Structural Characterization And The Mechanism Of Action Ganoderma Applanatum Polysaccharides On The Intestinal Barrier Function Protection

The intestinal barrier is one of the largest and most important internal barrier in vivo,which protects the host from toxic substances and microorganisms located in the intestinal lumen.Dysfunction of the intestinal barrier is associated with intestinal infections,IBD and other intestinal diseases.Dietary fiber is one of the main strategies to improve the integrity of the intestinal barrier.Edible mushroom polysaccharides,as the functional substance components of biogenic foods,can provide energy and nutrients to the host,and also can improve gut microbiota,regulate host immunity,and promote body’s health.As an important edible mushroom polysaccharide,Ganoderma applanatum polysaccharides(GAPs),its action mode and regulatory mechanism of promoting body health are still unclear.And the functional understanding and utilization of GAPs need to be further emphasized.Based on the above,this project investigated the protective effects of GAPs on intestinal barrier during the development and recovery of intestinal barrier damage,and seeked its mechanism in different periods.Firstly,we explored the impacts of GAPs on protecting the intestinal barrier by constructing a model of gut barrier damage.Secondly,we obtained the purified polysaccharides of GAPs by column chromatography and characterized the active structure.Thirdly,we investigated the alterations of active GAPs on the gut microbiota.Finally,we analyzed the mechanisms of intestinal barrier protection during the development and repair phases of intestinal barrier damage.The main details of this study as follows:(1)By constructing a DSS-induced intestinal barrier damage model,GAPs administration could reduce mortality during the recovery period of DSS mice,promote weight recovery,reduce colon length shortening,lower disease activity index(DAI)scores,promote colon tissue recovery and reduce the lymphocyte infiltration.GAPs administration also reduced the levels of intestinal inflammation-related enzymes and promoted the recovery from intestinal barrier damage.Meanwhile,GAPs administration increased the abundance of bacteria associated with polysaccharide degradation and short-chain fatty acids(SCFAs)production.In a mouse-injection model with intestinal damage caused by Salmonella typhimurium infection,GAPs supplement could increase the levels of MUC2 and tight junction proteins,maintain intestinal barrier homeostasis and prevent translocation of Salmonella typhimurium into the liver.Furthermore,GAPs could protect against intestinal inflammation,and decrease the levels of intestinal inflammation-related enzymes and pro-inflammatory factors.Besides,in ABx and DSS-treated mice,the promoting recovery from intestinal damaged by GAPs was performed in a gut microbiota-dependent manner.(2)The crude polysaccharides GAPs were purified using a Hi Trap DEAE FF column and Hi Load 16/20 Superdex 75pg column.The major fractions GAP1,GAP2,GAP3and GAP4were obtained.GAP1 and GAP2 exhibited protective effects on the intestinal barrier during Salmonella typhimurium invasion of Caco-2 cells.GAP1 and GAP2 prevented the disruption of adherens junctions by promoting the expression of intestinal barrier tight junction proteins.At the developmental phase of intestinal barrier damage caused by DSS,GAP1 and GAP2 were found to protect the intestinal barrier from dysfunction through attenuating body weight loss,reducing colon length shortening and DAI score,increasing structural integrity of the epithelial mucosal layer,decreasing focal infiltration of lymphocytes and evaluating the abundance of mucin/mucin-producing cells.Meanwhile,GAP1 and GAP2 could significantly reduce levels of inflammation-related enzymes and pro-inflammatory cytokines and increase levels of anti-inflammatory cytokines,mucins and tight junction proteins.Combined the molecular weight determination,monosaccharide composition analysis,fourier-transform infrared spectroscopy,and nuclear magnetic resonance analysis,the average M_w of GAP1 was calculated to be 1.9033×10~4 Da and composed of Man,Glc,and Gal.The primary structure of GAP1 might be defined as a→3)-β-Glcp-(1→4)-α-Glcp-(1→main chain,with 6→2)-α-D-Manp-(1→,6→4)-α-Glcp-(1→4)-α-Gal A-(1→,and 6→1)-α-D-Glcp-(6→side chains.The average M_w of GAP2 was calculated to be 2.0537×10~4 Da,and the primary structure of GAP2 might be defined as a→3)-β-Glcp-(1→main chain.(3)Analysis of colonic contents by 16S r RNA sequencing,short chain fatty acids,SCFA spectrometry and metagenomic sequencing,revealed that GAP1 changed the intestinal environment and enhanced alpha diversity in healthy mice.The relative abundance of Rikenellaceae RC9 gut group,Bifidobacterium,Oscillibacter and Negativibacillus were increased.Meanwhile,GAP1 put promoting effects on amino acid metabolism and the production of propionic acid,butyric acid,acetic acid and valeric acid.GAP2 affected beta diversity but had no significant difference in alpha diversity compared to the model group.Moreover,GAP2 administration could increase the relative abundance of Ruminococcaceae UCG＿014,Ruminiclostridium 6 and Ruminococcus 1 connected with the SCFAs production,and improve amino acid metabolism and the production of butyrate and acetate in mice.In comparison of the differences in gut microbiota between GAP1 and GAP2,the relative abundance of Clostridia bacterium,Ruminococcus sp.,Candidatus Saccharibacteria bacterium,Bacteroides stercorirosoris and Bacteroides oleiciplenu in the GAP2 group were higher than that in the GAP1 group,whereas,the relative abundance of Muribaculaceae bacterium Isolate-080,Prevotella sp.MGM2,Muribaculaceae,Phocaeicola vulgatus,Bacteroidaceae bacterium,Duncaniella dubosii,bacterium J10,Phocaeicola,Bacteroidetes bacterium and Muribaculaceae in the GAP1 group were higher than the GAP2 group.The differences between GAP1 and GAP2 in the enzyme families mainly focused on the GH and GT families.Correlation analysis showed that the proportion of Akkermansia muciniphila,Ruminococcus sp.and Clostridia bacterium were positively correlated with GT8,and Bacteroides fragilis,Bacteroides sp.,Bacteroides sp.CAG:927,Bacteroides sp.L10-4,Bacteroides uniformis,Muribaculum intestinale and Duncaniella sp.B8 were significantly correlated with GH31.GAP2 mainly affects lysine biosynthesis,the succinyl-DAP pathway and the aspartate-to-lysine pathway,whereas GAP1 mainly affects pyrimidine deoxyribonucleotide biosynthesis and the UDP-to-d TTP pathway.Metabolic enrichment analysis demenstrated that GAP2 could promote cysteine and methionine metabolism,aminoacyl-t RNA biosynthesis and histidine metabolism compared to GAP1.(4)GAP1 was found to improve immunity and kept intestinal barrier integrity by increasing energy metabolism,while GAP2 protected the intestinal barrier by increasing ATP metabolism,ameliorating mitochondrial dysfunction and preventing endoplasmic reticulum stress.In a DSS-induced GF mouse model of intestinal barrier damage,a gut microbiota-dependent protection of GAP1 and GAP2 against colitis was revealed.GAP1 administration in DSS-induced colitis significantly increased the abundance of Rikenellaceae RC9 and Lachnospiraceae NK4A136 and the levels of acetate,butyrate,propionate,isobutyrate and valeric acid.Colitis mice with GAP2 administration could significantly increase the abundance of Bacteroides and Alistipes and decrease the abundance of Escherichia＿Shiaella.Also,it could enhance the production of isovaleric acid,but slightly affect the production of other SCFAs.All these demonstrated GAP1 and GAP2 could alleviate intestinal barrier injury and colitis.Moreover,administration of GAP1 or GAP2 reduced focal infiltration of lymphocytes and increased the abundance of mucin/mucin,tight junction protein ZO-1 and occludin-producing cells.(5)The intestinal barrier damage repair model was constructed by DSS to evaluate the function of GAP1 and GAP2 in protecting the intestinal barrier.In mice with colitis,GAP2showed a promotion to restore intestinal barrier.During recovery from DSS-induced colitis,GAP2 intake significantly improved weight gain,colon length recovery and disease index.Also,GAP2 increased the number of local lamina propria intestinal glands,reduced connective tissue hyperplasia and focal infiltration of lymphocytes,elevated the abundance of cells producing mucin/mucin,tight junction protein ZO-1 and occludin,and promoted the repair of the intestinal barrier in colitis.Colonic transcriptome analysis revealed that GAP2 could increase the expression of adhesion-related factors.Western blot assay showed that the expression of adhesion-related proteins Cdc42,Rho,BAIAP2 and Par3 in GAP2 group was obviously increased.During the reestablishment of the intestinal barrier,GAP2 reduced the abundance of Escherichia＿Shiaella and Parabacteroides,and increased the proportion of SCFAs and its producing bacteria Ruminococcaceae UCG＿014,Alistipes and Lachnospiraceae NK4A136.