Studies On Immune-enhancing Activities Of Ploysaccharide-selenizing Ploysaccharide Compounds

Animal diseases have been harmful to healthy development of animal husbandry industry.Vaccines are often used to control animal diseases and it is a better immune effect that vaccines are combined with immune booster.But commonly used vaccines have strong toxic and side effects and other disadvantages.So it is urgent need to develop a new immune enhancer.In recent years,many studies have found that polysaccharides possessed immune-enhancing activity effects and that selenized 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 traditional Chinese medicine field.In present study,seven polysaccharides was extracted,and was prepared with three selenizing polysaccharides.18 polysaccharide-selenizing polysaccharide compounds were composed taking them as constituents according to 9:1 ratio of polysaccharide to selenizing polysaccharide and their immune-enhancing activities in vitro and in vivo were compared.The immune-enhancing activities of these compounds were subsequently compared in vitro using the MTT assay,the best three compounds of their immune-enhancing activity were screened out.Then the best compound was screened out from three compounds in vivo test.Study on the mechanism of imune-enhancing activity of the best conpound.The purpose of this study is to validate the the immune-enhancing activity of polysaccharide-selenizing polysaccharide compoundsselect a compound with the strongest activity and offer theoretical evidence for developing new-type immunopotentiators.The tests are divided into five parts as follows:Test 1.Preparation of seven polydsaccharides and their selenizing polysaccharide The Codonopsis pilosula polysaccharide(CPP),Chinese angelica polysaccharide(CAP),Garlic polysaccharide(GP),Lilium brownii polysaccharide(LP),Tractylodes macrocephala polysaccharide(AMP),Lycium barbarum polysaccharide(LBP),Schisandra chinensis polysaccharide(SCP)were extracted by water-decocting and one-step or stepwise ethanol precipitation method,respectively.CPP,CAP and GP were respectively separated proteion by Sevage method and Trichloroacetic acid method,,Purified by DEAE-52 column chromatography and Sephadex-G 200 glucose gel column and selenized by Nitric acid-Sodium selenite method..The carbohydrate contents of those seven polysaccharides were determined by phenol-sulfuric acid.Selenium content of three selenizing polysaccharide were determined by Hydride generation-atomic fluorescence spectrometry method.The results show that the polysaccharides yield and carbohydrate contents of CPP,CAP,GP,LP,AMP,LBP and SCP were 12.94%,13.84%,15.36%,33.68%,14.46%,12.71%and 63.42%,respectively.And the carbohydrate contents respectively were 68.2%,31.5%,61.3%,69.67%,56.0%,36.5%and 59.3%.The carbohydrate of sCPP,sCAP and sGP were 61.11%,50.90%and 52.2%,respectively.And selenium contents respectively were 11.86 mg-g-1;12.98 mg·g-1 and 11.36 mg-g-1.Test 2.Comparison of immune-enhangcing activities in vitro of polysaccharide-selenizing polysaccheeride compounds seven polysaccharides and three selenizing polysaccharides were composed 18 polysaccharide-selenizing polysaccharides compounds.On the basis of the safety concentrations of seven polysaccharides and three selenizing polysaccharides,the safety concentrations of 18 polysaccharide-selenizing polysaccharide compounds were detemined.18 polysaccharide-selenizing polysaccharide compounds were diluted with RPMI-1640 at five concentrations in safe concentrations were added into the cultured chicken peripheral blood lymphocyte in single or simultaneous with PHA,respectively.The changes of the lymphocyte proliferation(cellular A570 value and the lymphocyte proliferation rate)were determined by MTT method.These results showed that in single adding,the A570 values of all compounds at 1-5 concentration groups were higher than the cell control group,or significantly higher than the cell control at some concentration groups.The lymphocyte proliferation rates of CAP-sCPP,GP-sCPP and CPP-sCAP groups were 18.55%,15.38%and 15.18%..In simultaneous adding with PHA,the A570 values of all compounds at 1-5 concentration groups were higher than the PHA group,or significantly higher than the PHA control at some concentration groups.The lymphocyte proliferation rates of CAP-sCPP,GP-sCPP and CPP-sCAP were 18.28%,14.08%and 12.96%..These results indicated that 18 compouds could enhance the immune-enhancing activity at different levels.In general evaluation the actions of CAP-sCPP,CPP-sCAP and GP-sCPP were stronger.Test 3.Comparison of immune-enhangcing activities in vivo of polysaccharide-selenizing polysaccharides compounds To verify the immune-enhancing activity of 3 polysaccharide-selenizing polysaccharides(CAP-sCPP,CPP-sCAP and GP-sCPP)and screen out the best compoud,the immune-enhancing activity in vivo were compared by the immune response of ND vaccine assay.240 16-day-old chickens were averagely divided into seven groups.Eath group had 30 chichens.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 six compouds groups were intramuscularly injected respectively with 0.5 mL of CAP-sCPP,CPP-sCAP and GP-sCPP at high(10 mg·ml-1)and low concentrations(5 mg·mr-1),in vaccination control(VC)and blank control group,with equal volume of physiological saline,once a day for three successive days.On days of 7(D7),14(D14),21(D21)and 28(D28)after the first vaccination,the peripheral lymphocyte proliferation,serum ND antibody titer and the contents of IL-2,IL-6 and IFN-y were determined.These results showed that GP-sCPPH group at D7,GP-sCPPH and GP-sCPPL groups at D14,GP-sCPPH,CPP-sCAPH and GP-CPPL groups at D21,GP-sCPPH,CPP-sCAPL and GP-sCPPL groups at D28 could significantly promote lymphocytes proliferation and the lymphocyte proliferation rate of GP-sCPPH group was the highest at four time points after vaccination;The changes of serum HI antibody titer of GP-sCPPH,CAP-sCPPH,CAP-sCPPL and GP-sCPPL groups at D7,GP-sCPPH,CAP-sCPPH,CAP-sCPPL,CPP-sCAPH and GP-sCPPL groups at D14,GP-sCPPH group at D21 GP-sCPPH,CAP-sCPPH,CAP-sCPPL,GP-sCPPL,CPP-sCAPH and CPP-sCAPL groups at D28 were significantly higher than VC and BC groups,and GP-sCPPH group was the highest at at four time points after vaccination;The 3 compouds could significantly enhance IL-2,IL-6 and IFN-γ contents at differente time pionts in high or low concentrations,and the enhancement of GP-sCPPH group was the best at at four time points after vaccination.These results indicated that 3 compouds could enhance the immune-function of ND activity and GP-sCPP possessed the best efficacy at high dose and would be as a component drug of new-type immunoenhancer.Test 4.Verification of immune-enhancing activity of GP-sCPP compound To compare the immune-enhancing activity of 3 polysaccharides(GP-sCPP,sCPP and GP),the immune-enhancing activity in vivo were compared by the immune response of ND vaccine assay.150 16-day-old chickens were averagely divided into seven groups.Eath group had 30 chichens.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 six compouds groups were intramuscularly injected respectively with 0.5 mL of GP-sCPP(10 mg-mL-1),sCPP(2 mg-mL-1)and GP(8 mg·mL-1),in vaccination control(VC)and blank control group,with equal volume of physiological saline,once a day for three successive days.On days 7,14,21 and 28 after the first vaccination,the peripheral lymphocyte proliferation,serum ND antibody titer and the contents of IL-2,IL-6 and IFN-y were determined.These resulst showed that the A570 value of GP-sCPP group was significantly higher than VC and BC groups at four time points after vaccination,and its was significantly higher than sCPP at D28 and GP groups at D14,too.The lymphocyte proliferation rate of GP-sCPP group was the highestat four time points after vaccination.The changes of serum HI antibody titer of GP-sCPP group was significantly higher than VC and BC groups at four time points after vaccination,and its was significantly higher than sCPP group at D7 and GP groupat D14,too.The contents IL-2,IL-6 and IFN-y of GP-sCPP group were the highest at at four time points after vaccination.The contents of IL-2 and IL-6 of GP-sCPP group were higher than other groups,and the contents of IFN-y of GP-sCPP group was higher than sCPP and GP groups at some time pionts after vaccination.These results indicated that immune function of GP-sCPP is reliable and is significantly stronger than sCPP and GP.Test 5.Effects of GP-sCPP compound on chicken lymphocyte subpopulations and mRNA expression of IL-2 and IFN-γ In order to further study the action mechanism of GP-sCPP in imune-enhancing activity,the effects of three polysaccharides(GP-sCPP,sCPP and GP)on expression of IL-2 and IFN-y mRNA and subgroup of T cell of chicken peripheral lymphocyte was determined.The chicken peripheral blood lymphocyte were cultivated in adding GP-sCPP,sCPP and GP at three concentrations.After cultivation of 48 h,the cells were treated by cool PBS and were dyed by fluorescent dye.Finally,these cells were detected contents of CD4+and CD8+T cell by BD flow cytometry.After cultivation of 12 h,the cells were collected and total RNA was extracted.The expression of IL-2 and IFN-y mRNA was determined by fluorescence quantitative RT-PCR assay.The results showed that the content of CD4+T lymphocyte cell and ratio of CD4+/CD8+ of GP-sCPP at all concentrations were significantly higher Con A control group and sCPP and GP at high and medium concentration Groups were significantly higher Con A control group(P<0.05)·But the content of CD8+T lymphocyte cell were not significant difference beteen all groups.GP-sCPP at high and midium concentration Groups and sCPP at high concentration Group could significantly promote the expression of IL-2 mRNA,and GP-sCPP at all concentrations groups and sCPP at high concentration group and GP at high and medium concentrations groiups could significantly promote the expression of IFN-y mRNA.The results indicated that GP-sCPP could significantly promote CD4+T lymphocyte proliferation and expression of IL-2 and IFN-y and enhance ratio of CD4+/CD8+.Those factions were significantly stronger than sCPP and GP.So this could be section of mechanism of immune-enhancing of GP-sCPP.