Study On Phosphorylated Bush Sophora Root Polysaccharide Anti-Duck Viral Hepatitis And Hepatocyte Mitochondrial Damage

Bush Sophora Root polysaccharides(BSRPS)is extracted from the Bush Sophora Root,which is a traditional Chinese herbal medicine.Bush Sophora Root is the dry root and rhizome of the leguminous plant locust,which mainly produced in southwest Guangxi province of China.It is bitter and cold in nature,bitter in reducing and releasing heat,and has the effects of clearing heat,detoxification,detumescence and being beneficial to the pharynx.Modern pharmacologic studies have shown that BSRPS have the ability to anti-inflammatory and antimicrobial,anti-viral,antitumor,antioxidant and regulate immunity.However,in clinical practice,the BSRPS has the defects of poor water solubility,high effective concentration and strong herbal odor,which limits its wide application in clinical practice.In the previous studies,we used sulfuric modification to effectively improve the antiviral biological activity of BSRPS.However,the sulfation modification reaction is more intense,the conditions are demanding,and the process is dangerous.In order to effectively and safely improve the biological activity of BSRPS,improve its physical and chemical properties,and reduce the drug concentration,etc.,so that it can be widely used in clinic.In an attempt to modify BSRPS by polyphosphate method,Phosphated Bush Sophora Root polysaccharides(pBSRPS)were obtained.And the activity of BSRPS and pBSRPS against Duck hepatitis virus type I(DHAV-1)on duck embryonic hepatocytes(DEHs)in vitro and the mechanism of antiviral action in vitro were also investigated.Then the protective effects of the two drugs before and after modification on duck viral hepatitis liver injury were compared.Finally,the protective effects of BSRPS and pBSRPS on DHAV-1-induced mitochondrial damage in duck embryonic hepatocytes and liver tissues were compared.The aim of this study was to explore new ways of anti-DHAV-1 action and to develop new drugs.This experiment is divided into the following parts:Test I Phosphorylation modification of BSRPS and its effects on the ability of DHAV-1 infected with Duck Embryo Hepatocytes in vitroIn this experiment,crude polysaccharides from Bush Sophora Root were extracted by Decoction and alcohol sedimentation method,purified by sevega method to obtain BSRPS,phosphorylated by polyphosphate method,and then identified by Fourier infrared spectroscopy.The maximum safe concentration and the most effective antiviral concentration of BSRPS and its modifiers on Duck Embryonic Hepatocytes(DEHs)were determined.The results showed that the polysaccharide content of the prepared BSRPS was 89.56%by phenol sulfuric acid method.The polysaccharide content of phosphorylation modifier pBSRPS was 60.42%.The phosphate content of pBSRPS was 37.43%by Tris-ammonium molybdate colorimetric method.Infrared spectroscopy showed that the typical polysaccharide structure with characteristic absorption peaks of-OH,C-H,COO and C-O-C,the stretching vibration peaks of P=O,P-O-C,P-O-R were also detected,indicating that the phosphorylation modification was successful.The safe concentrations of BSRPS and pBSRPS on duck embryo liver cells were 2000 μg/mL and 15.63 μg/mL respectively,and BSRPS and pBSRPS had the highest viral inhibition rates on DHAV-1 infected duck embryo liver cells at 500 μg/mL and 15.6 μg/mL with 53.66%and 57.87%respectively,the latter was a little bit higher than the former.Test II Effects of pBSRPS on the proliferation ability of DHAV-1 on DEHsIn order to explore the mechanism of BSRPS and pBSRPS in resisting DHAV-1 infection on DEHs in vitro,DEHs were treated by adding virus first and then drug.Total RNA was extracted at the corresponding time points in each link of virus adsorption,replication and release.The relative gene expression of DHAV-1 in each link was detected by real-time PCR to compare the effects of the two drugs on the link of virus adsorption,replication and release.The results showed that both BSRPS and pBSRPS could significantly inhibit virus replication and release,and the inhibition of virus replication by pBSRPS was significantly better than that by BSRPS.The effect of the two drugs on inhibiting virus release was similar,and both had no inhibitory effect on virus adsorption.Test III Study on the therapeutic effect and anti-oxidative damage of pBSRPS in duck viral hepatitisIn order to study the therapeutic effect ofBSRPS and pBSRPS on DVH,240 feathered 6-day-old cherry valley ducklings were randomly divided into 4 groups:BSRPS group,pBSRPS group,VC group and BC group(isolated feeding).Except the BC group,the other 3 groups were intramuscular injected with 0.2mL DHAV-1,and the BC group was injected with the same volume of normal saline.After the challenge,the BSRPS group and the pBSRPS group mixed the drugs in drinking water and gave them 5 mg/day and 3 mg/day for each duck respectively for 3 days.The VC group and the BC group were free to drink water.Observing and recording the condition of ducklings regularly every day for one consecutive week,At 4 h,8 h and 54 h after challenge,5 ducks in each group were randomly captured for carotid artery blood sampling and liver sampling.Biochemical evaluation indexes of serum liver injury and pathological changes of liver tissues were detected to evaluate the hepatoprotective effect of drugs.SOD,CAT,iNOS activities and GSH,MDA contents in liver tissues were detected to evaluate the degree of oxidative stress in ducklings.The results:the final survival rates of BC group,VC group,BSRPS group and pBSRPS group were 100.00%,0.00%,26.7%and 37.8%,respectively.The resultsshowed that ducks infected with DHAV-1 in the control group developed severe oxidative stress,liver injury and died rapidly.BSRPSand pBSRPS could significantly improve the survival rate of ducklings,alleviated the changes of biochemical evaluation indicators of liver serum liver injury,increased the activities of antioxidant enzymes(CAT,SOD)and antioxidant content(GSH)in liver tissue,and reduced the accumulation of lipid peroxidation products(MDA).BSRPS and pBSRPS showed good anti-oxidation and liver protection effects,suggesting that the drugs may achieve liver protection by relieving oxidative stress in the liver tissues of ducklings.The effect of pBSRPS is better than that of BSRPS.Test Ⅳ Study on the protective effect of pBSRPS on mitochondrial damage of DEHs induced by DHAV-1 infection in vitroThe purpose of this study was to explore the effect of BSRPS and pBSRPS on relieving mitochondrial damage in liver cells caused by DHAV-1.BSRPS and pBSRPS were applied to hepatocytes infected with DHAV-1,and the mitochondrial membrane potential was detected,the ATP content in the cells was detected at different time points.Mitochondria in liver cells were extracted and the activities of SOD,GSH-Px,SDH,the levels of MDA,8-OHdG were detected,the cytoplasm of liver cells was extracted and the level of cytochrome C was detected to comprehensively evaluating mitochondrial damage.The results showed that after DHAV-1 infection,the ATP content of liver cells decreased,mitochondrial membrane potential disintegrated,antioxidant enzyme activity decreased,the accumulations of peroxidation products were raised,the levels of cytochrome C and 8-OHdG increased,SDH activity decreased.Both BSRPS and pBSRPS significantly improved these negative effects with similar effects.Test V Study on the effects of pBSRPS on alleviating liver mitochondrial damage induced by DHAV-1 infection in vivoTo study the protective effect of BSRPS and pBSRPS on the mitochondrial damage of hepatic cells induced by DHAV-1 infection.240 feathered 6-day-old cherry valley ducklings were randomly divided into 4 groups:BSRPS group,pBSRPS group,VC group and BC group(isolated feeding).Except the BC group,the other 3 groups were intramuscular injected with 0.2 mL DHAV-1 to challenge,and the BC group was injected with the same volume of normal saline.After the challenge,the BSRPS group and the pBSRPS group were given the drugs in drinking water at 5 mg/day and 3 mg/day,respectively,for 3 consecutive days,and the VC group and the BC group were free to drink water.At 4 h,8 h and 54 h after the challenge,5 ducks were grabbed in each group randomly,to observe its pathological changes under electron microscope,to detect its ATP content and to extract mitochondria for detecting its oxidative stress level.The results showed that DHAV-1 infection would destroy the mitochondrial morphology of the liver,cause severe oxidative stress,and cause mitochondrial dysfunction.Based on their antioxidant effects,BSRPS and pBSRPS alleviated mitochondrial damage and oxidative stress caused by DHAV-1 infection,and increased ATP content,ensure the normal function of duckling liver.The results were similar to those of in vitro experiments,and the effect of pBSRPS in vivo was better than that of BSRPS.