| Ulcerative colitis(UC)incidence rate is on an upward trend,which is severely threatening the health of human beings and has become a global public health issue.The study of food-borne functional factors to improve UC is of great significance for the development of functional food for the prevention and treatment of intestinal diseases.Dendrobium fimbriatum Hook belongs to Dendrobium of the family Orchidaceae,it has the functions of clearing heat,generating saliva,nourishing Yin and protecting the stomach.Modern pharmacological studies have shown that polysaccharides,as the main active components of Dendrobium species,play a potential role in preventing and improving UC,but its mechanism of action is still dimness.In this study,Dendrobium fimbriatum Hook polysaccharide(cDFPW1),which has clear chemical structure and immunomodulatory activity,was used as material to systematically evaluate the effects of oral cDFPW1 on intestinal mucosal function in mice.Based on the integrity of intestinal mucosa,this study investigated the intervention effect of cDFPW1 on dextran sulfate sodium(DSS)-induced UC and explored the molecular mechanism of cDFPW1 improving UC from the perspective of intestinal stem cells(ISCs)protecting the integrity of intestinal mucosa,in order to provide a scientific basis for the prevention of UC and the development of functional food by foodborne polysaccharides.The main results obtained are as follows:1.Oral administration of cDFPW1 protected the integrity of intestinal mucosa through regulating intestinal mucosal morphology,barrier function,immune response,oxidative stress,intestinal microflora and metabolites.(1)Compared with the control group,oral cDFPW1 significantly increased the colon length of normal mice and caused the appearance and structure of intestinal mucosa more intact.Analysis showed that cDFPW1 had the function of protecting the morphology of intestinal mucosa,making the microvilli(Mv)of intestinal epithelial cells arranged neatly,visible the tips,tightly connected intact,and increasing the number of goblet cells in a dose-dependent manner.(2)Oral administration of cDFPW1 could up-regulate the expression of tight junction protein Occludin and ZO-1 in intestinal epithelial cells,reduce the contents of endotoxin(EDT),diamine oxidase(DAO)and D-lactic acid(D-Lac)in serum,promote the secretion of mucin-2(MUC2),and inhibit the activity of myeloperoxidase(MPO),which indicated that cDFPW1 could enhance the physical and biochemical barrier functions of intestinal mucosa.Oral administration of cDFPW1 could up-regulate the expression of IL-1β,IL-4,IL-5,IL-10,IL-12,IL-13,IL-22 and TNF-α in intestinal mucosa,it suggested that cDFPW1 could mediate mucosal immune response.Oral administration of cDFPW1 could increase the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)in intestinal mucosa,and decrease the concentration of malondialdehyde(MDA),suggesting that cDFPW1 has the ability to enhance the antioxidant stress of intestinal mucosa.(3)The analysis of 16 S r RNA microbial sequencing and gas chromatography(GC)showed that oral cDFPW1 could up-regulate the abundance of beneficial bacteria Lactobacillus,Ileibacterium,Allobaculum and Akkermansia compared with the control group,and increase the content of acetic acid,propionic acid and n-butyric acid in feces.These results manifested that cDFPW1 could regulate the composition of intestinal flora and contribute to the production of short-chain fatty acids(SCFAs).2.Oral administration of cDFPW1 could effectively improve the integrity of intestinal mucosa and realize the effect of ameliorating UC in terms of repairing intestinal barrier function,reshaping intestinal flora,alleviating oxidative stress and inhibiting inflammatory response.(1)Intestinal mucosal morphology and intestinal barrier function analysis showed that compared with DSS group,cDFPW1 could improve the tendency to lose weight,inhibit the increase of disease activity index(DAI)score,reduce histopathological score,decrease the number of neutrophils,prevent the bleeding and shortening of colon,and effectively alleviate the intestinal mucosal injury caused by DSS,indicating that cDFPW1 could improve the symptoms of UC.Compared with DSS group,cDFPW1 significantly up-regulated the expression of Occludin and ZO-1,decreased the contents of EDT,DAO and D-Lac in serum,down-regulated the activity of MPO in colon mucosal tissue,improved intestinal permeability,and promoted the number of goblet cells and MUC2 secretion.These results suggested that cDFPW1 could repair the integrity of intestinal mucosa to resist UC.(2)Compared with DSS group,cDFPW1 could increase the diversity of intestinal microbial community,up-regulate the abundance of beneficial bacteria Romboutsia,Akkermansia,Odoribacter and Lactobacillus,reduce the abundance of harmful bacteria Burkholderia-Caballeronia-Paraburkholderia,Parasutterella and Acinetobacter,and promote the production of SCFAs.The results indicated that cDFPW1 could reshape intestinal flora and metabolites of mice to intervene with UC.(3)Compared with DSS group,cDFPW1 could not only restore the balance of Th17/Treg cells in colitis mice,but also down-regulate the levels of IL-1β,IL-6,IL-17 A,IL-17 F,IL-21 and IL-23 and up-regulate the levels of IL-5,IL-10,IL-22,IFN-γ,TNF-α and TGFβ1,suggesting that cDFPW1 could regulate intestinal inflammation.Compared with DSS treatment group,oral administration of cDFPW1 increased the activities of SOD,GSH-Px and CAT and significantly reduced the content of MDA in colonic mucosal tissues,suggesting that cDFPW1 has the effect of reducing oxidative stress.In addition,cDFPW1 enhanced Nrf2 expression and inhibited NF-κB translocation compared with DSS group.These results suggested that cDFPW1 could regulate the intestinal immune homeostasis to maintain the intestinal integrity and improve UC.3.cDFPW1 promoted ISCs regeneration through the upregulation of IL-22 mediated by intestinal mucosa lamina propria lymphocyte(LPLs)to protect the integrity of intestinal mucosa.(1)The digestion characteristics of FITC-cDFPW1 in mice were analyzed by ultra high performance liquid chromatography.The results showed that cDFPW1 was not degraded during digestion in the stomach or small intestine after oral administration but entered the large intestine as a prototype and gradually degraded.(2)The co-culture model of intestinal organoids and LPLs was constructed.cDFPW1 could directly ameliorate the intestinal epithelial injury induced by DSS was confirmed through the morphological characteristics of intestinal organoids,surface area,intestinal epithelial cell proliferation experiment,combined with the mice model of intestinal microbial depletion induced by DSS.(3)Laser confocal microscopy,RT-q PCR and western blot were used to confirm that cDFPW1 significantly increased the percentage of Lgr5-positive cells,promoted the protein expression of Lgr5 and up-regulated the m RNA expression of ISC markers(Lgr5,olfm4 and ascl2)in the intestinal organoid co-culture system containing LPLs.Similarly,animal experiments showed that cDFPW1 could promote the number of Lgr5 positive cells in the colon crypt.These results suggested that oral cDFPW1 could increase the number of ISCs and promote the regeneration of ISCs after DSS injury.(4)Cytokine antibody array,ELISA and antibody neutralization were used to determine that cDFPW1 could promote the ISCs-mediated intestinal epithelial regeneration through significantly up-regulated the expression of IL-22.Further,by adding cDFPW1 to the organoid system with or without LPLs,it was confirmed that the stimulation of IL-22 secretion by cDFPW1 was mediated by LPLs.The above studies suggested that cDFPW1 promoted ISCs regeneration through LPLs-mediated up-regulation of IL-22 to protect the integrity of intestinal mucosa,thus playing an important role in improving ulcerative colitis. |
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