Sulfated Polysaccharides From Pine Pollen On The Effects Of Immune Regulation Of Mice Macrophages

Former research of our laboratory had indicated that the sulfated polysaccharidesfrom Pinus massoniana pollen produced by hot water and60%ethyl alcohol(SPPM60) could increase the calcium concentration in spleen suspension of micesignificantly, and found that SPPM60can promote the intracellular free calciumconcentration ([Ca2+]i) in splenocytes. So we have investigated the effects of SPPM60to T lymphocytes and B lymphocytes respectively, and found that it could promote theproliferation, increase [Ca2+]i, and the immunocompetence of T lymphocytes and Blymphocytes via PI3K-PLC-IP3R-Ca2+signal pathway. We also drawn a conclusionthat the increasing of [Ca2+]iof T lymphocytes and B lymphocytes was caused bySPPM60→Receptor→PI3K→PLC→IP3→IP3R→CRAC mainly. As we know, Tolllike receptor4(TLR4) is an important receptor on B lymphocyte. Through theexperiment research further confirmed that the TLR4is the primary receptor ofSPPM60-D on B lymphocyte. We have many studies about the influence ofpolysaccharides on T, B lymphocyteshas. But up to the present it’s not clear that theinfluence of polysaccharides and sulfated polysaccharides from Pinus massoniana(PPM60and SPPM60) on mice macrophages and how did PPM60-D and SPPM60-Das one kind of big molecular weight compound affect macrophages. The aim of thisresearch is to investigate the effects of PPM60-D and SPPM60-D on micemacrophages (RAW264.7and mice peritoneal macrophages). Comparing the differenteffects between PPM60-D and SPPM60-D to macrophages, and preliminarily clarifythe active mechanism of PPM60-D and SPPM60-D to mice macrophages(RAW264.7and mice peritoneal macrophages) in vitro. The experiment was divided into thefollowing sections:1. Polysaccharides was extracted from Piuns massoniana pollen with hot waterand precipitated by20%,40%,60%and80%alcohol. Trifluoroacetic acid was usedto remove protein. Sephacryl S-400HR was used to separate the polysaccharides of precipitated by60%alcohol and got a homogeneous component, named PPM60-D.About14.87mg PPM60-D was derivated to sulfated polysaccharide (SPPM60-D) bychlorosulfonic acid-pyridine method and got white products of18.57mg. The degreeof the substitution was1.87tested by the barium chloride-gelatin turbidimetricmethod. There were characteristic absorption of sulfate ester bond (C-O-S and S=O)with infrared ray (IR) spectrum of SPPM60-D.2. Mice peritoneal macrophages were extracted by using peritoneal lavage. MTTmethod was used to detect the influences of different concentrations PPM60-D andSPPM60-D on the relative activity of the mice peritoneal macrophages. Aftertreatment with SPPM60-D in800μg/mL, relative activity of the mice peritonealmacrophages was the best. However, the effect of PPM60-D was not significant.MTT was also used to detect the influence of different concentrations of PPM60andSPPM60on the proliferation of macrophage RAW264.7. After treatment withSPPM60-D in800μg/mL, the proliferation of RAW264.7was stronger. And forPPM60, the proliferation function was relatively weaker.3. We tested the effects of PPM60-D and SPPM60-D on the [Ca2+]iin micemacrophage RAW264.7. The concentration of Ca2+in macrophages RAW264.7wasmeasured with Fura-2/AM double wavelength fluoremetry. SPPM60-D significantlyincreased [Ca2+]icomparing with control group in macrophages. TAK-242, U73122,2-APB, verapamil and LY294002could significantly lower the SPPM60-D elevated[Ca2+]iof macrophages to varying degrees. However, low molecular weight heparin(LMWH) could completely inhibit SPPM60-D caused [Ca2+]irising of macrophages4. In order to understand the relationship between the SPPM60and immuneregulation of mice macrophages, we detected the adhension, migration, phagocytosis,cytokine secretion and the NO synthsyzing of macrophages. TLR4is the importantreceptor on mice macrophages. To study the role of TLR4in macrophages we usedthe TAK-242(inhibitors of TLR4) to suppress the function of TLR4. The resultsshowed that SPPM60-D can promote the immune regulation of macrophagesobviously. What is more we known that the TLR4is the main receptor of SPPM60-Don mice macrophages. 5.In addition, we also detected the function of SPPM60-D on theimmunosuppressive macrophages.The results showed that SPPM60-D restored thefunction of immunosuppressive macrophages effectively including phagocytosis,cytokine (IL-1、IL-6、TNF-α) secretion and the NO synthasing.