~1H-NMR Based Metabolomics Analysis Of The Effects Of Sulfated Polysaccharides From Pine Pollen On RAW264.7 Cell

Because of there special structures, polysaccharides have a variety of biological activity. Many studies had shown that polysaccharide had a variety of activities, such as antiviral, anti-tumor and enhancing the immunity. Modified polysaccharide can change it's biological activity. Our laboratory had found that the sulfated polysaccharides from Pinus massoniana pollen named SPPM60-D restored the immune activity of immune inhibitory macrophage cell RAW264.7. However PPM60-D had a weaker effect. Researchers had found that sulfated polysaccharide from Pinus massoniana pollen called SPPM inhibited the proliferation of tumor cells in vivo and in vitro, at the same time, promoted the proliferation of T lymphocyte and B lymphocyte cells. And the effect was more significant than polysaccharides from Pinus massoniana pollen which named PPM. The aim of this research is to investigate the effects of SPPM60 on RAW264.7 cell by using nuclear magnetic resonance(NMR). My experiment is divided into six sections:1. Polysaccharides was extracted with hot water and precipitated by alcohol. Used Trifluoroaceticacid method to remove proteins. Separated the polysaccharides which were precipitated by 60% alcohol with Sephacryl S-400 HR. We named the homogeneous component as PPM60-D. Then sulfated modified the PPM60-D with chlorosulfonic acid-pyridine, and named SPPM60-D.2. Detected the influences of different concentrations of PPM60-D and SPPM60-D on the proliferation of RAW264.7 with MTT method. The results were as same as the previous laboratory research that SPPM60-D could significantly improve the proliferation of RAW264.7 at 800 ?g/mL, and the result was not significant with PPM60-D. Because SPPM60-D in the 400 ?g/mL and 800 ?g/mL could also significantly improve the proliferation of RAW264.7 and the difference between them was not significant, furthermore, the extraction process of polysaccharides was time-consuming and complicated,so we chose 400 ?g/mL SPPM60-D as the working concentration.3. In order to obtain the intracellular metabolites, cells were quenched using ice cold methanol. Cells were detached from the culture dish using a cell lifter. Intracellular metabolites were extracted using a dual phase extraction procedure with methanol chloroform and ultrapure water. After sonicated on ice, mixed the solution using vortex mixer and centrifugation, the aqueous phase was used for experiment. After that, removed the solvents by using a vacuum concentrator, and then reconstituted in D2 O. Remove the D2 O by freeze dying and reconstituted in D2 O with PBS. After centrifugation the supernatant was pipetted into a NMR tube.4. All the samples were detected by nuclear magnetic resonance spectromete(Bruker AVANCE ? 600). Thirty-five kinds of intracellular metabolites were identified through the human metabolome database(HMDB), Biological Magnetic Resonance Data Bank(BMRB) and the relevant literature, which were amino acid, choline, organic acids and alcohols.5. Processed data were imported into SIMCA-P+12.0 software(Umetrics Inc., Umea, Sweden) for multivariate statistical analysis and analyzed by partial least squares discriminant analysis(PLS-DA) and orthogonal partial least squares discriminant analysis(OPLS-DA). The results showed that the samples of SPPM60 group, lipopolysaccharides(LPS) group and control group distributed in different region. The contents of metabolites which were different between groups were confirmed by variable importance in the projection(VIP) value, the loading weights and the correlation coefficients(Pcorr). Compared with control group, the concentration of thirteen kinds of intracellular metabolites were increased in LPS group, including alanine, choline, proline, betaine and glycine, acetic acid, aspartic acid, creatine, uridine, threonine, leucine, ethanol and methanol, on the contrary, the concentration of five kinds of metabolites were decreased, including lactic acid, taurine, formate, trimethylamine oxide and glutamine. Compared with blank control group, twelve kinds of metabolites were increased, including betaine, proline, choline, glycine, alanine, creatine, acetic acid, aspartic acid, creatine phosphate, trimethylamine oxide, isoleucine and threonine in SPPM60-D group. Three kinds of intracellular metabolites were decreased, including lactic acid, taurine, and ethanol, respectively. The concentration of formate and trimethylamine oxide were increased in SPPM60-D group and ethanol were decreased LPS group had the opposite result. Pathway enrichment analysis showed that SPPM60 and LPS promoted the proliferation of macrophage by increasing protein synthesis, betaine metabolism and glycine, serine and threonine metabolism and decreasing taurine and hypotaurine metabolism. The result showed that LPS and SPPM60-D promoted the proliferation of RAW264.7 cell by promoting aerobic respiration process and reducing the process of glycolysis through the analysis of the different metabolites. But it's not clear that the specific mechanism of SPPM60 affect the RAW264.7 cell and the mechanism of the differences between two groups.