Study On Oxidative Stress And Histone Acetylation In Immune Cells Induced By PRRSV Infection

PRRSV infected can cause porcine reproductive and respiratory syndrome,infection of which induced immune suppression,and thus further results in immune suppression,secondary infection and other diseases,these diseases caused a great economic loss in swine husbandry.Numerous studies have shown that oxidative stress and histone acetylation in immune cells induced by virus infection are involved in virus proliferation and pathogenic process.The present study was to investigate PRRSV infection of immune cells in vitro or in vivo,and observe cellular oxidative stress and changes in histone acetylation,so as to study the pathogenesis of PRRSV and integrated control of the disease provide new ideas.Methods:(1)In order to explore the relationship between the concentration of virus infection,infection time and cellular oxidative stress,and establish the oxidative stress in vitro models of immune cells,the RAW264.7 cells,swine splenocytes and 3D4/2 cells was set as target cells,which were infected with 100?10-1?10-2?10-3 or 10-4 dilution PRRSV(TCID50=10-5.5/0.1 mL)in vitro,at 4h,8h,12h,24h and 48 hours post infection,the culture supernatant and the cells were harvested.The cell viability was measured by MTT,the NO level was detected by Griess regent,the ROS level was evaluated with DCFH-DA fluorescence probe,the GSH and GSSG levels were determined by fluorescence spectrometry,the activities of XOD,MPO and iNOS were measured by chemiluminescence assay.(2)To explore the relationship between the concentration of virus infection,infection time and cellular oxidative stress,and in order to establish an oxidative stress in vivo models of immune cells,the mice were set as target animals,which were infected with 100 or 10-1 dilution PRRSV by intranasally,orally and intraperitoneally joint infection,once a day for three days.On Day 7,Day 14 and Day 21 post infection,the mice were killed and spleen were isolated.The ROS level was evaluated with DCFH-DA fluorescence probe,the GSH and GSSG levels were determined by fluorescence spectrometry,the activities of XOD,MPO and iNOS were measured by chemiluminescence assay.(3)The RAW264.7 cells,swine splenocytes and 3D4/2 cells were deal with different concentrations of H2O2,at 4h,8h,12h,24h and 48 hours post treatment,the culture supernatant or the cells were harvested.The cell viability was measured by MTT,the ROS level was evaluated with DCFH-DA fluorescence probe.Establishment of H2O2-induced oxidative stress in vitro models of immune cells.(4)To investigate the relationship between reactive oxygen species and PRRSV-infected histone acetylation in immune cells,the RAW264.7 cells,swine splenocytes and 3D4/2 cells were used as target cells,the ceels were treated with H2O2 or PRRSV or both of them according to the established in vitro oxidative stress models of immune cells induced by PRRSV or H2O2.The activity of anti-superoxide anion,inhibitory capacity of hydroxyl radical and H2O2 levels were measured by chemiluminescence assay,The activity of HAT and HD AC were determined by ELISA assay,The protein expression levels of Ace-H3 and Ace-H4 were detected by Western-Blotting.Results:(1)The activities of XOD,MPO and iNOS,ROS production and NO levels in 10-2 dilution PRRSV infected RAW264.7 cells and swine splenocytes in vitro at 4h post infection and 10-1 dilution PRRSV infected 3D4/2 cells in vitro at 8h post infection were significantly increased,and GSH to GSSG ratio were significantly decreased.The findings suggested that PRRSV infection impacts the redox state of infected RAW264.7 cells,swine splenocytes and 3D4/2 cells.(2)The mice were successfully infected PRRSV.The activities of XOD,MPO and iNOS and ROS production in 100 dilution PRRSV infected mice at 14d post infection were significantly increased,and GSH levels and GSH to GSSG ratio were significantly decreased.The findings suggested that PRRSV infection impacts the redox state of infected mice.(3)The ROS production in 250?M H2O2 induced RAW264.7 cells,200?M H2O2 induced swine splenocytes and 150?M H2O2 induced 3D4/2 cells in vitro was significantly increased at 4h post treatment,and cell viability was significantly lowered.(4)The ability of anti-superoxide anion and inhibitory capacity of hydroxyl radical were significantly decreased in H2O2 or PRRSV infected cells,and H2O2 levels was significantly increased,resulting in reduced cellular antioxidant capacity.A more significant change was found when the cells were treated with both H2O2 and PRRSV.(5)The activity of HAT was significantly increased in H2O2 or PRRSV infected RAW264.7 cells,and the activity of HD AC was significantly decreased.As a result,the protein expression levels of Ace-H3 and Ace-H4 were significantly increased.A more significant change was found when the cells were treated with both H2O2 and PRRSV.(6)The activities of HAT and HD AC were significantly increased in H2O2 or PRRSV infected swine splenocytes and 3D4/2 cells.As a result,the protein expression levels of Ace-H3 and Ace-H4 were significantly increased.A more significant expression level was found when the cells were treated with both H2O2 and PRRSV.Conclusion:(1)PRRSV infection in RAW264.7 cells,swine splenocytes and 3D4/2 cells induced oxidative stress,and the oxidative stress in vitro models of immune cells which induced by PRRSV infection was successfully established.(2)The mice were successfully infected with PRRSV and oxidative stress was induced.(3)PRRSV infection in immune cells with oxidative stress promoted a significant increase in intracellular free radicals,and cellular free radical scavenging capacity significantly was reduced,the equilibrium between HAT and HDAC activity was further broken,which was due to high levels of protein expression of Ace-H3 and Ace-H4.