Effect On The Immunomodulatory Function Of Dendritic Cells And Researches On Biological Activity Of TBL

Lectins are proteins of non-immune origin that interact reversibly and specifically with carbohydrates. These proteins are widely distributed in animals and plants. Lectins exhibit a variety of biological activities, such as anti-insect, anti-fungal, anti-cancer, anti-viral and immunomodulatory activity. They have various different functions in nature, the most important of which is information mediation in biological systems. Recent studies have shown that some plant lectins have several applications within the plant body, such as in pathogen defense mechanisms, or outside the body, drug delivery, as well as a variety of diagnostic applications for a broad spectrum of diseases. However, there are few reports on the function that promotes maturation and proliferation of dendritic cells (DCs), and further study on the function of lectin on immune cells is warranted. The present study aims to purify and characterize lectin from tartary buckwheat seeds and study its properties as well as biological activities to determine its possible biomedical applications in promoting maturation, proliferation of peripheral blood DCs derived from healthy donors, to study the effect of inducing apoptosis in tumor cells, the effect on radiation protection and anti-tumor in vivo.A novel tartary buckwheat lectin (TBL) protein, purified from tartary buckwheat seeds, showed a single band with a molecular mass of 65 kDa in SDS-PAGE. The purified TBL hemagglutinated both human and animal erythrocytes and showed preference for human blood type O and the rabbit blood. TBL is active at up to 64℃, and it is metal ion, acid-and alkali-stable.TBL (50 μg/mL) combined with 5×10-5 mol/L rhIL-4 promotes maturation and proliferation of peripheral blood dendritic cells (DCs), which is stronger than that promoted by rhTNF-α (20 ng/mL). Exposure of DCs to 50 μg/mL TBL for 48 h resulted in extensive up-regulation of maturation markers CD83 and CD40. These TBL-DCs were capable of producing several pro-inflammatory cytokines such as interleukin-10 (IL-10) and interleukin-12 (IL-12).The rate of proliferationof the U937 and H22 cell line treated by TBL was identified by CCK-8 assays, apoptosis was tested by flow cytometry. Our results suggested that TBL inhibits tumor cell viability by inducing apoptosis with a concentration-dependent effect, but there were minimal effects on normal liver 7702 cells. The measurement of caspase activation assess showed TBL induced apoptosis was correlated with mitochondrial dysfunction, leading to the proteolytic activation of caspase-3, caspase-8 and caspase-9.We investigated the radioprotective effect of TBL, DCs transplantation and TBL-DCs transplantation. For studies of mice after exposed to a dose of 4 Gy of X irradiation, we found that the WBC, PLT counts, the thymus and spleen indexes and spleen lymphocyte transformation rates of mice in each treatment group increased sharply as in comparison with the model group, DCs transplantation TBL-DCs transplantation showed the best effects. On the other hand, TBL-DCs transplantation showed the best effects on the treatment of regulatory T cells, B lymphocytes and natural killer cell CD 16+ damage in mice. The result showed the bone marrow cell DNA content was increased after treated with TBL, and TBL-DCs transplantation, however apoptosis rate of bone marrow cells was decreased and the incidence of mutations was reduced after treated with DCs transplantation and TBL-DCs transplantation.These results suggested that there were marked radioprotective effects on mice, effective protection on hematopoietic and immune systems of mice after irradiation according to TBL-DCs transplantation treatment.The expression of DCs surface antigen Dectin-1, CD40, CD80, CD86 and MHCII was detected by flow cytometry. The secretion level IL-12, IFN-γ, IL-6, and IL-23 was tested by ELISA. We found that TBL could stimulate and promote maturation of DCs, partially through the dectin-1/Syk pathway, as shown by the up-regulation of CD40, CD80, CD86, and major histocompatibility complex Ⅱ (MHCII). TBL also enhanced the production of cytokines by DCs, including IL-12, IFN-γ, IL-6, and IL-23. Moreover, TBL-treated DCs showed an enhanced capability to stimulate T cells and promote T cell proliferation. Altogether, these results demonstrate that TBL are able to activate and maturate DCs, thereby up-regulating the immunostimulatory capacity of DCs, which leads to enhanced T cell responses. TBL showed great potential in enhancing immune response and inhibiting the growth of tumor as an immune enhancer.