| Former research of our laboratory had indicated that the sulfated polysaccharides from the 60% ethyl alcohol precipitate of the pinus massoniana pollen (PPM60) could promote the calcium ascension in spleen lymphocytes of mouse, there were three ways at least: initiates CICR through L-calcium channel of the membrane surface; the calcium release through IP3R in the endoplasmic reticulum; through store-operated calcium channel of the membrane.But it is not clear that how do the polysaccharides as one kind of big molecular weight compound affect on the cells or cause the concentration of calcium changing. The aim of our research is to probe into the mechanism of sulfated polysaccharides caused calcium rising in the lymphocytes, and the influence on cytokines in it, and try to make clear the immuno-enhancing mechanism in vitro. In the hope of providing theory support for development and utilization of the pinus massoniana pollen polysaccharides.Tests mainly divides into four parts as follows.1. Polysaccharide was extracted from piuns massoniana pollen with hot water and precipitated by 40%, 60% and 80% alcohol. Savage method was used several times to remove protein. We chosen Sephacryl S-400HR to separate the polysaccharides of precipitated by 60% alcohol and got three components: PPM60-A, PPM60-B, PPM60-C,which molecular weight were 1.44×105,1.2×105,7.26×104. We chosen the piuns massoniana pollen polysaccharides of 60% alcohol precipitated (PPM60) to become sulfated (SPPM60) by chlorosulfonic acid-pyridine method, the efficiency was 128%, and the the degree of its sulfate was 1.2264. There were characteristic absorptions of sulfate ester bond (C-O-S and S=O) showed with infrared ray (IR) spectrum of SPPM60. 2. The MTT method was used to detect the influence of different concentrations and different times of SPPM60 and PPM60 for the proliferation of splenocytes. After 48-hour treatment with 200μg/mL, the proliferation rate reached to 18.29% in the splenocytes. And for PPM60, the proliferation function was relatively weaker. After 72-hour treatment with 200μg/mL, the proliferation rate decreased significantly. For the separated of the three components and esters, the esterification of PPM60-A has improved greatly, SPPM60-A promote proliferative capacity increased by 10.84%.3. We tested the effects of the polysaccharides components on the concentration of free calcium in splenocytes. Within 5 min, SPPM60-A significantly increased [Ca2+]i compared with control group. TAK-242 and LY294002 could significantly lower SPPM60-A elevated [Ca2+]i in splenocytes. After adding U73122- a PLC inhibitor, could complete inhibit SPPM60-A caused [Ca2+]i rising of splenocytes.4. Enzyme immunosorbent adsorption experiment method was used to detect SPPM60 or PPM60 on the expression of IL-2, IL-4 in splenocytes. The results showed that SPPM60 in concentration of 200μg/mL stimulate 48 hours, the production of IL-2 and IL-4 had been increased in the culture medium. Compared with the control, TAK-242 completely inhibited the secretion of IL-2 and IL-4.The above results showed that: (1) PPM60 was weaker than SPPM60 in the proliferation of spleen lymphocytes. (2) Perhaps SPPM60 promoted the proliferation of splenocytes by increasing intracellular calcium concentration. (3) SPPM60 could promote [Ca2+]i by TLR4-PI3K-PLC signaling pathway. (4) SPPM60 could promote the production of IL-2, IL-4 in splenocytes through TLR4. |
