Immunomodulatory Effects Of Sulfated Polysaccharide And Its Ester From Pine Pollen On Mice T Lymphocytes

Former research of our laboratory had indicated that the sulfated polysaccharidesfrom the60%ethyl alcohol precipitate of the Pinus massoniana pollen （PPM60）could promote the calcium ascension in spleen suspension of mice, And found thatthe sulfated polysaccharides can promote lymphocyte [Ca²⁺]ito heighten which isassociated with TLR4-PI3K-PLC signal pathway. But it is not clear that how dothe polysaccharides as one kind of big molecular weight compound affect on the Tcells. The aim of our research is to probe into the mechanism of sulfated poly--saccharides caused calcium rising in the T lymphocytes, and the influence oncytokines in it,and try to make clear the immune-enhancing mechanism in vitro. Theexperiment was divided into the following sections.1. Polysaccharide was extracted from Pinus massoniana pollen with hot waterand precipitated by20%,40%,60%and80%alcohol. Trifluoroaceticacid methodwas used twice to remove protein. We chosen Sephacryl S-400HR to separate thepolysaccharides of precipitated by60%alcohol and got a homogeneous component.We chosen25mg piuns massoniana pollen polysaccharides of60%alcohol precipitated （PPM60） to become sulfated （SPPM60） by chlorosulfonic acid-pyridine methodand get the white product of31.6mg. With the barium chloride-gelatin turbidimetricmethod to determine the substitution degree was1.2. There were characteristicabsorptions of sulfate ester bond （C-O-S and S=O） showed with infrared ray （IR）spectrum of SPPM60.2. The mice spleen T lymphocytes was separated with nylon wool.The MTT method was used to detect the influence of different concentrations anddifferent hours of PPM60and SPPM60for the proliferation of T lymphocytes.After48-hour treatment with200μg/mL, the proliferation rate reached to15.7%in the T lymphocytes. And for PPM60, the proliferation function was relativelyweaker.3.We tested the effects of the polysaccharides components on the concentrationof free calcium in mice spleen T lymphocytes. Concentration of Ca²⁺in Tlymphocytes was measured with Fura-2double wavelength fluoremetry.SPPM60significantly increased [Ca²⁺]icompared with control group.2-APB andU73122could significantly lower SPPM60elevated [Ca²⁺]iin T lymphocytes.LY294002and verapamil also lower SPPM60elevated [Ca²⁺]iin T lymphocyteHowever, TAK-242had a little effects on SPPM60elevated [Ca²⁺]iin Tlymphocytes.4. Enzyme immunosorbent adsorption experiment method was used to detectSPPM60or PPM60on the expression of IL-2, IL-4in T cell culture supernatant. Theresults showed that SPPM60in concentration of200μg/mL stimulate48hours, theproduction of IL-2and IL-4had been increased in the culture medium.Compared withthe control, TAK-242can not inhibit the secretion of IL-2and IL-4, however,2-APBcan inhibit them.The above results showed that:（1） PPM60was weaker than SPPM60in theproliferation of T lymphocytes.（2） Perhaps SPPM60promoted the proliferation of Tlymphocytes by increasing intracellular calcium concentration.（3） SPPM60couldpromote [Ca²⁺]iby SOC channel and TCR/CD3-PI3K-PLC signaling pathway.（4）SPPM60could promote the production of IL-2, IL-4in T cell culture supernatant.