| In breeding of livestock and birds, it is of important significance to use immunopotentiators for enhancing immune function and preventing viral infectious disease. However, most of immunopotentiators commonly used in animals are chemical adjuvant with hidden danger of drug residue. The natural medicines display definite advantage. Many researches have showed that many Chinese herbal medicinal ingredients have immunoenhancement. Polysaccharides, as one of important active ingredients of Chinese herbal medicine, not only have antiviral activity but also can enhance immunity. As immunopotentiators, they possess some merits such as safety, low toxicity, and non-harmful residue in animal products and so on. In this study, four Chinese herbal polysaccharides were selected as object, and their antiviral and immune-enhancing action and mechanism were investigated through a series of experiments in vitro and vivo. The purpose is to offer the theory basis for developing polysaccharides as immunopotentiators. The details are divided into eight parts as follows:Test 1 The preparation of four Chinese herbal polysaccharides According to literatures, four Chinese herbal polysaccharides, astragalus polysaccharide (APS), isatis root polysaccharide (IRPS), Chinese yam polysaccharide (CYPS) and achyranthes root polysaccharide (ARPS), were selected as object and extracted by water-decoction and ethanol-precipitation method. The contents of four polysaccharides were measured by Vitriol-anthracene ketone. The total contents (%) of APS, IRPS, CYPS and ARPS were 65.8, 56.7, 73.5 and 54.9 respectively. The total extractive rates (g/Kg) of them were 37.4, 59.1, 53.6 and 48.9 respectively. The four polysaccharides were prepared for the whole study.Test 2 Effects of four polysaccharides on cell infectivity of NDV To evaluate the antiviral activity and mechanism of four polysaccharides, APS, IRPS, ARPS and CYPS were added into chick embryo fibroblast (CEF) monolayer before, after and simultaneous with new disease virus (NDV) respectively. The cell lesions were observed and the cells OD values were determined by MTr assay to evaluate the effects of four polysaccharides on the virus infecting the cell. The results showed that when the polysaccharides were added early to NDV, four Chinese herbal polysaccharides could decrease CPE lesions and inhibit virus to infect the cell in dosages dependent, and APS, IRPS were better than ARPS, CYPS. It Suggested that earlier administration should have better efficacy as clinical application.Test 3 Effects of four polysaccharides on proliferation and production of nitric oxide of splenic lymphocyte in chicken In order to study the action and mechanism four polysaccharides in enhancement of cellular immunity, the proliferation and secretion of nitric oxide were observed in splenic lymphocyte treated with four polysaccharides at five concentrations respectively. The lymphocytes proliferation was determined by MTT assay, NO concentrations and NOS activity were examined by a spectrophotometric assay based on the Griess reaction. The results showed that four polysaccharide at suitable dosages could stimulate lymphocyte proliferation and increase the production of NO and NOS activity in lymphocytes fairly. This is likely to be one of mechanisms of Chinese herbal polysaccharides to enhance the cellular immune responses.Test 4 Effects of four polysaccharides on mRNA expression of interleukin-4 and interferon-γin chicken peripheral lymphocyte In order to study the action and molecular mechanism of four Chinese herbal polysaccharides in immunoenhancement, according to Primer Premier software, two pairs of RT-PCR primers of interferon-γ(IFN-γ) and interleukin-4 (IL-4) were designed. The effects of four polysaccharides on mRNA expression level of IL-4 and IFN-γwere determined by fluorescence quantitative PCR assay. The results showed that the four polysaccharides could improve the mRNA expression level of IL-4 and IFN-γ, and APS, IRPS better than ARPS, CYPS. This might be one of molecular mechanisms of Chinese herbal polysaccharides in immunoenhancement.Test 5 Effect of four polysaccharides on peritoneal macrophages function, haemolytic plaque formation and hemolysin in mice and dosage screening In order to validate the effect of four polysaccharides on non-specific immunity and screen the suitable dosages. 102 mice were randomly assigned into equal seventeen groups; mices in 16 experimental groups were peritoneaUy injected respectively with four polysaccharides at four dosages, in control group, with saline, once a day for three successive times. On days 8, the phagocytotic test of intraperitoneal macrophages, the level of haemolytic plaque and serum hemolysin were measured in mice. The results showed that four polysaccharides could improve the function of intraperitoneal macrophages, raise the level of haemolytic empty spots and serum hemolysin, and the effects of APS at 30 mg.kg-1, IRPS at 20 mg'kg-1, ARPS and CYPS at 50 mg'kg-1 of dosage were the best.Test 6 Effects of four polysaccharides on antibody titer and immune organ index in vaccinated chickens In order to study the action and mechanism of four polysaccharides on specific-humoral and non-specific immunity, 450 14-day-old chickens were randomly assigned into equal nine groups and all chickens were vaccinated firstly with Newcastle Disease (ND)-Infectious Bronchitis (IB) live virus vaccine, secondly with ND-IB oil adjuvant vaccine at 28 days age. At the same time as first vaccination, the chickens in groups 1 to 8 were intramuscularly injected with astragalus polysaccharide, isatis root polysaccharide, achyranthes root polysaccharide and Chinese yam polysaccharide at high and low dosages, Group 9 (control group), with saline, once a day for three successive days, secondly with ND-IB oil adjuvant vaccine at 28 days of age. On days 7, 14, 21, 28, 35, 42, 49 and 56 after the first vaccination, serum ND-HI antibody titer were determind by Micro-method. On days 10, 20, 30, 40, 50 and 60 after the first vaccination, 5 chickens' weights of whole body, spleen, bursa and thymus were weighted for calculation of immune organ index. The results showed that APS and IRPS at low dosages, ARPS and CYPS at high dosages significantly enhanced the antibody titers and immune organ indexes, and APS and IRPS at low dosages were better. This indicated that Chinese herbal polysaccharides could improve the specific immunity in cooperation with vaccine and raise non-specific immunity by promoting immune organ development.Test 7 Effects of four polysaccharides on proliferation and subpopulation of lymphocyte in vaccinated chickens In order to study the action and mechanism of four Chinese herbal polysaccharides on cellular immunity in vivo, 450 14-day-old chickens were randomly assigned into equal nine groups and all chickens were vaccinated with Newcastle Disease (ND)-Infectious Bronchitis (IB) live virus vaccine. At the same time as the first vaccination, the chickens in groups 1 to 8 were intramuscularly injected with astragalus polysaccharide, isatis root polysaccharide, achyranthes root polysaccharide and Chinese yam polysaccharid at high and low dosages, Group 9 (control group), with saline, once a day for three successive days, secondly with ND-IB oil adjuvant vaccine at 28 days of age. On days 10, 20, 30, 40, 50 and 60 after the first vaccination, the peripheral T lymphocyte proliferation were determined by MTT method and the content of CD3+, CD3+CD4+ and CD3+CD8+ T cells were determined by flow cytometry and double-color-staining method. The results showed that four polysaccharides could significantly promote the proliferation of peripheral T lymphocyte and raise the content of CD3+ and CD3+CD4+ T cells and ratio of CD4+/CD8+. It might be one of the mechanisms on cellular immunity of Chinese herbal polysaccharides.Test 8 Effects of four polysaeeharides on mueosal immunity in vaccinated chickens In order to study the action and mechanism of four Chinese herbal polysaccharides in mucosal immunity, 450 14-day-old chickens were randomly assigned into equal nine groups and all chickens were vaccinated with Newcastle Disease (ND)-Infectious Bronchitis (IB) live virus vaccine. At the same time as the first vaccination, the chickens in groups 1 to 8 were intramuscularly injected with astragalus polysaccharide, isatis root polysaccharide, achyranthes root polysaccharide and Chinese yam polysaccharid at high and low dosages, Group 9 (control group), with saline, once a day for three successive days, secondly with ND-IB oil adjuvant vaccine at 28 days of age. On Days 10, 20, 30, 40, 50 and 60 after the first vaccination, five chickens were sampled randomly from each group for determination of secretory immunoglobulin A (SIgA) that is released into the intestinal mucus by ELISA assay, as well as the change of the SIgA positive cells in the intestinal mucosa and cecal tonsil by immunohistochemistry method. The results showed that four polysaccharides could raise the content of SIgA and increase the number of SIgA positive cells.The immunoenhancement of APS and IRPS at low dosages, ARPS and CYPS at high dosages were better. It demonstrated that four polysaccharides could enhance the immune function of mucosa as so to improve the ability of anti-infection.
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