Comparison Of The Treatment Effects Of Several Traditional Chinese Medicine Ingredients And Their Modified Products On Duck Virus Hepatitis And Their Mechanisms Study

In China,duck virus hepatitis(DVH),which can result in the widespread duckling death,is mainly caused by the duck hepatitis A virus type 1(DHAV-1).To date,licensed drugs except yolk antibody in clinic cannot treat DVH,thus the financial loss of duck breeding is disastrous.The research and development of anti-DHAV-1 drugs are therefore significant.In recent years,Chinese medicinal ingredient has become an important part of drug research and development because of the wide range of biological activities and stable sources.Polyporus polysaccharides(PPS),Astragalus polysaccharides(APS),Bush sophora root polysaccharides(BSRPS),and baicalin(BA)possess antiviral and liver protective activities.So,the anti-DHAV-1 effects of PPS,APS,BSRPS,and BA were tested in present work.Furthermore,a proper modification can enhance the biological activity and solubility of Chinese medicinal ingredient.In this study,the PPS,APS,and BSRPS were modified by the chlorosulfonic acid-pyridine method,and the sulfates sulfated Polyporus polysaccharides(sPPS),sulfated Astragalus polysaccharides(APS),and sulfated Bush sophora root polysaccharides(BSRPS)were respectively gained.At the same time,baicalin phospholipid complex(BAPC)was prepared by the reaction of BA and soy phospholipids.Then the anti-DHAV-1 abilities of APS,sAPS,BSRPS,sBSRPS,BA,and BAPC were ascertained by the in vitro tests.Meanwhile,the curative effects of these drugs on ducklings which infected by DHAV-1 as well as the anti-oxidative and immuno-enhancing effects were determined,which showed BSRPS,sBSRPS,BA,and BAPC were able to cure DVH.Afterwards,the key roles of anti-oxidative and immuno-enhancing in the curative effects of BSRPS,sBSRPS,BA,and BAPC were analyzed.Finally,the anti-DHAV-1 molecular mechanisms were studied.The particular assays are divided in to seven parts as follows:Experiment ?:The effects of four traditional Chinese medicine ingredients and their modified products on DEHs against DHAV-1 The aim of this assay was to detect the anti-DHAV-1 effects of PPS,sPPS,APS,sAPS,BSRPS,sBSRPS,BA,and BAPC in vitro.PPS,APS,and BSRPS were modified by the chlorosulfonic acid-pyridine method.At the same time,BAPC was prepared by the interaction of baicalin and soy phospholipids.Then the maximum safe concentrations of PPS,sPPS,APS,sAPS,BSRPS,sBSRPS,BA,and BAPC on DEHs were detected by the MTT method.After that,the inhibition effects of PPS,sPPS,APS,sAPS,BSRPS,sBSRPS,BA,and BAPC against DHAV-1 infecting DEHs were tested and the virus inhibition rates were calculated.The results showed the maximum safe concentrations of PPS,sPPS,APS,sAPS,BSRPS,sBSRPS,BA,and BAPC were 250 ?g·mL-1,125 ?g·mL-1,625?g·mL-1,7.813 ?g·mL-1,2000 ?g·mL-1,7.813?g·mL-1,156.25 ?g·mL-1,and 31.25 ?g·mL-1,respectively.Besides PPS,all the test drugs exhibited anti-DHAV-1 effects.APS,sAPS,BSRPS,sBSRPS,BA,and BAPC exhibited anti-DHAV-1 effects at multiple concentrations,however sPPS only showed anti-DHAV-1 effect at 125?g·mL-1.The inhibition abilities of APS,BSRPS,BA and their modified products on DHAV-1 infecting DEHs were siginificantly higher than those of PPS and sPPS.Experiment ?:The anti-DHAV-1 reproduction effects of APS,sAPS,BSRPS,sBSRPS,BA,and BAPC in vitro The aim of this assay was to determine the inhibition roles of APS,sAPS,BSRPS,sBSRPS,BA,and BAPC in the DHAV-1 reproduction.Both post-adding drug and pre-adding drug modes were used to detect the influences of drugs on the DHAV-1 adsorption by Realtime-PCR method.At the same time,the influences of drugs on the DHAV-1 replication and release were also tested by Realtime-PCR method.The results showed both APS and sAPS were able to inhibit the virus replication and sAPS did better.Both BSRPS and sBSPRS inhibited DHAV-1 replication while sBSRPS also inhibited DHAV-1 release.BA could inhibit DHAV-1 replication and release;BAPC could inhibit DHAV-1 adsorption,replication and release.Experiment ?:The effects of APS,sAPS,BSRPS,sBSRPS,BA,and BAPC on ducklings against DHAV-1 infecting The aim of this assay was to ascertain the anti-DHAV-1 effects in vivo.Ducklings at three days old were intramuscular injected with DHAV-1.Then the ducklings were treated by APS,sAPS,BSRPS,sBSRPS,BA,and BAPC at the dosage of 3,3,4,2,3,and 3 mg per duckling one day,respectively.And the death of ducklings at the 12nd,the 24th,the 36th,the 48th,the 72nd,the 96th,the 120th,and the 144th h were recorded.Afterwards,the DHAV-1 gene expression in the blood was detected.And the levels of ALT,AST,ALP,LDH,TP,ALB and GLO were also determined.The results showed APS,sAPS,BSRPS,sBSRPS,BA,and BAPC were able to inhibit the DHAV-1 reproduction in ducklings.BSRPS,sBSRPS,BA,and BAPC reduced the mortality rates of ducklings however APS and sAPS did not.BSRPS,sBSRPS,BA,and BAPC alleviated the liver injuries of ducklings.APS and sAPS also alleviated the liver injuries of ducklings to some degree while the effects were not ideal.Experiment ?:The effects of APS,sAPS,BSRPS,sBSRPS,BA,and BAPC on anti-oxidative and immuno-enhancing of ducklings infected by DHAV-1 The aim of this assay was to study the anti-oxidative and immuno-enhancing effects of APS,sAPS,BSRPS,sBSRPS,BA,and BAPC in the treatments of ducklings which were infected by DHAV-1.Ducklings at three days old were intramuscular injected with DHAV-1.Then the ducklings were treated by APS,sAPS,BSRPS,sBSRPS,BA,and BAPC at the dosage of 3,3,4,2,3,and 3 mg per duckling one day,respectively.After that,the activities of SOD,CAT,and GSH-Px as well as the MDA,IL-2,and IFN-? were tested.The results showed DHAV-1 decreased the activities of SOD,CAT,and GSH-Px while increased the MDA content in ducklings.APS could enhance the SOD activity and reduce the MDA content;sAPS could enhance the CAT activity and reduce the MDA content;BSRPS,sBSRPS,BA,and BAPC could enhance the SOD,CAT,and GSH-Px activities and reduce the MDA content.APS and sAPS could not promoted the IL-2 and IFN-? secretion.But BSRPS,sBSRPS,BA,and BAPC significantly promoted the IL-2 and IFN-? secretion.Furthermore,the immuno-enhancing effects of sBSRPS and BAPC were respectively higher than those of BSRPS and BA.In sum,the anti-oxidatie and immuno-enhancing effects of BSRPS,sBSRPS,BA,and BAPC were higher than those of APS and sAPS.Experiment ?:The verification of the anti-oxidative and imuno-enhancing effects of BSRPS,sBSRPS,BA,and BAPC The aim of this assay was to explore the key roles of the anti-oxidative and immuno-enhancing effects of BSRPS,sBSRPS,BA,and BAPC in the treatments of ducklings which were infected by DHAV-1.Ducklings were intramuscular injected with DHAV-1 and treated by BSRPS,sBSRPS,BA,and BAPC at the same time intervened by hinokitiol and FK506.The levels of GSH-Px,SOD,CAT,MDA,IL-2 and IFN-? were detected and the mortality rates of ducklings in each group were calculated.The results showed hinokitiol significantly reduced the activities of GSH-Px,SOD,and CAT as well as enhanced the MDA contents.Under the pro-oxidation,the curative effects of BSRPS and BA were blocked,which indicated the anti-oxidative effects of BSRPS and BA were important to the anti-DHAV-1 abilities.FK506 significantly reduced the levels of IL-2 and IFN-y.Under the immunosuppression,the curative effects of BSRPS,sBSRPS,BA,and BAPC were blocked,which indicated the immuno-enhancing effects of BSRPS,sBSRPS,BA,and BAPC were important to the anti-DHAV-1 abilities.Experiment ?:The inhibition effects and mechanisms of BSRPS,sBSRPS,BA,and BAPC on DHAV-1 gene synthesis The aim of this assay was to study the influences and mechanisms of BSRPS,sBSRPS,BA,and BAPC on DHAV-1 gene synthesis.The influences of BSRPS,sBSRPS,BA,and BAPC on DHAV-1 gene synthesis were detected after the inhibition of protein translation by the cycloheximide.The results showed BSRPS,sBSRPS,and BAPC were able to inhibit the DHAV-1 gene synthesis while BA could not.Then the influences of BSRPS,sBSRPS,and BAPC on 3D protein stabilities were detected by Western Blot.The results showed BAPC reduced the 3D protein stability however BSRPS and sBSRPS did not.Hsp70 was important to the DHAV-1 gene synthesis which was certified by RNA interference.The influences of BSRPS,sBSRPS,and BAPC' on Hsp70 protein expressions were determined by Western Blot.And the results showed BSRPS,sBSRPS,and BAPC inhibited DHAV-1 gene synthesis via suppressing the Hsp70 protein expressions.Experiment ?:The inhibition effects and mechanisms of BSRPS,sBSRPS,BA,and BAPC on DHAV-1 protein translation The aim of this assay was to study the influences and mechanisms of BSRPS,sBSRPS,BA,and BAPC on DHAV-1 protein translation.The influences of BSRPS,sBSRPS,BA,and BAPC on DHAV-1 protein translation were analyzed with the VP1 protein expressions after 5 hours for DHAV-1 entering DHEs by Western Blot.In the meantime,the influences of BSRPS,sBSRPS,BA,and BAPC on DHAV-1 IRES activities were detected by Dual-Luciferase Reporter Assay System.The results showed BSRPS,sBSRPS,BA,and BAPC inhibited DHAV-1 protein translation at the same time inhibited DHAV-1 IRES activities.It indicated BSRPS,sBSRPS,BA,and BAPC inhibited DHAV-1 protein translation via suppressing the DHAV-1 IRES activities.The results of this study showed that APS,sAPS,BSRPS,sBSRPS,BA,and BAPC exhibited inhibition abilities on DHAV-1 reproduction.However,APS and sAPS did not decrease the mortality rates of ducklings which were infected with DHAV-1.This might be that the anti-oxidative and immuno-enhancing abilities of APS and sAPS in the course of therapy were weak.BSRPS,sBSRPS,BA,and BAPC significantly decrease the mortality rates of ducklings which were infected with DHAV-1.And in the course of therapy,they exhibited strong anti-oxidative and immuno-enhancing effects.Additionally,the results of intervening tests showed the anti-oxidative and immuno-enhancing abilities of BSRPS and BA were important to their therapy,and the immuno-enhancing abilities of sBSRPS and BAPC were important to their therapy.RNA synthesis and protein translation were two important links of DHAV-1 replication.BSRPS and sBSRPS inhibited DHAV-1 gene synthesis via inhibiting Hsp70 protein expression.BAPC inhibited DHAV-1 gene synthesis via inhibiting Hsp70 protein expression and 3D protein stability.Furthermore,BSRPS,sBSRPS,BA and BAPC also inhibited DHAV-1 protein translation via inhiting DHAV-1 IRES acitivities.