Studies On The Anti-tumor And Immunomodulatory Effects Of Pine Pollen Polysaccharide On H22 Tumor-bearing Mice Before And After Sulfated

Pinus yunnanensis polysaccharides and sulfated polysaccharides have been studied at the cellular level in vitro.Previous experiments in the laboratory showed that pine pollen polysaccharides and sulfated polysaccharides can promote macrophage proliferation and activate their immune effects in vitro,and indicate it is possible to inhibit the proliferation of human hepatoma cell HepG2 by cell cycle arrest.The anti-tumor effect and immune effect of pine pollen polysaccharide and sulfated polysaccharide were combined with in vitro and in vivo experiments to establish a mouse H22 hepatocellular cancer transplantation model,and the mouse serum was subjected to nuclear magnetic resonance analysis combined with metabolome.To analyze the effects of pine pollen polysaccharides and esterified polysaccharides on the cytokines and serum metabolites in H22 cells and tumor-bearing mice before and after the action of pine pollen polysaccharides and esterified polysaccharides,and to explore the anti-tumor effect of pine pollen before and after esterification.1.Extraction,detection and purification of Pinus yunnanensis polysaccharide.The polysaccharide was extracted by hot water extraction,the protein was removed by trichloroacetic acid,and the precipitate was precipitated with different concentrations of ethanol to obtain a polysaccharide precipitated from 60% ethanol,and named as PPM60.The polysaccharide content was measured and the polysaccharide content was determined to be 44.02%.The PPM60 was separated and purified by atmospheric pressure preparative chromatography.Five polysaccharide fractions were obtained.The purity of the obtained five polysaccharide fractions was also determined by atmospheric pressure chromatography.Finally,the third component with appropriate purity and activity was selected and named it as PC.2.The PC is sulfated.The method used for sulfation of PC was chlorosulfonic acid-pyridine method,and the degree of sulfate substitution was measured by barium sulfate turbidimetry.Theresult showed that the degree of sulfate substitution was 1.7,and the PC which successfully sulfated and it was named SPC.The detection of PC and SPC by infrared spectroscopy showed that the sulfation of PC was successful.3.The effects of PC and SPC on proliferation,migration and cell cycle of H22 cells were investigated by cell expriment.The cell proliferation activity was measured by MTT assay.The results showed that both PC and SPC could significantly inhibit the proliferation activity of H22 cells at 800 ?g/mL(p<0.01),and the inhibition of H22 proliferation activity by PC and SPC also showed differences(p<0.01),so the subsequent experiments were explored.The concentration of 800?g/mL was selected;PC could inhibit the migration ability of cells,but there was no significant difference.SPC could significantly inhibit the migration ability of cells compared with the control group and PC group(p<0.01);SPC was compared with the control group and PC group,in comparison,the number of cells arrested in the G2 phase increased significantly(p<0.05).Compared with the control group,the G2 phase cells increased but did not differ.The expression of cell cycle-associated protein mRNA was detected by real-time PCR,and cyclinA in the SPC group.The expression levels of cyclinA(p<0.01)? cyclinB(p<0.01)and CDK1(p<0.05)were significantly decreased,and the expression of P21 was significantly increased(p<0.01).4.To explore the anti-tumor effect of PC and SPC in H22 tumor-bearing mice.The H22 hepatocellular cancer transplantation model was established by BALB/C mice.The intraperitoneal injection concentration of PC and SPC was 3.125 mg/kg.The formal experiment was divided into four groups,namely healthy control(HC).Model control(MC),polysaccharide group(PC),esterified polysaccharide group(SPC),intraperitoneal administration for 10 days,mouse serum was taken for cytokines TNF-?,IFN-? and IL-6.Determination and nuclear magnetic resonance analysis,and calculation of tumor weight,tumor inhibition rate and organ index.In addition,1H-NMR technology was used to detect the metabolites in serum of mice from different groups,so as to find out the differences in serum metabolites of mice from different groups.Our research shows that Pinus yunnanensis polysaccharides and sulfated polysaccharides have certain immunomodulatory and anti-tumor effects.At the same concentration,sulfated polysaccharides have significant effects on cell proliferation,migration ability and cell cycle compared with polysaccharides.It indicated that the effect of sulfated polysaccharides on tumor cells was significantly better than that of polysaccharides.By flow cytometry and real-time PCR analysis,SPC could not induce apoptosis of H22 cells,but inhibited cell proliferation and migration by causing G2/M phase arrest.In the tumor-bearing mice,the SPC inhibition rate was 24.48%,which was significantly higher than the 1.17% inhibition rate of the PC group.Although the average tumor weight of the SPC group was significantly reduced,there was no significant difference after the statistical analysis.Determination of TNF-? and IL-6 and IFN-? levels in serum and nuclear magnetic resonance analysis.Compared with PC group,serum of TNF-?,IL-6 and IFN-? were significantly different in SPC group.The PC group had no significant effect on the metabolic disorders caused by tumor-bearing mice,especially the reduction of bile acid synthesis.However,the SPC group was able to reverse the decrease of bile acid synthesis and other amino acid metabolism disorders caused by tumor-bearing mice,making the sulfated polysaccharide group metabolic levels approach the metabolism of normal mice.