Immunoenhancement Effect Of Ginsenoside Rb1 On Chicken Infectious Bursal Disease Attenuated Vaccine And Its Mechanism

Vaccination is the main measure to prevent animal diseases,but the immune organs of the body are damaged by vaccination methods,times,immune stress and other factors in the process of immunization,which leads to the failure of effective immune protection after vaccination.Vaccine adjuvant is an effective means to solve the above problems.Studies have found that ginsenosides have high health and medicinal value,and have immunomodulatory and anti-inflammatory effects,and can significantly improve humoral immune response and cellular immune response.Therefore,In this study,ginsenoside Rb1 was used as a new adjuvant to enhance the immunity of IBD attenuated vaccine in vitro and in vivo.On this basis,the immunopotentiating effect of ginsenoside Rb1 regulating IL-4 on chicken IBD vaccine was studied.In addition,in order to improve the bioavailability,ginsenoside Rb1 calcium phosphate nanoparticles were prepared by encapsulating ginsenoside Rb1 in calcium phosphate nanoparticles,and the immunopotentiating effect and mechanism of the nanoparticles on chicken IBD vaccine were further investigated.The main research contents and results are as follows:1.Safety evaluation of ginsenoside Rb1 in vitroTo study the safe concentration of ginsenoside Rb1 in vitro.In this study,different concentrations of ginsenoside Rb1 alone or together with IBD attenuated vaccine were added to chicken spleen lymphocytes,and the effects of ginsenoside Rb1 on the proliferation of chicken spleen lymphocytes stimulated by Con A and LPS were detected by MTT assay.The results showed that compared with the control group,the proliferation ability of chicken spleen lymphocytes was the strongest when ginsenoside Rb1 was added alone or together with IBD attenuated vaccine at the concentrations of 1.5625μg/m L,3.125μg/m L and 6.25μg/m L.The safe concentration of ginsenoside Rb1 was 1.5625μg/m L-6.25 μg/m L.2.Immunopotentiation effect of ginsenoside Rb1 on chicken IBD attenuated vaccineFor the sake of research the effect of ginsenoside Rb1 on the immune enhancement of IBD vaccine,240 SPF chickens were randomly divided into 7 groups and immunized with different doses of ginsenoside Rb1(0.5 mg/kg BW、1mg/kg BW、2mg/kg BW)and IBD vaccine,respectively.On the 7th,14 th and 21 st day after combined immunization,the titers of IBDV-specific antibodies and the expression levels of IL-6,IFN-γ and IL-4in serum were detected by ELISA;At the same time point,splenic lymphocytes,spleen and bursa of Fabricius were aseptically isolated,and the proliferation level of splenic lymphocytes and the expression level of immune-related molecules m RNA in tissues were detected by CCK-8 and RT-q PCR.Finally,the protective efficacy was evaluated by intranasal challenge 28 days after immunization.The results of ELISA showed that compared with the control group,ginsenoside Rb1 at different doses could significantly increase the titer of anti-IBDV specific antibody and the level of cytokines in chicken serum(P<0.01);CCK-8 was used to detect the proliferation of spleen lymphocytes after immunization.The results showed that the proliferation of spleen lymphocytes of chickens immunized with different doses of Rb1 and IBD attenuated vaccine was significantly higher than that of the control group(P<0.01).The results of RT-q PCR showed that ginsenoside Rb1 significantly up-regulated the m RNA expression levels of CD40,CD80,CD86,MHCII,TGF-β,TNF-α,IL-6 and IL-4 in spleen and bursa of Fabricius.The results of immunity evaluation showed that ginsenoside Rb1 could improve the protective efficiency of IBD attenuated vaccine.3.Effect of ginsenoside Rb1 on intestinal immunity of chickens immunized with IBD attenuated vaccineIn order to study whether ginsenoside Rb1 can enhance the intestinal mucosal immunity of IBD vaccine in chickens,the m RNA expression levels of mucosal immune-related molecules in duodenum were detected by RT-q PCR on the 7th day after immunization;On the 7th,14 th and 21 st day after immunization,the number of Ig A +cells in lamina propria and intestinal intraepithelial lymphocytes(IELs)in the duodenum was observed by immunohistochemistry.Specific s Ig A and total s Ig A in intestinal lavage fluid were detected by ELISA at the same time.The results showed that ginsenoside Rb1 significantly up-regulated the m RNA expression levels of CD40,CD80,CD86,MHCII,TGF-β,TNF-α,IL-6,IL-4,plg R,Ig A and CCR-9 in chicken intestinal tissue(P<0.01);The results of ELISA and immunohistochemistry showed that oral administration of ginsenoside Rb1 significantly increased the content of total and specific s Ig A in the intestine,and the number of Ig A + cells in the lamina propria and IELs in the duodenal mucosa(P<0.01).4.Synergistic effect of ginsenoside Rb1 and IL-4 on the immune enhancement of chicken IBD attenuated vaccineFor further study the synergistic effect of ginsenoside Rb1 and IL-4 on the immune enhancement of chicken IBD attenuated vaccine,SPF chickens were randomly divided into groups and immunized with ginsenoside Rb1/IL-4 and chicken IBD attenuated vaccine.On the 7th,14 th,21st,28 th and 42 nd days after immunization,the levels of specific antibody,Ig G2 a and Ig G1 and the levels of TNF-α,IL-6 and IFN-γ were detected by ELISA;At the same time,spleen lymphocytes and chicken bone marrow-derived dendritic cells were isolated in sterile medium,and the expression of surface molecules on chicken dendritic cells and the proliferation of spleen lymphocytes were detected.The results showed that compared with the control group,ginsenoside Rb1/IL-4 combined with IBD attenuated vaccine significantly increased the titers of anti-IBDV specific antibodies and antibody subtypes and the levels of cytokines in chicken serum(P<0.01).At the same time,the proliferation of chicken spleen lymphocytes was significantly enhanced(P<0.01).and the expression of CD80 and MHCII on the surface of dendritic cells was increased.5.Immunopotentiation of Ginsenoside Rb1 Calcium Phosphate Nanoparticles on Chicken IBD Attenuated VaccineIn order to improve the bioavailability thereof,the ginsenoside Rb1 nanoparticles with high stability and good immunogenicity are obtained,In this study,ginsenoside Rb1/IL-4 calcium phosphate nanoparticles(GRb1/IL-4@CS/Ca P)were prepared by encapsulating Rb1 and IL-4 in calcium phosphate nanoparticles and characterized.Then,the immunopotentiating effect of IBD attenuated vaccine was evaluated.On the 20 th day,chicken femurs were collected to prepare bone marrow-derived cells.the expression of MHCII and CD80 on the surface of bone marrow-derived dendritic cells and the secretion levels of TNF-α and IL-1β were detected by flow cytometry.On the 28 th and 42 nd day after immunization,the titers of specific Ig G,Ig G2 a and Ig G1,and the secretion levels of TNF-α、IL-1α、IL-6 and IL-4 were detected by ELISA.On the 7th,14 th and 21 st day after immunization,the number of Ig A + cells in lamina propria and intestinal intraepithelial lymphocytes(IELs)in duodenum were observed.The contents of total s Ig A and specific s Ig A in intestinal lavage fluid and duodenal tissue were detected by ELISA,and the m RNA expression level was detected by RT-q PCR.The characterization results of nanoparticles showed that GRb1/IL-4 @ CS/Ca P exhibited irregular flower-like cubic structure under cryoscanning microscopy,the particle size increased from 3121 nm ±72.46 nm to 4265 nm ± 72.92 nm,and the potential decreased from +6.87± 0.27 to-22.71 ±0.89.The PDI and Zeta potential of the nanoparticles did not change significantly within28 days,and the PDI was less than 0.3.The results of ELISA showed that GRb1/IL-4@CS/Ca P could significantly increase the levels of IBDV-specific Ig G,Ig G2 a and Ig G1 antibodies,and the levels of TNF-a、IL-1α、IL-6 and IL-4(P<0.01),and could more effectively promote the maturation and cytokine secretion of chicken bone marrow-derived dendritic cells after encapsulation.In addition,GRb1/IL-4@CS/Ca P group could also increase the contents of total s Ig A and specific s Ig A in intestinal tract,and the number of Ig A+ cells in epithelial lamina propria and the number of IELs in duodenal mucosa also showed an increasing trend.The m RNA expression levels of CD40,CD80,CD86,MHCII,TGF-β,TNF-α,IL-4,IL-6,plg R,Ig A and CCR9 in chicken duodenum were significantly up-regulated(P<0.01).To sum up,ginsenoside Rb1 can significantly increase the level of specific antibodies and the expression of cytokines in serum,enhance the proliferation of splenic lymphocytes,and significantly increase the m RNA expression of immune-related genes in spleen,bursa of Fabricius and duodenum.Oral administration of ginsenoside Rb1 can significantly increase the secretion of intestinal IELs cells and lamina propria Ig A,thereby improving intestinal mucosal immunity.The results showed that ginsenosides had immunopotentiating effect.In addition,the ginsenoside Rb1 is used in cooperation with IL-4,and the ginsenoside Rb1/IL-4 calcium phosphate nanoparticles prepared by coating Rb1/IL-4 in the calcium phosphate nanoparticles can be used as an immunopotentiator to improve the humoral immune response of an organism,effectively promote the secretion of cytokines and the maturation of bone marrow-derived dendritic cells,Significantly increase the secretion of intestinal IELs and lamina propria Ig A and the expression level of immune-related gene m RNA in duodenal tissue,and further verify the immunopotentiation of IBD vaccine in chickens.These results indicate that ginsenoside Rb1 has the potential to be developed as a new immune adjuvant.