| Baicalin is the main effective component of traditional Chinese medicine Scutellaria baicalensis.Many studies had shown that baicalin had antibacterial,antioxidant,antiviral,mitochondrial protection and other functions.But its solubility and absorption were poor,and its bioavailability was low,which posed a great challenge to clinical treatment.Physical and chemical modification can change the structure and function of natural medicine ingredients,which make it possible for the clinical.The aim of this study is to explore the structural modification of baicalin to improve its bioavailability,and to discuss the antiviral effect and the pathway of baicalin and its modification.To obtain phosphorylated baicalin,the polyphosphate method was used to screen the optimal modification conditions in three conditions: reactive temperature,p H and reactive time.Then we studied the anti-DHAV-1activities and the mechanism of protecting hepatocytes and mitochondria about the two medicines in vitro and vivo.The differences between the modified and unmodified medicine were compared.The test was divided into four parts.Test I: Phosphorylation medification of baicalin and its anti-DHAV-1 activities on DEHsUsing polyphosphate method,orthogonal experiments were designed to screen the optimal modificative conditions by three factors: reactive time,reactive temperature and p H.The anti-DHAV-1 activities of BA and pBA on DEHs in vitro were detected by MTT method.The results showed that there was no significant difference in the total amount of product between BA and pBA.The optimal phosphorylation conditions were 80℃,8 h and p H 9.5.The surface structure of pBA was changed compare with BA: the skeleton structure of BA was retained,and the organic phosphate,phosphate ester and pyrophosphate bond were added.The highest virus inhibition rate was 60.16% in BA group and 107.11% in pBA group,respectively.BA and pBA showed virus inhibition at 36 h and 24 h after virus infection respectively.The antiviral effect of pBA appeared earlierly.Test II: Study on the antioxidative and therapeutic effect of phosphorylated baicalin against DVHThis experiment aims to study the effects of BA and pBA anti-DHAV-1 and their antioxidative stress.244 Cherry Valley ducks were randomly divided into 4 groups: blank control group(BC group),virus control group(VC group),BA treatment group(BA group)and pBA treatment group(pBA group).The model of DVH was established by inoculating DHAV-1 in VC,BA and pBA groups.At 1 h after challenged,BA group and pBA group were oral administrated with BA and pBA with the dosage of 5 mg/d and 3 mg/d for successive 3days,respectively.At 4 h,8 h and 54 h after challenged,blood samples were taken from 5feathers in each group randomly to determine the biochemical indexes related to serum liver function.Liver tissues were taken and HE staining was used to observe the histopathological changes.The activities of antioxidant enzymes(including SOD,CAT,i NOS and GPX)and the level of lipid peroxide(MDA)were detected in liver tissue.The results showed that there was no significant change in liver pathological changes,serum biochemical indexes,liver antioxidant enzymes and lipid peroxide level in each group at 4 h after chanllenged.At 8 h after chanlleged,swelling and petechia of liver appeared.The levels of serum ALT and AST increased and and the level of serum TG decreased significantly in each group,respectively.Characteristic pathological changes and death peak at 24 h appeared.The ducks in pBA group,BA group and VC group stopped dying at 24 h,36 h and 108 h,respectively.The liver pathological changes in VC group were obvious at 54 h after challenged.There were significant differences in VC group in the serum biochemical indexes,liver antioxidant enzymes and lipid peroxidation level.The BA group and pBA group were significantly improved.Hence,ducks died rapidly due to DHAV-1,resulting in sever liver injury and oxidative stress.BA and pBA could reduce the mortality of ducks and alleviate the liver dysfuntion and lipid metabolism caused by oxidative stress.Test III: The protective effect of phosphorylated baicalin on mitochondrial injury induced by DHAV-1 infectionIn order to explore the protective effect of pBA on mitochondrial injury of hepatocytes induced by DHAV-1 infection,the vivo and vitro experiments were conducted.244 six-dayold pre-immune Cherry Valley ducks were randomly divided into 4 groups: BC group,VC group,BA group and pBA group.Ducklings in VC group,BA group and pBA group were intramuscularly injected with 0.2 m L of DHAV-1 virus solution,while in the BC group was injected with the same volume of saline.After challenged,ducklings in BA group and pBA group were administrated with BA and pBA at 5 mg/feather and 3 mg/feather respectively by drinking water daily for 3 days.At 4 h,8 h and 54 h after challenged,liver tissues were taken from 5 feathers in each group randomly to observe the changes of mitochondrial ultrastructure and detect the content of ATP,the activity of mitochondrial oxidative stress(including SOD,CAT,i NOS,GPX)and the level of lipid peroxidation(MDA).DEHs were prepared and divided into 4 groups: BC group,VC group,BA group and pBA group.DHAV-1 virus solution was used for pretreatment in VC,BA and pBA groups.BA solution with the concentration of 19.53 μg/m L was added in BA group and pBA solution with the concentration of 125 μg/m L was added in pBA group.The BC group and VC group were added with the same volume of MM.Then mitochondria were extracted to detect the activities of MMP,SDH,COX and the oxidative damage indexes including MDA,SOD and GPX at 12 h,24h and 36 h after challenged.The results indicated that due to the infection of DHAV-1,the morphology of mitochondria was destroyed and the content of ATP in the liver of ducks decreased significantly,as well as severe oxidative stress and mitochondrial dysfunction appeared.Based on the antioxidant effects,BA and pBA alleviated mitochondrial damage and oxidative stress caused by DHAV-1,and increased ATP content.Meanwhile,MMP,SOD,GPX,SDH and COX activities were decreased caused by DHAV-1 infection.The MDA content was increased.Baicalin and its phosphorylated modification reversed the above indexes.Baicalin regulated mitochondrial dysfuntion induced by DHAV-1 through antioxidative effect.Test IV: The effect of phosphorylated baicalin on Nrf2/ARE signaling pathwayTo investigate the mechanism of mitochondrial damage induced by DHAV-1 and the protect of phosphorylation medification of baicalin,in DEHs,the Nrf2/ARE signaling transductive pathway related genes(including SOD,GPX-1,NQO1,HO-1 and Nrf2)were detected by real-time q PCR and the levels of related proteins(including SOD,GPX and Nrf2)were compared by Western blot.Meanwhile,the protein expression of Nrf2 in liver tissue of ducklings infected with DHAV-1 was detected.The results suggested that DHAV-1 infection reduced the levels of relative m RNA expressions,including SOD,GPX-1,NQO1,HO-1 and Nrf2 in DEHs.The relative protein expressions of SOD,GPX and Nrf2 were also reduced.The expression levels of these genes and proteins could be regulated with the administration of BA and its modification,and the effect of modification was better.However,the expression of Nrf2 was decreased in the pBA group.BA and pBA protected mitochondria by activating Nrf2/ARE signaling pathway in DEHs.The effect of pBA on the activation of Nrf2 in the liver tissue of ducklings was not obvious,which may be related to the dosage of pBA.In conculusion,pBA was obtained by phosphorylation of BA.Compared with BA,pBA has stronger alleviating effect on oxidative stress and mitochondrial dysfunction in duck liver caused by DHAV-1 infection.At the same time,it was found that pBA may be regulated by activating Nrf2/ARE signaling pathway to activate antioxidant pathway. |
