Establishing oxidative stress model of RAW264.7 cells induced by porcine circovirus type 2 in vitro

ObjectiveThe present study investigated the relationship between infection dose,infection time of porcine circovirus type 2(PCV2) and dynamic changes of reactive oxygen species(ROS) levels in PCV2 infected RAW264.7 cells,so as to provide a reference for the establishment of in vitro oxidative stress model of RAW264.7 cell.Method PCV2 virus liquid of 5×106/mL was prepared,and was 10,102,103 and 104 times diluted(10-1,10-2,10-3,10-4 dilutions) before use.RAW264.7 cells were infected with five different concentrations of PCV2 for 2 hours,and then virus liquids were discarded.The cells were cultured in DMEM after adding 5% FBS for 4,8,12,24 and 48 h.Cell supernatant or cells were collected and levels of nitric oxide (NO),reactive oxide species (ROS),reduced glutathione (GSH),oxidized glutathione (GSSG),xanthine oxidase (XOD),myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) were determined.ResultAfter 10-1 PCV2 infected RAW264.7 cells,levels of NO and ROS increased while GSH levels and GSH/GSSG ratio decreased.Activities of XOD,MPO and iNOS also increased.These findings suggested that 10-1 PCV2 infection could change cellular redox state and induce cells to produce oxidative stress.Conclusion PCV2 infection can induce oxidative stress in RAW264.7 cells.10-1 PCV2(5×l02/mL) infection in vitro on RAW264.7 cells for 4-24 h is the optimum condition for establishing oxidative stress model.