Sulfated Polysaccharides From Pine Pollen On The Effects Of [Ca²⁺]_i And Antibody Production Of B Lymphocytes

Former research of our laboratory had indicated that the sulfated polysaccharides frompinus massoniana pollen produced by hot water and60%ethyl alcohol (SPPM60) couldsignificantly promote the calcium ascension in spleen suspension of mice, and found thatSPPM60can promote the intracellular free calcium concentration ([Ca2+]i) and level of IL-2and IL-4in splenocytes which is associated with TLR4-PI3K-PLC signal pathway. In addition,we investigated the effects of SPPM60to T lymphocytes, and found that it can promote theproliferation,[Ca2+]iand the level of IL-2and IL-4of T lymphocytes viaPI3K-PLC-IP3R-Ca2+signal pathway. We confirmed that the large increasment of [Ca2+]iiscaused by extracellular free calcium through Ca2+release activated Ca2+(CRAC) channel, butTOLL like receptor4(TLR4) is not a receptor of SPPM60on T lymphocyte. But it’s not clearthat how did PPM60and SPPM60as one kind of big molecular weight compound affect Blymphocytes. The aim of this research is to investigate the effects of PPM60and SPPM60onB lymphocytes. Compare the differences between PPM60and SPPM60to B lymphocytes,and preliminarily clarify the mechanism of effects of SPPM60to B lymphocytes in vitro. Theexperiment is divided into the following sections:1. Polysaccharides was extracted from piuns massoniana pollen with hot water andprecipitated by20%,40%,60%and80%alcohol. Trifluoroaceticacid method was usedtwice to remove protein. We chose Sephacryl S-400HR to separate the polysaccharides ofprecipitated by60%alcohol and got a homogeneous component (PPM60-D). Then chose25mg PPM60-D to become sulfated polysaccharide (SPPM60-D) by chlorosulfonicacid-pyridine method and got white products of31.6mg. Determine the substitution degreewas1.2with the barium chloride-gelatin turbidimetric method. There were characteristicabsorptions of sulfate ester bond (C-O-S and S=O) showed in infrared ray (IR) spectrum ofSPPM60-D.2. The mice spleen B lymphocytes was separated with nylon wool. MTT method wasused to detect the influences of different concentrations and different hours of PPM60-D and SPPM60-D on the proliferation of B lymphocytes. After48-hour treatment withSPPM60-D in200μg/mL, the B cell proliferation rate reached to13.88%. And for PPM60-D,the proliferation rate was7.13%.3. We tested the effects of PPM60-D and SPPM60-D on the [Ca2+]iin mice spleen Blymphocytes. The concentration of Ca2+in B lymphocytes was measured with Fura-2/AMdouble wavelength fluoremetry. SPPM60-D and PPM60-D both significantly increased[Ca2+]icompared with control group in B lymphocytes. The effects of SPPM60-D is betterthan PPM60-D. TAK-242, U73122,2-APB, verapamil and LY294002could significantlylower the SPPM60-D elevated [Ca2+]iof B lymphocytes to varying degrees. However, lowmolecular weight heparin (LMWH) could completely inhibit SPPM60-D caused [Ca2+]irisingof B lymphocytes.4. We used plaque forming assay (PFC) and quantitative hemolysis spectrophotometry(QHS) to test antibody production of B lymphocytes stimulated by PPM60-D and SPPM60-D.The results showed that no matter immune in vivo or in vitro, SPPM60-D could significantlypromote the antibody production of B lymphocytes and had a fast effect in vitro. HoweverPPM60-D had a weaker effect.The above results showed that:(1) PPM60-D and SPPM60-D could promote theproliferation,[Ca2+]iand antibody production of B lymphocytes significantly, but the effectsof PPM60-D was weaker.(2) Perhaps SPPM60-D promoted the proliferation and antibodyproduction of B lymphocytes by increasing intracellular calcium concentration.(3)SPPM60-D could promote [Ca2+]iby store-operated Ca2+entry (SOCE) andTLR4-PI3K-PLC-IP3R-Ca2+signaling pathway.