Study On Flavonoids From Polygonum Hydropiper Linn On The Inflammatory Response Of PCV2 Infected Porcine Alveolar Macrophages And Its Molecular Mechanism

FEA,ethyl acetate partial flavonoids that from Polygonum hydropiper Linn.is a natural active ingredient extracted occurring in the whole grass of Polygonum hydropiper Linn and has shown various biological activities such as scavenging oxygen free radicals,anti-inflammation and anti-bacterial.Porcine circovirus type 2（PCV2）is a very small and non-enveloped with single-stranded DNA virus and is thought to be the major causative agent of post-weaning multisystemic wasting syndrome（PMWS）,which induces immune suppression in the body,resulting in immunization failure,secondary infections and other diseases,which has caused huge economic losses to the pig industry in the world.In the present study,a virus infection inflammatory model was established to observe actions of FEA on the free radical levels,redox state and inflammatory factors with PCV2 infected the PAMs（3D4/2 cells）in vitro,and then clarified the molecular mechanism of bioactive flavonoids regulating inflammatory response.The results will provide a new idea for the development and utilization of FEA and the prevention and treatment of animal viral diseases.Methods:（1）In order to explore the relationship among the concentration of virus infection,infection time and levels of inflammatory factors,we established the inflammation models of 3D4/2 cells infected with 10⁰、10^-1、10^-2、10^-3 dilution PCV2（TCID50=10³/0.1 mL）in vitro,at 4h,8h,12h,24h post infection,the culture supernatant and the cells were harvested.The cell viability was measured by CCK8,the NO level was detected by Griess regent,the ROS level was evaluated with DCFH-DA fluorescence probe,the GSH level was determined by fluorescence spectrometry,the activities of XOD,MPO and iNOS were measured by chemiluminescence assay,the levels of IL-1beta,IL-6,TNF-alpha,IL-10,IFN-gamma,IL-8,MCP-1,COX-1,COX-2 and other inflammatory factors were measured by ELISA.（2）To investigate the anti-inflammatory effect of FEA in PCV2 infected 3D4/2 cells in vitro.The cell safety concentration of FEA was determined by CCK8,FEA was used at concentration of 100,50 and 25μg/mL in 3D4/2 cells infected with PCV2 in vitro with 8h respectively,the culture supernatant and the cells were harvested.The NO level was detected by Griess regent,the ROS level was evaluated with DCFH-DA fluorescence probe,the GSH levels were determined by fluorescence spectrometry,the activities of XOD,MPO and iNOS were measured by chemiluminescence assay,the levels of IL-1beta,IL-6,TNF-alpha,IL-10,IFN-gamma,IL-8,MCP-1,COX-1,COX-2 and other inflammatory factors were measured by ELISA in order to observe the regulation of FEA on the inflammatory response in 3D4/2 cell with PCV2infected.（3）To investigate the molecular mechanisms of anti-inflammatory effects of FEA in PCV2-treated 3D4/2 cells.FEA was used at concentration of 100,50 and 25μg/mL in 3D4/2 cells infected PCV2 in vitro with 8h respectively,and the cells were harvested.Relative expressions of TNF-α,IL-1β,IL-6,IL-8,IL-10,COX-2,iNOS,c-fos,c-jun,c-myc,P65,AKT,P38 MAPK,and ERK mRNA of the Pig lung were measured with a SYBER green detection system by using CFX96 Real-Time PCR Detection System.（4）To further investigate the molecular mechanisms of anti-inflammatory effects of FEA in PCV2-treated 3D4/2 cells.FEA was used at concentration of 100,50 and 25μg/mL in 3D4/2 cells infected PCV2 in vitro with 8h respectively.After incubated,the cells were collected and washed twice with cold PBS,then total protein in cell was extracted.Western-Bloting was used to detect the expression of PI3K/AKT,p44/42,p38,NF-κB and their corresponding protein phosphorylation.Results:（1）There is no significant change to cell viability after 10⁰dilution of PCV2 infected 3D4/2 cells in vitro.The activities of XOD,MPO and iNOS,ROS production and NO levels in 10⁰ dilution PCV2 infected3D4/2 cells in vitro at 8 h post infection were significantly increased,and the GSH level were significantly decreased,the levels of cytokine IL-6,TNF-α,IFN-γ,MCP-1,COX-1 increased significantly.The findings suggested that PCV2 infection impacts the levels inflammatory factors and the redox state of 3D4/2 cells in vitro.（2）PCV2 infected was no significant effect on the activity of 3D4/2 cells,whereas FEA treatments at 25μg/mL significantly increased viability of 3D4/2 cells for 8 h,and no significant change in the treatment of 100μg/mL、50μg/mL FEA.FEA treatments at concentration from 25μg/mL to 100μg/mL for 8h reduced the release of NO,ROS,TNF-alpha,IL-1bet,IL-6,IL-10,COX-1,COX-2,IL-8,IFN-gamma and MCP-1,and decreased the activity of iNOS,MPO and XOD of inflammatory mediators and upgraded GSH levels in PCV2 infected3D4/2 cells.（3）PCV2 infection significantly increased the expression of TNF-α,IL-6,IL-8,IL-10,c-fos,c-jun,c-myc,P65,AKT,P38 MAPK,ERK mRNA,extremely significant increased the expression of IL-1β,COX-2 and iNOS mRNA in 3D4/2 cells,the transcription of IκB mRNA was clearly decrease;FEA treatments at concentration from 25μug/mL to100μg/mL downregulation the expression of IL-1β、TNF-α、IL-6、IL-8、IL-10、c-fos、c-jun、c-myc、P65、AKT、P38 MAPK、ERK mRNA,and the expression of IκB mRNA was obviously increased in 3D4/2 cells with PCV2 infected.（4）As compared to control group,3D4/2cells treated with PCV2 alone revealed significant increases the protein expression of P-AKT、P-P38、P-ERK、P-P65、P-c-jun、P-c-fos and c-fos、c-jun as well as c-myc,significantly decreased IκBαlevel.After the cells were treated by FEA for 8h,the expression of phosphorylated protein of AKT was extremely significant reduced with 25μg/mL A and 100μg/mL FEA,and significantly reduced AKT phosphorylation protein levels with 50μg/mL FEA treatment;25μg/mL,50μg/mL,100μg/mL FEA treatments significantly decreased p-p38、P-ERK、P-c-jun、c-fos、c-jun and c-myc levels,and significantly promoted the recovery of IκBαlevel and P65 level;the expression level of P-c-fos was significantly reduced with 50μg/mL、100μg/mL FEA treatment.Conclusion:（1）Increase in the level of reactive oxygen species,the related enzyme activities affecting free radical production,the overproduction of inflammatory factors were obtained in 3D4/2 cells infected by PCV2 in vitro,the inflammatory model was successfully established.（2）25μg/mL,50μg/mL,100μg/mL FEA could reduce the release of TNF-α、IL-1β、IL-6、IL-10、COX-1、COX-2、IL-8、IFN-γand MCP-1 and the production of NO and ROS as well as down-regulation of inflammatory mediators such as iNOS,MPO,XOD activities and elevated GSH levels in PCV2 infected 3D4/2 cells,possibly by inhibiting MAPK and PI3K/Akt signaling pathways,NF-κB pathway or AP-1 to play anti-inflammatory effect.