The Immunosuppressive Effects And Mechanisms Of Daphnetin On Mouse Lymphocytes

Immunosuppressive agents are an important drug class that is valuable for the treatment ofvarious human diseases. These drugs have been considered to be useful in controlling andtreatment of autoimmune diseases and transplantation rejection. Although there are a variety ofimmunosuppressive drugs such as cyclosporin A, tacrolimus(FK506), Methotrexate(MTX),Azathioprine(AZA) and Rituximab, the toxicity is the major obstacle in the widely use of theseimmunosuppressive drugs. Therefore, new and safe immunosuppressive drugs againstautoimmune diseases and transplantation rejection are eagerly awaited. Traditional Chinesemedicines have been reevaluated by clinicians, because these medicines have fewer side effectsand because they are more suitable for long-term use as compared to chemically synthesizedmedicines. The have particular advantages in treating diseases. Their active components drewgreat attention worldwide. In this dissertation, immunosuppressive activity and mechanism in vitroand in vivo of Chinese medicine monomers were researched; it would provide evidence forclinical application as an immunosuppressant.To screen immunosuppressive activity of Chinese medicine monomers by using mouse spleenlymphocyte proliferation test in vitro. Cytotoxicity and spleen lymphocytes proliferation weremeasured by MTT assay. Eight kinds of Chinese medicine monomers respectively in differentconcentration have acted on mice spleen lymphocytes (which stimulated by ConA and LPS) for48h in vitro. The result found that Fisetin could effectively suppress T lymphocytesoverproduction, Gossypol and Daphnetin could effectively suppress T and B lymphocytesoverproduction and possessed immunosuppressive activity on dosage. Among the three Chinesemedicine monomers, DAP was found to be the least toxic of the monomers tested. In the presentstudy, we picked up Daphnetin, with a lower toxicity and the potent immunosuppressive activityto do the further research.The present study aimed to determine the immunosuppressive effects and the underlyingmechanisms of DAP in mice. As described in the previous chapter, DAP could suppress ConA-or LPS-induced splenocytes proliferation in vitro, cellular proliferation is regulated primarily byregulation of the cell cycle, which consists of distinct sequential phases (G0/G1, S, G2/M). Thedistribution of the cell-cycle was analyzed by propidium iodide (PI) staining together withflowcytometry analysis. We investigated the effects of DAP on cell-cycle progression ofLymphocytes. Flow cytometry analysis of the cell-cycle distribution revealed that the cell numberin sub-G0/G1was increased while the cell numbers in S and G2/M was decreased as theconcentration of DAP increased. The result showed that DAP blocked DNA synthesis at G0/G1stage to inhibit mouse lymphocytes mitosis and cell over-proliferation.Cytokines are important modulators and effectors in the immune system. In particular, multipleproinflammatory cytokines have been proved to be closely associated with many autoimmunediseases. In this study, we used ConA as T cell mitogens, and selected IL-2, IFN-γ as Th1cytokines and IL-4, IL-6as Th2cytokines to test the effect of DAP on modulating the Th1andTh2cytokines. The result showed that DAP significantly inhibited cytokines production and themRNA expression induced by ConA, suggesting that the inhibition of cytokine production isresulted from the suppression of mRNA expression. These findings indicated that theimmunosuppressive action of DAP may relate to its inhibition of production and secretion of thecytokines.Intracellular Ca2+is a quintessential intracellular messenger, and many of its cellular effects aretransduced by calmodulin. The calcium/CaM complex regulates several downstream targetsincluding protein kinases and phosphatases. Immunosuppressant of important components of theCa2+signaling pathway revealed that ConA-induced NFAT activation depends on aCa2+/calmodulin/calcineurin pathway whereas ConA-induced NF-kB activation depends on aCa2+/calmodulin/CamK Ⅱ pathway that is also affected by calmodulin. In order to studyimmunosuppressive action molecular mechanism, we further investigated the effect of DAP onCaN-NFAT and CaMK Ⅱ-NF-κB signal transduction pathways in ConA-stimulated murinesplenocytes. Our results showed that in ConA group, after activation by both [Ca2+]i and CaM,CaN and CaMKII dephosphorylated its cytoplasmic substrate, NFAT and NF-κB, then theactivated NFAT and NF-κB translocated from the cytosol into the nucleus. As expected, daphnetininhibited the ConA-induced NFAT and NF-κB translocation into the nucleus and prevented T cellactivation.To provide objective evaluation index on clinical therapeutic of DAP, the DNFB-induced delayed type hypersensitivity (DTH) model was established by painting0.5%DNFB on shavedabdomen and was used to evaluate the immunosuppressive effect of DAP in vivo. The impact ofDAP on the thymus index, spleen index, T lymphocytes proliferation, cytokine, CD4+T and CD8+T cell numbers and tissue edema of mouse ear were recorded. Compared with the DTH group,DAP could decrease the thymus index and spleen index. Lymphocytes from drug-treated DTHmice had reduced proliferation capacity when compared to DTH mice. Histological observationindicated DAP markedly reduced the infiltration of lymphocytes, DAP treatments also reduced theCD4+T and CD8+T cell numbers after DNFB treatment. These findings demonstrated theimmunosuppressive effect of daphnetin on DTH model induced by DNFB.The mice were immunized subcutaneously with OVA to establish an OVA-sensitization micemodel. We also investigated the effect of different concentrations of DAP on B lymphocytesproliferation, cytokine and OVA-specific IgG, IgGl, IgG2a antibody titers in OVA-sensitizationmice model. In this study, our findings demonstrated that DAP significantly inhibited Blymphocytes proliferation, cytokines and the OVA-specific IgG, IgGl, IgG2a antibodies secretion.These findings indicated that DAP could decrease immune responses in the OVA-sensitizationmodel.Collectively, DAP has inhibitory effect on humoral and cellular immunity, directly inhibits theproliferation and secretory function of T and B lymphocytes. DAP had strong immunosuppressiveactivity in vitro and in vivo, suggesting a potential role for DAP as an immunosuppressive agent.This study established the groundwork for further research on DAP and provide foundation forreasonable application of DAP in clinic and improve clinical value.