Effect Of Panax Notoginseng Saponins On Oxidative Stress Induced By PCV2 Infection In Immune Cells

Panax notoginseng saponins(PNS),is an active ingredient of Panax notoginseng.It has been confirmed that PNS has a wide range of pharmacological activites such as immunomodulatory activity and free radicals scavenging activity.Porcine circovirus type 2(PCV2)infection could induce the immune cells damage and result in immune suppression,which caused a serious economic losses to livestock.Immune cells,as an important immune defense of the immune system,its redox state and histone acetylation directly affected the regulation of the body's immune response.The objective of this study is to investigate the effect of PNS on oxidative stress and histone acetylation induced by PCV2 infection in immune cells in vitro or in vivo,and aims to provide theoretical basis for development of PNS and prevention of the diseases involved in PCV2 infection.Metheod:(1)To purify the Panax notoginseng saponins(PNS),macroporous resin was applied after extraction by 70%ethanol,and the content was determined by ultraviolet spectrometry.(2)To establish a PCV2 infected model in vitro,the RAW264.6 cells,3D4/2 cells and swine splenic lymphocytes were incubated with PCV2,and examined by PCR.(3)To investigate the correlation between PCV2 virus concentration and oxidative stress,and establish a oxidative stress model in vitro,RAW264.6 cells,3D4/2 cells and swine splenic lymphocytes were incubated with series concentrations of PCV2(104 TCID50/0.1 mL),to observe the levels of NO,ROS,GSH,SGGS,cell viability,and the activities of XOD,MPO,iNOS.(4)To investigate the effect of PNS on oxidative stress induced by PCV2 infection in immune cells,RAW264.6 cells,3D4/2 cells and swine splenic lymphocytes were treated with different concentrations of PNS after incubated with 10-1 PCV2,to observe the production of ROS,NO,XOD,MPO,iNOS,cell viability and the ratio of GSH to GSSG.(5)To investigate the effect of PNS on histone acetylation in immune cells with PCV2 infection in vitro.RAW264.6 cells,3D4/2 cells and swine splenic lymphocytes were treated with PNS at dose of 200 ?g/mL after incubated with 10-1 PCV2 to observe the levels of H2O2,inhibitory capacity of hydroxyl radical,anti-superoxide anion radical activities,and the levels of HAT,HDAC,Ac-H3,Ac-H4.(6)To investigate the best proper way to establish experimental infection of PCV2 in mice.Mice were inoculated with different concentrations of PCV2 for different days,and evaluated by PCR.(7)To establish in vivo models of immune cells with oxidative stress,mice were inoculated with 100 PCV2(once daily on Day 1 to Day 3)for different days,and the splenic lymphocytes were obtained to measure the levels of ROS,T-GHS,GSH,GSSG,and the activities of XOD,MPO,iNOS.(8)To investigate the effect of PNS on oxidative stress and histone acetylation in the spleen of PCV2-infected mice.Mice were inoculated with 100 PCV2(once daily on Day 1 to Day 3)for different days,and the infected mice were obtained PNS at dose of 50,100,or 200 mg/kg body weight once daily on Day 4 to Day 6.The splenic lymphocytes were obtained to measure the levels of H2O2,ROS,T-GHS,GSH,GSSG,HAT,HDAC,Ac-H3,Ac-H4,and the activities of XOD,MPO,iNOS,inhibitory capacity of hydroxyl radical,and anti-superoxide anion radical activities.Result:(1)After purification,the extraction ratio of saponins was 7.5%and the purity of PNS was 64.42%by UV spectrophotometry.(2)The secretion of NO in RAW264.7cells,3D4/2 cells and swine splenic lymphocytes with PCV2 infected in vitro was significantly increased,and ROS production,GSSG level and XOD,MPO,iNOS activities were significantly upregulated post infection.The GSH level and the ratio of GSH/GSSG were significantly downregulated post infection.The findings showed that PCV2 infection impacts the redox state of infected immune cells,PCV2 at concentration of 10-1 and infection time at 12 h were the best practices to set up in vitro models of immune cells from oxidative stress.(3)PNS at concentration of 50?200 ?g/mL significantly reduced the levels of NO,ROS,H2O2,GSSG,and the activities of XOD,MPO,iNOS in PCV2 infected immune cells in vitro,and significantly increased the inhibitory capacity of hydroxyl radical,anti-superoxide anion radical activities,GSH levels,and the ratio of GSH to GSSG in PCV2 infected immune cells in vitro.The result showed that PNS enhanced antioxidant capacity of immune cells,and promoted the viabilities of infected cells in vitro,and PNS at concentration of 200 ?g/mL could more effectively protect immune cells against oxidative stress from PCV2 infection.(4)HAT level was significantly elevated and HDAC level was significantly reduced,accompanied with the downregulation of Ac-H3 level in RAW264.7 cells with PCV2 infection in vitro.Treatment with PNS post PCV2 infection increased HDAC level,and the level of Ac-H3 was significantly reduced in RAW264.7 cells.HAT level was significantly increased and HDAC level was significantly reduced,accompanied with the upregulation of Ac-H4 level in 3D4/2 cells infected with PCV2 in vitro.Treatment with PNS post PCV2 infection showed significantly upregulation of HDAC level,and significantly downregulation of Ac-H4 level.The level of HAT was significantly increased,and Ac-H3,Ac-H4 levels were significantly increased in swine splenic lymphocytes with PCV2 infection in vitro.Treatment with PNS post PCV2 infection showed significantly upregulation of HDAC level,and significantly downregulation of HAT level,accompanied with the downregulation of Ac-H3,Ac-H4 levels.(5)In this study,significantly increased the levels of ROS and GSSG,elevated XOD,MPO,iNOS activities were found in PCV2-infected mice,and the level of T-GSH and GSH were significantly decreased.The result demonstrated that PCV2 infection impacts the redox state of splenic lymphocytes in PCV2-infected mice,and mice were inoculated with 100 PCV2 post 7 d was the best way to establish in vivo models of immune cells with oxidative stress.(6)PNS significantly decreased the productions of ROS,H2O2,GSSG,as well as the activities of XOD,MPO and iNOS.The inhibitory capacity of hydroxyl radical,anti-superoxide anion radical activities,and the levels of T-GSH,GSH were significantly increased when the PCV2-infected animals were treated with PNS.The results showed that PNS promoted antioxidant capacity of splenic lymphocytes in PCV2-infected mice,and PNS at dose of 200 mg/kg body weight had effectively effect against oxidative stress induced by PCV2 infection.(7)PCV2 infection significantly increased the level of HAT,and significantly decreased the level of HDAC in the spleen of PCV2-infected mice,resulted in the increase of Ac-H3 level.PNS elevated the level of HD AC,reduced the level of HAT,and significantly inhibited the level of Ac-H3 in the spleen of PCV2-infected mice.Conclusion:(1)PCV2 infection induced oxidative stress and histone acetylation in RAW264.7 cells,3D4/2 cells and swine splenic lymphocytes in vitro.The effect of PNS against PCV2 infection by regulating PCV2-induced oxidative stress,and inhibited of oxidative-related histone acetylation induced by PCV2 infection in RAW264.7 cells,3D4/2 cells and swine splenic lymphocytes in vitro.(2)PCV2 infection induced oxidative stress and histone acetylation in the splenic lymphocytes in PCV2-infected mice.The effect of PNS against PCV2 infection by regulating oxidative stress,along with inhibiting of oxidative-related histone acetylation induced by PCV2 infection in mice.These findings demonstrated that oxidative stress is closely related to histone acetylation and effects of PNS on oxidative stress and histone acetylation induced by PCV2 infection in immune cells were confirmed.