma Induces Reactive Oxygen Species by Epigenetically Modifying the Expression of NADPH Oxidase Family Genes in Keratinocytes

We have previously shown that non-thermal dielectric-barrier discharge (DBD) plasma induces the generation of reactive oxygen species (ROS) in cells; however, the underlying mechanism has not been elucidated. This study aimed to identify the mechanisms through which DBD plasma induces the expression of NADPH oxidase (NOX) family members by epigenetic modification in human keratinocytes (HaCaT). Cell exposure to DBD plasma in 10% oxygen and 90% argon resulted in the generation of ROS, triggering oxidative stress that manifested in various forms, including lipid membrane peroxidation, DNA base modification, and protein carbonylation. DBD plasma upregulated the expression of NOX1, NOX5, and DUOX2 at the mRNA and protein levels; and siRNAs targeting NOX1, NOX5, and DUOX2 attenuated the generation of DBD plasma-induced ROS. DBD plasma upregulated the transcriptional activators TET1, MLL1, and HAT1 and downregulated the transcriptional repressors DNMT1, EZH2, and HDAC1. Additionally, DBD plasma increased the binding of transcriptional activators and decreased the binding of transcriptional repressors to the DUOX2 promoter. Methyl-specific polymerase chain reaction and bisulfite sequencing indicated that DBD plasma decreased methylation at the DUOX2 promoter. These results suggest that DBD plasma induces ROS generation by enhancing the expression of the NOX system through epigenetic DNA and histone modifications.