The Antiviral And Immune-enhancing Activities Of Polysaccharide-sulfated Polysaccharide Compounds And Its Mechanism

At present,the research of new type antivirus preparations and immunopotentiators to prevent and control animal disease is very imminent.Polysaccharides and their derivatives exhibit many kinds of biological activities,such as anti-virus,anti-tumor,anticoagulation,enhancing immunity and so on.Many studies confirmed that sulfated modification could further enhance the biological activity of polysaccharide and produce new pharmaceutical value.The compound of Tradional Chinese medicine is the most effective measure to prevent and treatment of animal diseases.In present study,firstly,seven polysaccharides(PS)and seven sulfated polysaccharides(sPS)were prepared.Secondly,the 126 compounds of three ratios(PS9-sPS1,PS7-sPS3 and PS6-sPS4)were prepared with PS and sPS-The antiviral activities of these compounds were subsequently compared in vitro using the MTT assay,the best five compounds of their antiviral activity were screened out.Thirdly,the best compound SP9-sCP1 was screened out from five compounds by their antiviral and immune-enhancing activity,and study on the mechanism of antiviral and immune-enhancing activity of SP9-sCP1.The purpose of this study is to validate the probability of PS-sPS compound to raise the bioactivity of PS and sPS of their antiviral and immune-enhancing activity,screen the best compound,and provide the materials for developing antivirus preparations and immunopotentiators from polysaccharides.The tests are divided into seven parts as follows:Test 1.Preparation of seven polysaccharides and their sulfated polysaccharide The Epimedium polysaccharide(EP),Astragalus polysaccharide(AP),Codonopsis pilosula polysaccharide(CP),Chinese angelica polysaccharide(CA),Solomonseal polysaccharide(SP),Garlic polysaccharide(GP)and Isatis tinctoria polysaccharide(IP)were extracted by water-decocting and one-step ethanol precipitation method,respectively.The carbohydrate contents of those seven PS were determined by phenol-sulfuric acid.The results show that the carbohydrate contents of seven PS were from 47.31%to 93.39%.The sEP,sAP,sCP,sCA,sSP,sGP and sIP were prepared by chlorosulfonic acid-pyridine method,which the optimization of sulfation modification conditions of improving the antiviral activity of sPS were described as previously mentioned.The carbohydrate contents of those seven sPS were determined by phenol-sulfuric acid.The sulfate contents of those seven sPS were determined by Ultrasonic-acidic barium chromate spectrophotometry.And the structures were determined with Fourier transform-infrared spectroscopy.The results show that the carbohydrate contents of seven sPS were from 29.63%to 77.30%,the sulfate contents of seven sPS were from 31.23%to 43.63%,and the spectra of seven sPS contained two characteristic absorption bands for sPS,such as a large S=O stretching vibration and a symmetrical C-O-S stretching vibration,which indicated that seven sPS were successfully in sulfated.Test 2.The antiviral activity comparison of PS-sPS compounds In order to screen out the best antiviral activity of PS-sPS compound,the safety concentrations of seven PS and seven sPS were determined for Chicken embryo fibroblast(CEF)by MTT method.Then seven PS and seven sPS combined to PS-sPS compound,at three ratios according to the carbohydrate content,PS9-sPS1,PS7-sPS3 and PS6-sPS4,respectively,for a total of 126 compounds were prepared.Then 126 compounds at five concentrations within safety concentration were added into CEF monolayer by post-adding polysaccharide of Newcastle disease virus(NDV).The effects of 126 compounds on cellular infectivity of NDV were compared by MTT method.The results show that certain concentrations of 126 compounds could inhibit significantly the virus to infect the cells in three models.The comprehensive comparison of the highest virus inhibitory rate of these compounds,CP7-sCA3(86.90%),EP7-sAP3(82.00%),SP9-sCP1(78.30%),EP7-sCA3(77.10%),CA9-sEP1(65.80%)were the first five compounds than the others.These five compound maybe the best compound of antiviral activity.Test 3.The antiviral activity verification of PS-sPS compounds To verify the best combination of 5 PS-sPS compounds according to antiviral activity,the five PS-sPS compounds at three ratios according to the carbohydrate content,PS9-sPS1,PS7-sPS3 and PS6-sPS4,respectively,for a total of 15 compounds were prepared.The effects of 15 compounds on cellular infectivity of NDV were compared by MTT method.The results demonstrated that SP9-sCP1,CP7-sCA3,EP7-sAP3,CA9-sEP,and EP7-sCA3 presented higher activities then other compounds,and SP9-sCP1 displayed the highest virus inhibition rate.Then the antiviral activities of three dosage of SP9-sCP1 were subsequently observation of the virus structure with transmission electron microscopy,the virus antigen expression by immunofluorescence assay,the effects of CEF cell apoptosis and cell cycle by Flow Cytometry assay.The result displayed that SP9-sCP1 can directly damage the structure of NDV with time-effect and dose-effect relationship,significantly inhibited viral antigen expression and apoptosis and cell cycle post infection of NDV,and the high dose of SP9-sCP1 possessed the best efficacy.Test 4.The immune-enhancing activity comparison of PS-sPS compounds In order to screen out the best immune-enhancing activity of PS-sPS compound,the 5 compounds,SP9-sCP1,CP7-sCA3,EP7-sAP3,CA9-sEP1 and EP7-sCA3 at five concentrations within safety concentration were added into the peripheral blood lymphocytes,respectively,singly or synergistically with PHA.The changes of peripheral blood proliferation were determined by MTT assay.The results showed that in single adding 5 compounds,the A570 values in SP9-sCP1,CP7-sCA3,EP7-sAP3 and CA9-sEP1 groups at 7.813 to 0.488 ?g·mL-1,EP7-sCA3 at concentration of 3.906 to 0.488 ?g·mL-1 were significantly higher than the corresponding cell control group(P<0.05).The lymphocyte proliferation rate in SP9-sCP1 group was the highest(37.13%),were significantly higher than those in other groups(P<0.05).In simultaneous adding with PHA assay,5 compounds at certain concentration were significantly higher than the corresponding cell control group.And the lymphocyte proliferation rate in SP9-sCP1 group was the highest(24.31%),were significantly higher than those in other groups(P<0.05).These demonstrated that SP9-sCP1 possess better immune-enhancing activity than other 4 compounds.Test 5.The immune-enhancing activity verification of PS-sPS compounds To verify the immune-enhancing activity of 5 PS-sPS compounds,the immune-enhancing activity in vivo were compared by the immune response of ND vaccine assay.21016-day-old chickens were averagely divided into seven groups.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of each vaccination,the chickens in 5 PS-sPS compounds groups were injected respectively with 0.5 ml(5 mg·ml-1)of 5 PS-sPS compounds,respectively,in vaccination control(VC)and BC group,with 0.5 ml of physiological saline for one time.On 7th,14th,21st,28th day after the first vaccination,the peripheral lymphocyte proliferation,serum ND antibody titer and the contents of IL-2,IL-6 and IFN-?were determined.The result showed that 5 PS-sPS compounds could significantly promote lymphocyte proliferation,except EP7-sCA3 group,and the lymphocyte proliferation rate in SP9-sCP1 group was the highest(49.85%),and significantly higher than those in other groups(P<0.05).The 5 PS-sPS compounds could significantly enhance serum antibody titer and IL-2,IL-6 and IFN-? contents,and the enhancement of SP9-SCP1 group was better than those in other groups.Test 6.Effects of SP9-SCP1 on NP mRNA expression of NDV In order to further study the action mechanism of SP9-SCP1 in antiviral activity,the effects of three dosages of SP9-SCP1 on mRNA expression of NP gene of NDV were determined.SP9-SCP1 at three concentrations(7.813,3.906,1.953 ?g-mL-1)were added into the CEF cell on 48h post infection of NDV,respectively,the cells were harvested and total RNA was extracted,then reverse transcribed into cDNA.The specific of PCR products was evaluated by 2.5%agarose gel electrophoresis of PCR products of target fragments.The mRNA expression of NP gene was evaluated by fluorescence quantitative RT-PCR assay.The results showed that PCR products of target fragments were specific,without nonspecific amplification products.SP9-SCP1 could inhibit the relative expression of NP mRNA.In SP9-sCP1 group at three concentrations were down-regulated the fold changes of NP mRNA relative expression with a dose-dependent manner.Test 7.Effects of SP9-sCP1 on mRNA expression of IL-2,IL-6 and IFN-? in chicken lymphocyte In order to further study the action mechanism of SP9-sCP1 in immune-enhancing actions,the effects of three dosages of SP9-sCP1 on mRNA expression of IL-2,IL-6 and IFN-? of chicken peripheral lymphocyte were determined.SP9-sCP1 at three concentrations(7.813,3.906,1.953 ?g-mL-1)were added into the chicken peripheral blood lymphocyte respectively and cultured 12 h,the cells were harvested and total RNA was extracted,the mRNA expression of IL-2,IL-6 and IFN-? were evaluated by fluorescence quantitative RT-PCR assay.The results of the fold changes of IL-2,IL-6 and IFN-? mRNA relative expression showed that in SP9-sCP,group at 7.813 and 3.906?g-mL-1 could up-regulated IL-2 mRNA relative expression,at 7.813 ?g·mL-1 could up-regulated IL-6 mRNA relative expression,and at three concentration could up-regulated IFN-? mRNA relative expression.The results indicated that SP9-sCP1 could up-regulated IL-2,IL-6 and IFN-? mRNA relative expression,and SP9-sCP1 may be used as an effective immunopotentiator.