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-?? on murine spleen lymphocytes in vitro, and discuss its possible mechanism.Methods: 1. The investigation of immunoregulatory effect: The effects of LEGPS-?on the proliferation of resting and ConA-induced murine spleen lymphocytes were observed with MTT method in vitro. The effect of LEGPS- ? on cell cycle was detected with propidium iodide stain and flow cytometry. Different concentrations of LEGPS- ? were cultured with resting and ConA-induced lymphocytes, the cytokine about TNF-? 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- ?.Result: LEGPS-? enhanced the proliferation of resting lymphocytes ?dose range3.125?100?g/ml? and ConA-induced lymphocytes ?dose range 0.781?100?g/ml?with dose-dependent. LEGPS-? ?100?g/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- ? ?25?100?g/ml?had no effect on the excretion of TNF-? in resting lymphocytes, but in lymphocytes induced by ConA with dose-dependent. LEGPS-? ?6.25?100?g/ml? had no effect on the secretion of IL-2 in resting lymphocytes, but in ConA-induced lymphocytes with dose-dependent.LEGPS-? ?100?g/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- ? 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- ? 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-? 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-? and IL-2 in ConA-induced lymphocytes. The mechanism of action may be related to calcium signal transduction pathway in lymphocytes.