Intervention Effect Of Flavonoids From Polygonum Hydropiper L. On Inflammatory Response Induced By Pseudorabies Virus Infection In Swine Alveolar Macrophages

The part of ethyl acetate of Flavonoids of Polygonum hydropiper L.（FEA）is one of the extracts of the active ingredient flavonoids of Chinese herbal medicine.Flavonoids have many biological effects such as anti-inflammatory,anti-viral,anti-oxidation and immune regulation.Porcine pseudorabies virus（PRV）is the causative agent of pseudorabies（PR）in pigs.It can infect pigs of all ages,mainly causing respiratory diseases,inflammation,reproductive disorders and respiratory syndrome in pigs.It even interferes with the body’s immune regulation system defense function.In this study,we investigated the effects of FEA on the inflammatory factor levels of immune cells（porcine alveolar macrophages）induced by PRV infection in vitro and the regulation of gene expression and protein expression in inflammation-related pathways.The purpose of this study is to explore the development and utilization of polygonum hydropiper L.and the prevention and adjuvant treatment of FEA for viral infectious diseases,and provide reference for the research of natural drugs in the prevention and control of animal viral diseases.Methods:（1）PK15 cells were used to amplify PRV,and virus TCID₅₀was calculated.3D4/2 cells were infected with PRV(10^-2～10^-6)with different dilution gradients for 2 h,then the supernatant was discarded,and new culture medium was added to continue the culture.Cultured for 4 h,8 h,12 h,24 h,48 h,samples of cells or supernatant were collected for determining levels of TNF-α,IL-1β,IL-6,IL-8,IL-10,IFN-γ,MCP-1,ROS,GSHand NO,and the activities of iNOS,XOD,MPO,COX-1and COX-2,to find the PRV infection concentration,infection time on 3D4/2 cells according to ROS levels and inflammatory factors and enzyme activities.In order to establishing a model of immune cell inflammation in vitro.（2）FEA in the concentration of 100μg/mL,50μg/mL,and 25μg/mL was applied to 3D4/2 cells infected with 10^-3PRV,and rutin standard control group and LPS positive control group were set at the same time.Samples were collected at 8 h,12 h,24 h,and 48 h to determine the values of NO,ROS,GSH,TNF-α,IFN-γ,IL-1β,IL-6,IL-8,IL-10and MCP-1,and activities of COX-1,COX-2 XOD,MPO and iNOS,to investigate the intervention effect of FEA on the inflammatory response induced by PRV-infected immune cells（3D4/2 cells）.（3）The 3D4/2 cells were infected by10^-3PRV and were treated with FEA at concentrations of 100μg/mL,50μg/mL,and 25μg/mL.Real-time PCR analysis of mRNA expression of TNF-α,IFN-γ,IL-1,IL-6,IL-8,iNOS,COX-1,COX-2,c-Jun and c-fos at the level of gene transcription were carried out.Western blot was used to detect the phosphorylation of p65,p38,AKT,ERK1/2,c-Jun proteins in NF-κB and MAPKs pathways.Results:（1）The TCID₅₀of PRV was determined to be 10^-7.5/0.1 mL.PRV infection significantly increased the levels of IL-1β,IL-6,IL-8,IL-10,MCP-1,TNF-αIFN-γand ROS,and the activities of COX-1,COX-2,XOD,iNOS and MPO,decreased intracellular GSH levels in 3D4/2 cells.（2）,FEA treatment at100μg/mL,50μg/mL,and 25μg/mL decreased theproduction of IL-1β,IL-6,IL-8,IL-10,MCP-1,TNF-α,IFN-γ,COX-1,COX-2,XOD,iNOS,MPO and ROS and increased the levels of GSH in PRV infected 3D4/2 cells in different time.（3）FEA treatment at 100μg/mL,50μg/mL,25μg/m inhibited mRNA expression levels of TNF-α,IFN-γ,IL-1,IL-6,IL-8,iNOS,COX-1,COX-2,c-jun,c-fos after virus infection.The values of TNF-α、IL-1、IL-6、iNOS、COX-1、COX-2、c-jun、c-fos were statistically decreased when compared with the PRV group.（4）Compared with the blank control group,PRV infection significantly increased the phosphorylation levels of p65,p38,ERK1/2,AKT,and c-jun proteins in 3D4/2 cells..FEA treatment at 100μg/mL,50μg/mL,and 25μg/mL significantly increased down-regulated the phosphorylation levels of p65,p38,ERK1/2,AKT,and c-jun proteins in PRV-infected cells,.The difference between the drug groups and the PRV group was statistically significant.Conclusion:Infection of 3D4/2 cells by PRV in vitro can significantly induce the expression of ROS and inflammatory cytokines in immune cells,and caused the inflammatory reaction.FEA effectively interfered with the inflammatory response of 3D4/2 cells induced by PRV infection in vitro,down-regulated the expression levels of inflammatory cytokines and ROS,reduced the increase of inflammatory cytokine mRNA levels induced by PRV infection,and the phosphorylated expression of p65,p38,ERK1/2,AKT,and c-jun proteins in 3D4/2 cells in an inflammatory state was inhibited by FEA treatments.The results indicated that PRV infection could induce inflammatory response in 3D4/2 cells.FEA played a regulatory effect on inflammation induced by PRV infection,and its regulatory mechanism may be related to signaling pathways such as NF-κB and MAPKs.