Screening Of Anti-duck Virus Hepatitis Flavonoids Prescriptions And Its Machanisam

There are less antivirual chemical drugs could be used for the veterinary clinic at present.Duck virus hepatitis(DVH)is an acute and rapid transmission disease for yong ducklings.It would cause huge economical damage once the clinical cases of DVH emerged in clinic because no effective anti-DVH drugs have been found so far.Hence,it is of urgence to develop high efficiency anti-DHAV drugs.The flavonoids of traditional Chinese medicine possess antiviral,antioxidantive activities and immunological enhancement effect.We screened out several flavonoids with good anti-DHAV actitvities in vitro to compose twelve flavonoids prescriptions according to the formulating traditional Chinese medicine prescription.The optimal prescription was selected by comparing the anti-DHAV activities of these prescriptions in vitro and in vivo.The mechanism of the optimal prescription was studies by comparing the changes of the evaluation biomarkes of hepatic injury,evaluation indices of oxidantive injury,and the secretion of several cytokines.The antiviral activity in vitro and the stimulative effect for lympcyte proliferation in vitro also were tested as the mechanism of the optimal prescription.The particular assays are divided in to six parts as follows:Experiment 1:The screen of the anti-DHAV flavonoids in vitro This study is designed to screen single flavonoids with good anti-DHAV acvitities in vitro.The maximal safe concetrations of the six kinds of flavonoids and one kind of polyphenol on the duck embryonic hepatocytes(DEHs)were measured by the MTT assay.Two test modes were used to measure the anti-DHAV activities of these seven ingredients.Mode A is pre-adding drugs and mode B is post-adding drugs.The A570 values and the maximal virus inhibitive rates were considered as the indicators of the activites of anti-DHAV.The results showed all these ingredients possess anti-DHAV activities on DEHs,especially propolis flavones(PF),presented the strongest anti-DHAV activity in both of the two test modes.The antivirual activities of polydatin(PD)and apigenin(AG)were relative equilibrium on the two modes,and the activities of silymarin(SM)and Chrysanthemi Indici Flos flavonesWild Chrysanthemum Indici flavones(CF)were stonger in the mode A than the mode B.Experiment 2:The screen of the anti-DHAV flavonoids prescriptions in vitro This study is designed to screen flavonoids prescriptions with good anti-DHAV activities in vitro.The maximal safe concentrtions of the twelve kinds of flavonoids prescriptions on the duck embryonic hepatocytes(DEHs)were measured by the MTT assay.Two test modes were used to measure the anti-DHAV activities of these presciptions.Mode A is pre-adding drugs and mode B is post-adding drugs.The A570 values and the maximal virus inhibitive rates were considered as the indicators of the activites of anti-DHAV.The results showed the prescriptions of AG-EF-PD,AG-PD-PF,AG-SM,BCEN,BL-NF-PD,BL-PD,NF-PD-PF,PD-PF and PF-PR-SM possess anti-DHAV activities on DEHs.While the prescriptions of AG-PD,AG-PF-PR and BL-EFshowed no anti-DHAV activitiy on DEHs.Experiment 3:The screen of the anti-DVH flavonoids prescriptions in vivo This study is designed to screen flavonoids prescriptions with good anti-DVH activities in vivo.Ducklings were intramuscular injected DHAV to created DVH models,and the flavonoids prescriptions with good anti-DHAV activities in vitro were used for treating these ducklings.The survival rate of the ducklings was considered as the indicator of the anti-DVH activities in vivo.The results showed the prescriptions of AG-PD-PF,BCEN,BL-PD,NF-PD-PF and PD-PF increased the survival rates of the ducklings that were infected with DVH.The prescriptions of AG-PD-PF,BCEN,BL-PD,NF-PD-PF and PD-PF possessed anti-DVH activities in vivo,and the BCEN was the best one.Experiment 4:The therapeutic effect of the optimal prescription on DVH This study is designed to explore the therapeutic effect of the optimal prescription on DVH.Ducklings were intramuscular injected DHAV to create DVH models,and the optimal prescription BCEN was used for treating these ducklings.The therapeutic effect of BCEN was studied by comparing the survival rates,evaluation biomarkers of hepatic injury(including the visual pathological changes of the hepar and the evaluation biomarkers of the hepatic injury),the dynamic changes of the inflammatory cytokines(IL-6 and IL-8)and the DHVA gene expressions in blood of the BC,VC and BCEN groups.The results showed BCEN increased the survival rates of the ducklings significantly,improved the level of IL-6,IL-8 and HGF obviously,decreased the DHAV gene expression in blood,and alleviated the hepatic injury.Experiment 5:The anti-oxidative and immunological regulative effects of the optimal prescription This study is designed to explore the anti-oxidative and immunological regulative effects of the optimal prescription.The serum oxidantive injury indices(MDA,iNOS,SOD,CAT and GSH-Px)and the immune function evaluation indices(Ab,INF-?and IL-2)of ducklings were measured to evaluate the anti-oxidative and immunological regulative effects of BCEN in the course of DVH.The correlation analysis among the hepatic injury evaluation indices,oxidative injury evaluation indices and the cytokines was applied to evaluate the comprehensive effect of BCEN through the course of DVH.The results showed BCEN decreased the serum MDA and iNOS levels significantly,and increased the activities of the SOD,CAT and GSH-Px significantly.BCEN promoted the secretion of Ab notably,improve the immune function to anti-DVH.BCEN could alleviate the injury of hepar through the anti-oxidative effect,and it could promote the secretion of various cytokines to mobilize the immune system,which made the response to the invasion of DHAV more quickly,thus improve the protective rate of ducklings.Experiment 6:The anti-DHVA proliferation and lymphocyte proliferation stimulative effects of BCEN in vitro This study is designed to explore the anti-DHVA proliferation and lymphocyte proliferation stimulative effects of BCEN in vitro.The effects of BCEN on DHAV adsorption,replication and releasion were measured by the RT-PCR methods.The MTT assay was applied to assess the stimulative effect for T and B lymphocyte proliferation of BCEN in vitro.The results showed that BCEN could inhibit the releasing of DHAV from DEHs significanlty.BCEN could cooperate with PHA and LSP,or separately stimulate the proliferation of T and B lymphocyte.The stimulative effect for B lymphocyte was stronger than that for the T lymphocyte.