Effects Of Pine Pollen Polysaccharides And Esterified Polysaccharides On Colitis In Mice By Regulating Th17/Treg

The polysaccharides and sulfated polysaccharides of pine pollen have good immune activity,which can help T cell proliferation and differentiation of different subgroups,thereby stimulating the production of anti-inflammatory factors and inhibiting the release of pro-inflammatory cytokines.Therefore,we speculate that pine pollen may affect Th17/Treg balance,improve intestinal immunity,and thereby improving UC.In this experiment,a model of ulcerative colitis induced by DSS chemical reagents was selected to explore the effects of pine pollen polysaccharides and sulfated polysaccharides on the balance of Th17/Treg in mice under the three dimensions of intestinal immunity,serum metabolomics and metagenomics effect.This experiment has the following parts:1.The monosaccharide composition analysis of Pinus yunnanensis pollen polysaccharide and the preparation of pine pollen esterified polysaccharide.The pine pollen polysaccharide was made by boiling water extraction and alcohol precipitation method,and the crude polysaccharide extracted and processed with 60%ethanol is named PPM60.Its monosaccharide composition is galactose,glucose,xylose,mannose,rhamnose and an unknown monosaccharide.The third component of crude polysaccharide separated by Sephacryl^TM S-400 was selected and named PPM60-III for subsequent experiments.The chlorosulfonic acid-pyridine method was used to esterify PPM60-III.The degree of substitution of the esterified polysaccharides（SPPM60-III）was determined to be 1.21 by the Ba SO₄ turbidimetric test.FTIR is used to detect the signs of esterified polysaccharides.2.The animal model of ulcerative colitis induced by DSS chemical reagents was selected to further reveal the improvement and cellular mechanism of polysaccharide and sulfated polysaccharide.C57BL/6 mice,freely ingested drinking water containing 3%DSS,were modeled for one week,and were randomly divided into four groups:healthy control group（HC）,model group（DSS）,polysaccharide group（PM）and sulfated polysaccharide group（SPM）.Results showed that compared with the HC group,there are significant differences in weight loss in the DSS group.The colon tissue is significantly damaged,the DAI index is increased,and the appearance of the colon is significantly shortened.After calculation,the above characteristics meet the standards of the ulcerative colitis model.In the PM and SPM group,the increase in the DAI index was significantly suppressed,the shortening of the colon was effectively suppressed,the intestinal injury was improved,and the trend of weight loss was effectively controlled,.It indicated that PPM60-III and SPPM60-III have an effect on DSS-induced colitis It has a positive effect on the body and accelerate the recovery,SPPM60-III has a better improvement effect.3.To study the immunoregulatory effects of PPM60-III and SPPM60-III on the balance of Th17/Treg cells in colitis mice.The results showed that in the DSS group,Th17 cells increased significantly,and Treg cells decreased significantly.Compared with the DSS group,after treatment,the percentage of Th17 cells in the PM group decreased slightly,and the SPM group decreased significantly,which was almost close to the level of the HC group.Compared with the DSS group,the content of Treg cells in the PM group increased significantly,and the SPM group had a more significant increase.It has a significant effect on cytokines.Compared with HC group,the concentration of pro-inflammatory cytokines IL-1β,IL-6,TNF-αand IL-17A in DSS group increased significantly,while the concentration of anti-inflammatory cytokines,IL-10 and TGF-βdecreased significantly.It shows that during the onset of colitis in mice,there is a severe inflammatory reaction in the colon tissue.IL-17A is a unique inflammatory factor secreted by Th17 cells.The trend of IL-17A in the treatment group is the same as that of Th17 cells.The concentration of IL-1βand IL-6 in mice has a downward trend,and the downward trend in the SPM group is more significant.The concentration of IL-10 and TGF-βanti-inflammatory cytokines in the PM group and SPM group showed a rising trend.IL-10 was secreted by Treg cells,and the trend of changes in each group was the same as the number of Treg cells.Among them,the SPM group increased significantly.Has obvious statistical significance.It shows that PPM60-III and SPPM60-III can help the secretion of anti-inflammatory cytokines IL-10 and TGF-βby regulating the balance of Th17/Treg,inhibit the secretion of pro-inflammatory cytokines,and achieve a good immune regulation effect.The SPM group had a more significant effect on colitis.4.Serum NMR analysis.Used ¹H-NMR technology to compare the differences in serum metabolites.Analysis of data showed that the four groups can be completely separated.Compared with HC group,the DSS group has 9 differential metabolites:L-glutamine,and succinic acid.L-methionine,undecanoic acid,α-lipoic acid,acetylcholine,cholesterol sulfate and caffeine.Compared with DSS,PM group has 9 different metabolites of 6-hydroxynicotinic acid,glutenedioic acid,dopamine,acetyl-L-carnitine,caffeine,xanthine,acetylcholine,indole-3-formaldehyde and quinolinic acid.Compared with DSS,the SPM group has 9 different metabolites of 6-hydroxynicotinic acid,trigonelline,L-carnosine,niacin,caffeine,hypoxanthine,acetylcholine,indole-3-formaldehyde and quinolinic acid.We speculate that in the inflammatory process of UC,due to the metabolism of alanine,aspartic acid and glutamate,the metabolism of cysteine and methionine,the tricarboxylic acid cycle,the biosynthesis of aminoacyl-t RNA,nitrogen metabolism and arginine Biosynthesis has been changed to varying degrees,led to inflammation.PPM60-III and SPPM60-III regulate the level of amino acid metabolism.Among them,PPM60-III regulates tyrosine metabolism,and SPPM60-III had regulated histidine metabolism and alanine metabolism,restoring purine metabolism and glycerophospholipid metabolism to a certain extent increase the level of nicotinate and nicotinamide metabolism and caffeine metabolism to inhibit inflammation and improve the pathological condition of UC.5.16S r DNA amplicon sequencing technology,to study the diversity of the intestinal flora in the colon,and to explore the changes in the colonic intestinal flora of PPM60-III and SPPM60-III in the process of colitis.The results showed that the intestinal flora in the HC group was relatively stable and the diversity was in a balanced state.The types and numbers of the intestinal flora in the DSS group increased.PPM60-III and SPPM60-III can make the types and numbers of the intestinal flora continue.It was speculated that the diversity of the intestinal flora was increased to maintain the stability of the intestine when colitis occurs.The main strains of HC group were Lactobacillus and Lactobacillus reuteri.This experiment carried out a correlation analysis of the intestinal flora in the mouse colon,and found that compared with the HC group,the DSS group of Escherichia coli,Escherichia-Shigella,Alistipes,Dubosiella and Blautia increased,and lactic acid The abundance of beneficial bacteria such as Lactic acid bacteria and Bifidobacterium was significantly reduced.In the PM group,the abundance of Akkermansia and Escherichia-Shigella was increased,and the number of Clostridium perfringens was significantly increased.In the SPM group Among them,the abundance of Bacteroides acidogenicus and Escherichia coli was restored to near normal levels,and the abundance of Bifidobacterium was increased.The results of this experiment show that PPM60-III and SPPM60-III have a significant improvement and repair effect on ulcerative colitis induced by DSS,which is specifically manifested in preventing the shortening of the colon and regulating the Th17/Treg balance,thereby regulating the inflammation in the colon.The reaction of factors can improve the degree of intestinal damage and affect the homeostasis of the intestinal flora through serum metabolism.