Immunomodulatory Effects Of Proanthocyanidin A-1 From Rhododendron Spiciferum

Spiciferum are shrubs of Ericaceae and Rhododendron, the main production area is westen part of GUIZHOU, central and south east region of YUNNAN in China. Proanthocyanidin A-1 (PAA-1) extracted from Rhododendron spiciferum in ethylacetate is a Dimer compound of Proanthocyanidins. Proanthocyanidins (PAs) are general of Polyphenols. Recently researchs have found that PAs have many medical benefits including antioxidant, cardiovascular protection, anti-cancer, anti-platelet aggregation, immunomodulation and protection of liver and kidney function. So development of PAs drugs has great clinical prospects. Researchers have focused on potent antioxidant capacity, there have few reports related to immunomodulatory effects of PAs.In the present studies, the immunomodulatory effects of PAA-1 were investigated in vitro. Different concentrations of 5, 25, 50 or 100 mg/L PAA-1 alone or PAA-1 plus Con A, or anti-CD3 mAb, or PAA-1 plus LPS can stimulate cell proliferation of plenocytes. A number of studys reported that potentiation of Con A, anti-CD3 mAb or LPS-induced proliferation of immunocyte by a certain compound suggested whose important effect in immune function. The evaluation of substances that either promote or inhibit immunocyte proliferation is crucial to the study of immunomodulatory and drug discovery. Con A and anti-CD3 mAb act directly on T cells, while LPS acts on B cells. T cells are involved in cell-mediated immunity whereas B cells are primarily responsible for humoral immunity. Therefore, PAA-1 could enhance both cellular immunity and humoral immunity.NK cells are a part of innate immune system and play a major role in defending the host from both tumors and virally infected cells. Different concentrations of PAA-1 significantly enhanced NK function to kill YAC-1 target cells. PAA-1 also can enhance the nonspecific immunity.To research the mechanism of stimulating cell proliferation of plenocytes of PAA-1 from cell level, effects of PAA-1 to ratio of two main lymphocyte T subsets (CD4+ cells or T helpers, and CD8+ cells or T cytotoxic/suppressors) in mice were detected by flow cytometer. CD4 T cells recognize antigens presented by major histocompatibility complex class II (MHC II) and mediate both cellular immune response through Th1 cells and humoral immune response through Th2 cells. CD8 recognizes antigens presented by MHC I and mediates cellular immune responses through cytotoxic T cells. In our study, a significant up regulation of the CD4+, CD8+ and CD4+CD8+ double positive cell populations after PAA-1 treatment was found. It means that PAA-1 possess immune-stimulating capacity.We found that PAA-1 has a significant stimulatory effect on inducing the release of Th1 cytokines (IL-2, IL-12, IFN-γ, and TNF-α) and decreasing the release of Th2 cytokines (IL-4, IL-10) with Con A by ELISA kit in vitro. The results showed that PAA-1 can exert the effect of immunological regulation by up-adjusting the Th1/Th2 arms of the immune system. PAA-1 might be useful for the correction of Th2 dominant pathological disorders.Macrophages play an important role in immunity. Different concentration PAA-1 can enhance proliferation of peritoneal macrophages and macrophage phagocytic function on neutral red significantly. Phagocytic activity is accompanied by the production of reactive oxygen (O^2-) and NO as well as the release of lysosomal enzymes such as acid phosphatase (AP) and these products is involved in killing and digesting microbial pathogens. NO plays an important role in immunity through bactericidal and antitumor.In vitro PAA-1 can increase NO secreted by macrophage. The higher reduction in NBT assay represented higher activity of oxidase enzyme, reflecting the stimulation of phagocytes in proportional to intracellular killing. The concentration of 50 mg/L PAA-1 can stimulate macrophage secret more O^2- to reduce NBT dye. For lysosomal enzyme activity, transformation of p-NPP to the colored compound by the acid phosphatase of the stimulated macrophages was correlated to degranulation in phagocytosis. For lysosomal enzyme activity, the 50 mg/L concentration PAA-1 exhibited the maximum potency. These results showed that PAA-1 can active macrophage, and increase Macrophage phagocytosis.In vivo different concentrations of 0, 5, 10, 15 mg/kg PAA-1 were fed to 11-day chickens for ten days, HI antibody titer against NDV in chicken and immune organs index were detected in clinical Trials. The results showed that PAA-1 can enhance antibody level of ND and immune organs index of chickens.All of the above tests provided scientific and experimental foundations for developing new kind immune adjuvant of PAA-1.