Adjuvant Activity And Mechanism Of Polysaccharide From Isatis Indigotica

Indigowoad Root is the root of Isatis Indigotica belongs to the mustard family. It is thetraditional Chinese medicine and has a lot of active fractions, such as flavones,polysaccharides, alkaloid, lignin and so on. Total polysaccharide from Isatis Indigotica(named IIP) is one of the main active materials of Indigowoad Root. The previouspharmacological research indicated that IIP could have the activities of immunologicalregulation, anti-inflammatory, anti-septicize, and anti-tumor. IIP-A and IIP-B were twocomponents from IIP. In this study we determined the vaccine adjuvant activities of IIP-A,IIP-B, and their further separated compositions, IIP-A-1, IIP-B-1and IIP-B-2. H1N1influenza virus split vaccine and the recombinated hepatitis B surface antigen (HBsAg)were used as antigens to immunize Balb/c mice by intramuscular injection. The titer andsubclass of serum antibody of mice were determined by ELISA for evaluating their effecton humoral immunity. The proliferation of splenic lymphocyte was measured by MTT.Flow cytometry was used to determine percentage of splenic lymphocyte population andlymphocyte subpopulation. The secretion of lymphocyte cytokines IFN-γ and IL-4wasmeasured through commercialized ELISAkits.The results of IIP combined with H1N1influenza virus split vaccine showed thatIIP-A-1could significantly accelerate the production of antibody against H1N1. In the micesera, higher titers of IgG2a, IgG2b and IgG3were obtained. IIP-B-1and IIP-B-2alsoassisted the antibody production and promoted more titers of IgG2b and IgG3in the sera.However, their effects were lower than that of IIP-A-1. When IIP and the antigen wereindividually injected in different positions, no obvious difference was observed in antibodytiters compared with those from mice received antigen without any adjuvant. But theconcents of CD3~+T cell, CD8~+T cell and the ratio of CD3~+/CD19~+(CD3~+/CD19~+) werepromoted significantly in the polysaccharides groups. IIP-A could promote the secretion ofIFN-γ compared with group H1N1without adjuvant. Results of weight and organcoefficient showed that IIP have no observable toxicity. Combined with HBsAg, a recombinated hepatitis B surface antigen, the adjuvant’sactivities of IIP-A and IIP-B were also evaluated. The results showed that IIP-A and IIP-Bcould induce the production of HBsAg specific antibody in serum after one time’simmunization. After the second immunization IIP-A could significantly promote theproduction of specific antibody in mouse. Compared with the result from alum group, theserum antibody contained more titers of IgG1and IgG3, the same levels of IgG2a and IgA.IIP-A and IIP-A-1could sustain the high level of serum antibody for the longer time thanthose from the group of HBsAg control, even they exhibited less effect than alum. IIP-Bcould promote the proliferation of splenic lymphocytes from immunized mice, which wereinduce by ConA or LPS, with the significantly difference compared with alum treatment.IIP could promote percentage of CD3~+T cell, CD4~+T cell and CD8~+T cell compared withimmunization with HBsAg alone, but less effective than alum adjuvant. IIP could promotethe secretion of IFN-γ but decrease the IL-4level.The effect of IIP on immune function of normal mice showed that IIP itself had noimmunogenicity. It had no effect on T lymphocyte and B lymphocyte proliferation inducedby ConA and LPS. IIP could increase the percentage of CD3~+, CD4~+, CD8~+, CD19~+cellsubsets and the ratio of (CD3~+/CD19~+) of splenic lymphocytes, indicating that IIP couldimprove the cellular immune functions. When mice treated with polysaccharides only,IIP-A inhibited the secretion of splenic lymphocyte cytokine IFN-γ and thymic lymphocytecytokine IL-4and IIP-B inhibited IFN-γ secretion but increased IL-4level.In the summary, being a candidate of adjuvant, IIP could improve both humoral andcellular response on Balb/c mice. It had two-ways regulation on cytokine secretion towardsnormal and immunized mice. Being a good immunopotentiators, IIP has a good potencityto be researched as a new kind of vaccine adjuvant.