The Study On The Anti-duck Hepatitis A Virus Activities And Autophagic Mechanisms Of Chrysanthemum Indicum Polysaccharide And Codonopsis Pilosula Polysaccharide And Their Phosphorylated Products

Duck viral hepatitis(DVH)prevailed in China is mainly contributed by duck hepatitis A virus type1(DHAV-1),ducklings within three-week old are usually vulnerable.This highly contagious virus would lead to acute symptom and high mortality rate,and results in enormous economic losses in poultry industries.So far there is no effective antiviral drugs to treat DHAV-1 infection in clinic,while the exertion of egg-yolk antibody put the animals in the risk of cross infection,and once failed,greater economic losses would be inevitable.Polysaccharides from Traditional Chinese Medicine(TCM)possess wide bioactivities.Based on the Traditional Chinese Veterinary Medicine(TCVM)theory,the pathogenesis of DVH lies in the evil heat and wind in the liver,and syndrome type of DVH belongs to highheating wind syndrome.Therefore,clearing heat and cooling blood,nourishing yin and stopping phlegm should be the treatment principle.Herb Chrysanthemum indicum possesses various effects in dispelling wind,calming liver,clearing the heat and toxic,has been used to treat hepatitis for a long time.Chrysanthemum indicum polysaccharide(CIPS)is one of the main ingredients in herb Chrysanthemum indicum and thus was chosen as one of the tested drugs.Codonopsis pilosula has effects of invigorating spleen-stomach and replenishing qi and promoting fluid,its main ingredient is Codonopsis pilosula polysaccharide(CPPS),shows good antioxidative and antitumor activities,but no antiviral activity is reported.DVH leads to serious inflammation and acute death,belongs to excessive syndrome.Excessive syndrome should be treated with purgation,and no Qi deficiency was observed,therefore,Qi tonifying drug is not suitable for treating DVH.Thus,CPPS was chosen as the comparison.Meanwhile,researches demonstrated that chemical modification can change the bioactivities of polysaccharides,even give them new bioactivities.In this dissertation,CIPS and CPPS were chosen as the tested polysaccharides,and their anti-DHAV-1 activities and mechanisms were studied.To verify the effect of chemical modification on polysaccharides' bioactivities,sodium trimetaphosphate and sodium tripolyphosphate(STMP-STPP)method was used to prepare phosphorylated Chrysanthemum indicum polysaccharide(pCIPS)and phosphorylated Codonopsis pilosula polysaccharide(pCPPS),and the effects of modification on polysaccharides' antiviral activities,as well as the autophagic mechanisms,were studied and compared.The research content can be divided into following six parts:Experiment I.The phosphorylation modification of CIPS and CPPS and their effects on DHAV-1 replication in vitro.This experiment aimed to research the effects of CIPS,pCIPS,CPPS and pCPPS on DHAV-1 infected duck embryonic hepatocytes(DEHs).STMPSTPP method was used to prepare pCIPS and pCPPS.Phosphate radical content and polysaccharide content were also measured.Meanwhile,infrared spectroscopy analysis was exerted to analyze the structure of CIPS,pCIPS,CPPS and pCPPS.The maximum safe concentration and most effective anti-DHAV-1 concentration of these drugs on DEHs were measured by MTT method,then quantitative polymerase chain reaction(qPCR)was used to research the polysaccharides effects on virus replication.Results showed that the maximum safe concentration of CIPS,pCIPS,CPPS and pCPPS on DEHs was 625 ?g/mL,19.532?g/mL,625 ?g/mL and 39.063 ?g/mL,respectively.CIPS,pCIPS and pCPPS showed antiDHAV-1 activities and their most effective antiviral concentration was 39.063 ?g/mL,19.532?g/mL,19.532 ?g/mL,respectively.CIPS,pCIPS and pCPPS can inhibit DHAV-1 gene replication,the relative virus gene expressions were 0.655,0.217,0.443,respectively,pCIPS showed the best effects.The results suggested that phosphorylated modification enhanced the antiviral activities of polysaccharides.Experiment ?.Therapeutic effects of CIPS and CPPS and their phosphorylated products on DHAV-1 infected ducklings.To observe the therapeutic effects and of CIPS,pCIPS,CPPS and pCPPS on DHAV-1 infected ducklings,and their effects on ducklings'hepatic injury,6-day-old Cherry Valley ducklings were intramuscular injected with 0.2 mL of DHAV-1 except those in blank control(BC)group.The infected ducklings were randomly divided into 4 groups with drugs treatment and 1 group without treatment.In treatment groups,ducklings were dosed by drinking according to following dosage:3 mg each for CIPS and CPPS,2.5 mg each for pCIPS and pCPPS.Infected ducklings were dosed consecutive 3 days,while duckling BC group and virus control(VC)group were fed with water.The death in each group was recorded regularly.The blood was collected to calculate DHAV-1 gene expression level,as well as the levels of alanine transaminase(ALT),aspartate transaminase(AST),total protein(TP),albumin(Alb)and globulin(GLO).Results showed that in CIPS,pCIPS and pCPPS treatment group,DHAV-1 gene expression level decreased significantly,ALT and AST levels decreased while TP,Alb and GLO levels increased,compared with those in VC group.What's more,the death toll was lower than that in VC group.Abovementioned indices in pCIPS treatment group performed better than that those in CIPS group.Those results suggested that CIPS,pCIPS and pCPPS inhibited DHAV-1 replication in ducklings,raised the ducklings' survival rate,alleviated hepatic injury caused by virus infection,pCIPS performed best therapeutic effect.Experiment ?.DHAV-1 infection induced autophagy on DEHs.The experiment aimed to explore the mechanisms of DHAV-1 gene replication in DHEs and provide theoretical basis for researching antiviral mechanisms of polysaccharides.DEHs was infected with DHAV-1,then western blot was used to observe expression levels of autophagy related protein light chain 3(LC3)II and ubiquitin-binding protein p62(p62).At the same time,EGFP labeled LC3 expressing plasmid was transfected into DEHs to observe the localization of LC3-II by using confocal microscope.Then autophagy inducer rapamycin and inhibitor wortmannin were introduced to DEHs which subsequently infected with DHAV-1,qPCR was used to analyze the effect of autophagy on virus gene expression level.Meanwhile,virus was inactivated by ultraviolet(UV)irradiation to see if virus without the capacity of gene replication can still induce autophagy on DEHs.Results showed the increase in LC3-II and p62 expression levels after DHAV-1 infection in DEHs,suggested that virus infection facilitated autophagosomes formation and impaired the degradation of autophagosomes.Rapamycin treatment promoted virus gene replication while wortmannin treatment reduced virus gene expression level.UV-inactivated virus failed to induce autophagy in DEHs.Those results indicated that the initiation of autophagy relies on virus gene replication,and autophagy in turn can facilitate virus gene replication.To further explore how the autophagy is induced by DHAV-1,western blot was used to analyze the expression of phosphoinositide 3-kinase Class III(PI3KC3)and its product phosphatidylinositol 3-phosphate(PI3P).Results showed that DHAV-1 infection upregulated the expression of PI3KC3 and raised the concentration of PI3P in DEHs,and thus promoted the formation of autophagosomes.Experiment ?.Effects of CIPS and CPPS and their phosphorylated products on DHAV-1 induced autophagy on DEHs.Based on the results from Experiment III,the effects of polysaccharides on autophagy was studied in this experiment.DEHs were infected with DHAV-1,followed by treating with CIPS,pCIPS,CPPS and pCPPS.Western blot was used to analyzed LC3-II protein expression level.Result showed LC3-II expression increased in DHAV-1 infected DEHs,and decreased by treating with CIPS,pCIPS and pCPPS.This indicated that these three polysaccharides inhibited virus induced autophagosomes formation.To further study polysaccharides' effects on autophagy,rapamycin was used to induce autophagy on DEHs,and then CIPS,pCIPS,CPPS and pCPPS were added to cells to detect the LC3 localization,LC3-II expression level and p62 expression level.Results showed that rapamycin treatment led to the punctate gathering of LC3 protein,the increase of LC3-II and the decrease of p62,suggesting that autophagy was induced.CIPS,pCIPS and pCPPS treatment reduced the punctate distribution of LC3 in DEHs cytoplasm,decreased the LC3II expression level and increased the p62 expression level.This indicated that these polysaccharides worked well in inhibiting rapamycin induced autophagy.PI3KC3 expression level was further evaluated by using western blot and results showed that CIPS,pCIPS and pCPPS treatment downregulated PI3KC3 expression level and PI3P concentration in DEHs,phosphorylated polysaccharides performed better than polysaccharides without modification.The results suggested that antiviral polysaccharides,especially pCIPS,inhibited autophagosomes formation by downregulating PI3KC3 activity.CPPS showed upregulation effects on PI3KC3 expression and PI3P concentration.Experiment V.Effects of CIPS and CPPS and their phosphorylated products on autophagy in DHAV-1 infected ducklings' tissues.The experiment aimed to further confirm the effects of polysaccharides on autophagy by in vivo experiments.Six-day-old ducklings were intramuscular injected with DHAV-1(0.2 mL for each)and rapamycin(2mg per kilogram weight,two hours before and after being dosed)to induce autophagy,respectively,followed by orally dosing drugs according to following dosage:3 mg each for CIPS and CPPS,2.5 mg each for pCIPS and pCPPS.Blank control group was set at the same time.Six hours later,the liver,kidney,brain,and spleen were collected,and western blot was conducted to detect the LC3-II expression levels in those tissues.Enzyme-linked immunosorbent assay(ELISA)was used to measure the PI3P concentrations in liver,kidney,brain,spleen and blood.Results showed that DHAV-1 and rapamycin induced autophagy in ducklings' liver,spleen and kidney,while CIPS,pCIPS and pCPPS treatment significantly inhibited autophagy initiation in those three tissues.Autophagy initiation didn't be detected in brain.ELISA results showed that DHAV-1 infection raised the PI3P concentration in liver,spleen,kidney and blood,and PI3P concentration decreased after CIPS,pCIPS,pCPPS treatment.These results suggested that CIPS,pCIPS and pCPPS could inhibit autophagy in tissues under similar mechanisms with those on DEHs.Experiment VI.Effects of CIPS and CPPS and their phosphorylated products on IFN? signaling pathway.Autophagy is closely related to host innate immunity.The tested polysaccharides inhibited autophagy initiation and inspired by this phenomenon,the effects of the polysaccharides on IFN-? signaling pathway were explored in this experiment.qPCR was used to measure the IFN-? gene expression levels in DHAV-1 infected and polysaccharides treated DEHs,and ELISA method was used to detect the IFN-?concentration.Results showed that DHAV-1 facilitated the IFN-? transcription.However,the production of IFN-? was impaired by virus.Polysaccharides treatment didn't show impact on IFN-? concentration.Proteins in IFN-? downstream signaling pathway were further analyzed by western blot and results showed that DHAV-1 can also inhibit the phosphorylation of Signal transducer and activator of transcription 1(STAT1),inhibit the transcription of IFN-stimulated genes(ISGs).CIPS,pCIPS and pCPPS treatment upregulated the phosphorylation of STAT1 and further promoted the transcription of several ISGs like protein kinase R(PKR)and 2'-5'-oligoadenylate synthetase(OAS).pCIPS showed the best effect in facilitating ISGs transcription.Among the three polysaccharides,pCIPS and pCPPS also upregulated the expression level of STAT1.The results in this study suggested that phosphorylated modification improved CIPS and CPPS's activity of inhibiting DHAV-1 gene replication.CIPS,pCIPS and pCPPS treatment raised the survival rate of DHAV-1 infected ducklings and alleviated hepatic injury caused by virus,pCIPS showed the best therapeutic effect.Viral gene replication is one of the most important parts in virus life cycle,mechanism research found that DHAV-1 could induce autophagosomes formation and utilize autophagosomes to facilitated their own gene replication,while CIPS,pCIPS and pCPPS inhibited autophagosomes formation by decreasing cellular PI3P concentration and thus play a role in inhibiting virus gene replication.In vivo experiment also suggested that CIPS,pCIPS and pCPPS inhibited autophagy initiation in liver,spleen and kidney.Autophagy can inhibit host innate immunity.Research found that DHAV-1 infection induced IFN-? transcription but impaired the production of IFN-?.All polysaccharides failed to promote the transcription of IFN-?,but CIPS,pCIPS and pCPPS,especially pCIPS,facilitated the phosphorylation of STAT1,the downstream protein of IFN-?,and further upregulated the transcription of ISGs like PKR and OAS,which can directly play a role in antiviral process.