Effects Of Pine Pollen Polysaccharides And Sulfated Polysaccharides On Colitis And Intestinal Flora In Mice

Pine pollen polysaccharide and sulfated polysaccharidecan regulate immune function and stimulate splenic lymphocytes to secrete anti-inflammatory factors.The fluorescent polysaccharide tracing experiments showed that the pollen polysaccharide can enter the rat intestinal crypt epithelial cells through the cell surface receptor Toll like receptor 4(TLR4).Therefore,we speculate that pine pollen may affect intestinal immunity and improve intestinal related diseases.In this experiment,using DSS-induced mouse ulcerative colitis as a model,the effects of pine pollen polysaccharides and sulfated polysaccharides on inflammation in mice were investigated through three levels of intestinal immunity,serum metabolomics and fecal metagenomics.This experiment mainly includes the following parts:1.The monosaccharide composition and sulfate modification of Pinus yunnanensis pollen polysaccharide were performed.The polysaccharide of pine pollen was extracted by the method of water boiling and ethanol precipitation.The precipitated polysaccharide by 60%ethanol was named PPM60.The monosaccharide was mainly composed of galactose,glucose,xylose,mannose,rhamnose and an unknown monosaccharide.The polysaccharide of pine pollen was sulfated with pyridine-chlorosulfonic acid,and the degree of substitution was1.45 by the method of BaSO4 turbidimetric.The results of FTIR also showed that the polysaccharide was sulfated successfully.2.DSS induced ulcerative colitis model in mice,to investigate the effect of polysaccharide and sulfated polysaccharide on colitis mice.C57BL/6 mice were divided into four groups:healthy control group(HC),model group(DSS),polysaccharide group(PM)and sulfated polysaccharide group(SPM).The results showed that compared with HC group,the weight of DSS group mice was reduced,DAI index was increased,colon length was significantly shortened,mucosal tissue was seriously damaged,and a large number of inflammatory cells were infiltrated,which indicated that the colon lesions of mice were successful.The results showed that PPM60 and SPPM60 could repair the colon damage and improve the development and pathological changes of colitis.3.The effects of polysaccharide and sulfated polysaccharide on the intestinal immunity of colitis mice were studied by detecting the levels of serum C-reactive protein(CRP)and cytokines in colon tissue.The results showed that in DSS group,the level of serum CRP increased significantly,the level of anti-inflammatory cytokines including IL-2,IL-10 and IL-13 decreased,and the level of pro-inflammatory cytokines including IL-1β,IL-6 and TNF-αincreased.PPM60 and SPPM60 can improve this trend,and polysaccharide has better effect on inhibiting the level of pro-inflammatory cytokines,and sulfated polysaccharide has better effect on anti-inflammatory cytokines.4.The changes of serum metabolome in mice were analyzed by ~1H-NMR.The results of multivariate statistical analysis showed that the four groups of mice had the trend of separation,and when the two groups of mice were compared,they could get a good distinction.HC group and DSS group identified eight possible differential metabolites such as N-acetyl-l-alanine,Acetic acid,L-Fucose,Lactic acid,Taurine,Betaine,Acetylcholine and Allose.DSS and PM groups identified seven possible differential metabolites including N-acetyl-l-alanine,Acetic acid,Lactic acid,Fructose-6-phosphate,Allose,D-Xylose and L-carnitine.DSS group and SPM group also identified seven possible differential metabolites,including Betaine,Glycerophosphate,L-serine,D-Xylose,Acetylcholine,Lactic acid and Allose.5.The 16S rDNA amplicon sequencing technology was used to analyze the flora of feces and cecum contents of mice,and to study the changes of species diversity and the effects of PPM60 and SPPM60 on the changes of intestinal flora.The results showed that in the DSS group,the species diversity decreased,the abundance of harmful bacteria increased,the abundance of beneficial bacteria decreased,and the flora was imbalanced.PPM60 and SPPM60 can restore species diversity,and have a down-regulation effect on the genus of Aerococcus and Akkermania,and up-regulation effect on Lactobacillus and Parasutterella.The results showed that polysaccharide and sulfated polysaccharide can improve the ulcerative colitis induced by DSS,including ameliorating colon shortening,repairing intestinal injury,alleviating intestinal inflammation,regulating serum metabolome and regulating the balance of intestinal flora.