The Immunoregulatory Effects And Mechanism Of Polysaccharide From Lappula Echinata Gilib In Mice In Vitro

Objective: To study on the immunoregulatory effects of polysaccharide from Lappula echinata Gilib （LEGPS-Ⅱ） on murine spleen lymphocytes in vitro, and discuss its possible mechanism.Methods: 1. The investigation of immunoregulatory effect: The effects of LEGPS-Ⅱon the proliferation of resting and ConA-induced murine spleen lymphocytes were observed with MTT method in vitro. The effect of LEGPS- Ⅱ on cell cycle was detected with propidium iodide stain and flow cytometry. Different concentrations of LEGPS- Ⅱ were cultured with resting and ConA-induced lymphocytes, the cytokine about TNF-α and IL-2 in the supernatant of lymphocytes were assayed by MTT method and enzyme-linked immunoabsorption assay （ELISA） separately.2. The investigation of mechanisms: Laser scanning confocal microscope （LSCM） of the calcium fluorescent indicator dye Fluo-3/AM were used to determine the concentration of [Ca²⁺]i in the lymphocytes influenced by LEGPS- Ⅱ.Result: LEGPS-Ⅱ enhanced the proliferation of resting lymphocytes （dose range3.125～100μg/ml） and ConA-induced lymphocytes （dose range 0.781～100μg/ml）with dose-dependent. LEGPS-Ⅱ （100μg/ml） changed the cell-cycle distribution of lymphocytes and increased the proportion of S-phase and G2/M-phase cells, the proliferation index （PI） increased from 14.60% to 23.90%. LEGPS- Ⅱ （25～100μg/ml）had no effect on the excretion of TNF-α in resting lymphocytes, but in lymphocytes induced by ConA with dose-dependent. LEGPS-Ⅱ （6.25～100μg/ml） had no effect on the secretion of IL-2 in resting lymphocytes, but in ConA-induced lymphocytes with dose-dependent.LEGPS-Ⅱ （100μg/ml） increased the concentration of [Ca²⁺]i in resting murine spleen lymphocytes. Compared with the baseline, the elevation amplitude is about（180.6±5.73）%. LEGPS- Ⅱ also increased the concentration of [Ca²⁺]i in lymphocytes after treatment with EGTA and Verapamil, but the elevation amplitude was much smaller than in the non-treatment group. After treating the cells with heparin and procaine to block IP3 and Ryanodine receptors, respectively, LEGPS- Ⅱ increased the concentration of [Ca²⁺]i in these cells, but both of the elevation amplitude were less than in the non-treatment group.Conclusion: LEGPS-Ⅱ expresses its immunoregulatory effects by improving the DNA synthesis, making the cell-cycle progress into mitosis phase, stimulating lymphocytes proliferation, and enhancing the secretion of TNF-α and IL-2 in ConA-induced lymphocytes. The mechanism of action may be related to calcium signal transduction pathway in lymphocytes.