| Porcine circovirus type 2(PCV2)is the primary causative agent of porcine cireovirus-associated disease(PCVAI)),which includes PNDS,post-weaning multi-systeniic wasting syndrome(PMWS),respiratory disease,and reproduction disorders.It causes huge economic losses on global swine industry.Astragalus polysaccharide(APS),as one kind of biological macromolecule extracted from Astragalus,has antioxidant,anti-inflammation and antiviral activities.However,it is unknown whether APS affects PCV2 replication and its underlying mechanism.This thesis mainly studied and discussed for the effect of APS on PCV2 replication in vivo and in vitro.We tried to explain the possible mechanism from oxidative stress,NF-Kappa 13 and endoplasmic reticulum stress about the inhibition of APS on PCV2infection,which provided theoretical guidance for the prevention and control of PCVD,and the application of APS in pig production.Experiment 1.Effects of APS on PCV2 infection in vivo and vitroTo study the effects of astragalus polysaccharide(APS)on porcine circovirus virus type 2(PCV2)replication,we respectively applied PK15 cells and mice to simulate PCV2 infection in vitro and in vivo.In vitro,PK15 cells were treated with APS and PCV2 infection simultaneous,or added APS before PCV2 infection.In vivo,50 2-week-old mice were randomLy divided into five groups,including Con,PCV2,100 mg APS/kg +PCV2,200 mg APS/kg +PCV2 and 400 mg APS/kg+ PCV2.During the APS group after treatment with APS for 14d,and then injected each mice with 1000 TCID50 PCV2.The trial lasted 28 days.In vitro,our results showed that adding APS before PCV2 infection decreased significantly PCV2 DNA copies,the number of infected cells.In vivo,our results showed that comparing with PCV2 infection control group,APS groups weakened pathological injury in mice lung,spleen and liver;significantly reduced the number of PCV2 DNA copy and Cap protein expression in lung tissues;and immunohistochemistry showed that APS decreased PCV2 infection.Experiment 2.APS inhibits PCV2 replication by inhibiting oxidative stress and blocking NF-?B PathwayAPS inhibits PCV2 replicatioa In order to study the mechanism of APS inhibiting PCV2 replication,we measured the oxidative stress indices,NF-? B activation,and the effects of the oxidative stress activator BSO,NF-?B activator LPS and inhibitor BAY 11-7082 on APS-inhibited PCV2 replication.The results showed that adding APS before PCV2 infection,MDA level,ROS level and NF-?B activation in PK15 cells and increased significantly GSH contents and SOD activity compared to control without APS.Oxidative stress induced by BSO could eliminate the effect of PCV2 replication inhibition by APS.LPS,as a NF-? B activator,could attenuate the effect of PCV2 replication inhibition by APS.In conclusion,APS inhibits PCV2 replication by decreasing oxidative stress and the activation of NF-? B signaling pathway.Experiment 3.APS inhibits PCV2 infection by inhibiting endoplasmic reticulum stressAPS inhibits PCV2 infection.In order to future study the mechanism of APS inhibiting PCV2 replication,we measured the oxidative stress indices,endoplasmic reticulun stress(ERS)related GRP78 gene and protein expression,apoptosis gene(GADD153/CHOP)and protein CHOP expression induced by ERS.Then,we measured the effects of ERS activator TM on APS-inhibited PCV2 replication.The results showed that comparing with PCV2 control group,TAOC activity,GRP78 gene and protein expression,GADDI 53/CHOP gene and protein expression decreased significantly in APS treatment groups.Furthermore,our results showed that TM attenuated the effect of APS on PCV2 infection in mice.In addition,we carried out the corresponding tests in vitro,the results of which has the same trend to that in vivo.These results indicated that APS suppressed PCV2 infection by inhibiting endoplasmic reticulum stress. |
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