| Objective:Inflammatory Bowel Diseases(IBD)is a group of chronic recurrent intestinal inflammation,and has become an important public health problem with the increasing incidence.Most of the drugs currently used in clinical treatment of IBD can only alleviate the disease with some serious side effects.Hence,the lack of treatment response raises challenges to the advancement of IBD therapy.Studies have found that edible and medicinal mushroom polysaccharides(MPs)plays important roles in the prevention and treatment of IBD.Helvella leucopus,an edible and medicinal wild fungi,is distributed in Populus euphratica forest in Tarim Basin,also called the Bachu mushroom by the local residents because of the Bachu County,is a potential resource of traditional Chinese medicine with high nutritional and medicinal value.However,the relationship between the modulation of gut microbiota and prevention of colitis by Bachu mushroom hopolysaccharides is had not been studied.Thus,the aim of this study was to characterize the structure of purified Helvella leucopus polysaccharide(p-HLP),to clarify whether p-HLP could improve the symptoms of dextran sodium sulfate(DSS)-induced ulcerative colitis and modulate gut microbial composition,and to explore the possible inflammation regulation mechanism.Method:1)The crude polysaccharide from Bachu mushroom was obtained by water extraction and alcohol precipitation,and the non-polysaccharide impurities in the crude polysaccharide were preliminarily purified by deproteinization、decolorization and dialysis to obtain the refined polysaccharide of Bachu mushroom.Subsequently,gel purified system occupied with on-line detection(RI-502 SHODEX)was used to fractionation and further purification.After purification,a homogeneous polysaccharide from Bachu mushroom was obtained by on-line detection.2)The monosaccharide composition,molecular weight range and glycosidic linkages of p-HLP were identified by ion chromatography(IC),high performance liquid chromatography(HPLC),gel permeation chromatography-refractive index-multiangle laser light scattering(GPC-RI-MALS)and GC-MS,the primary structure of polysaccharide from Bachu mushroom was also studied.3)A mouse model of ulcerative colitis was established in C57BL/6 mice induced by dextran sulfate sodium(DSS).25 male C57BL/6 mice were randomly divided into blank control group(NC group),model group(DSS group),low-dose p-HLP group(DSS+p-HLP(L)),high-dose p-HLP group(DSS+p-HLP(H))and p-HLP-only treatment group(p-HLP group),with 5 mice in each group(n=5).After the formal intervention,the weight of the mice was measured and the disease activity index(DAI)was evaluated every day;the mice were killed on the 8th day,and the colon length and spleen weight were measured and recorded;the histopathological analysis and histological score were performed by HE staining.4)q RT-PCR was used to investigate the effects of p-HLP on the m RNA expression levels of inflammation assotiated factors and mediators in colon tissue of UC mice,including the anti-inflammatory cytokines(IL-10),pro-inflammatory cytokines(IL-6,IL-1β,TNF-α)and pro-inflammatory mediators(i NOS and COX-2).5)The intestinal contents of mice with ulcerative colitis were collected,and the genomic DNA was extracted and purified.The subsequent high-throughput DNA library was sequenced with the Illumina miseq second generation sequencing technology platform,and the V4 region of 16S r RNA gene of 20 samples from5 groups was analyzed statistically and bioinformatically.Then,the effects of p-HLP on the abundance,evenness and diversity of intestinal microflora in mice with ulcerative colitis were investigated byαdiversity,βdiversity and LEfse analysis.Results:1)The protein of Bachu mushroom was removed by sevag method,and the organic matter was removed by ethanol precipitation method.Then the extract was freeze-dried to obtain refined polysaccharide of Bachu mushroom.The yield was 4.25%.The refined polysaccharide was further purified by gel purification system and the symmetrical part of the single peak was detected by RI-502 SHODEX,indicating that HLP was homogenous purified polysaccharide.The yield of homogeneous purified polysaccharide(p-HLP)from Bachu mushroom was 2.8%.2)The results of p-HLP monosaccharide composition analysis showed that the monosaccharide composition of p-HLP was composed of mannose(Man),glucose(Glu),rhamnose(Rha),galactose(Gal),fucose(Fuc),xylose(Xly),galacturonic acid(Gala),glucuronic acid(Glc A),guluronic acid(Glu A)and arabinose(Ara)with a molar ratio of 43.68:38.16:9.34:4.35:0.88:0.84:0.79:0.74:0.72:0.50,respectively;The weight-average molecular weight(Mw),number-average molecular weight(Mn),and Z-average molecular weight(Mz)of p-HLP were calculated to be39.14×10~8、18.95×10~8 and 93.84×10~8 Da,respectively.The polydispersity coefficient was2.07(Mw/Mn)or 4.95(Mz/Mn).The molecular configuration of p-HLP showed that the p-HLP configuration was nearly spherical.p-HLP was mainly composed of eleven different linkage residues,including nonreducing terminals of rhamnose(T-D-Rha),mannose(T-D-Man),glucose(T-D-Glc)and galactose(T-D-Gal),the molar ratios of which were 6.05%,5.50%,8.48%and 0.70%,respectively.Other saccharide residues,including 4-Glcp,2-Manp,6-Manp,3,6-Manp,4,6-Galp,6-Glcp and 3-Glcp with molar ratios of 31.60:28.71:8.22:4.29:2.74:2.15:1.58,respectively.The molar ratio of the above sugar residues was consistent with the monosaccharide composition.According to the above characterization results,it is concluded that p-HLP,isolated and purified from Bachu mushroom,is a novel polysaccharide.These findings demonstrate that the structure and biological activity of p-HLP should be further studied.3)The results of DSS induced ulcerative colitis mice model showed that compared with NC group,the body weight of DSS group showed a significant decreases with time,accompanied with severe diarrhea with stool bleeding,and DAI disease activity index was higher(P<0.05).Meanwhile,it was found that the length of colon in DSS group was significantly shortened,the colonic mucosa was congested,and the spleen index was significantly increased in DSS group compared to NC group(P<0.01).Supplementation of p-HLP could alleviate typical symptoms of UC including weight loss,colon shortening,and spleen index was returned to near normal levels in both low/high dose of p-HLP administration group(P<0.01).At the same time,HE staining showed that DSS-treatment had induced the loss of goblet cell,severe damage of crypt structure,as well as infiltration of inflammatory cells in the muscular layer in colon tissue.However,administration of p-HLP,especially treated with high dose of p-HLP(50 mg/kg)relieved these histological changes,including relative intact surface epithelium,glandular structure or regeneration of crypts,less inflammatory cell infiltration,and mild submucosal edema,the colonic tissues close to normal pathological morphologies,showed that significant protection against colon damage induced by DSS.In addition,there was no significant difference between NC group and p-HLP-only group,which indicated that p-HLP at the dose of 100 mg/kg had no side effects on normal mice.4)DSS induction increased the m RNA expression level of proinflammatory cytokines(IL-1β,IL-6 and TNF-α).After intervention with different doses of p-HLP(10 and 50 mg/kg),the level of TNF-αdecreased significantly(P<0.01);for IL-1βand IL-6,low dose of p-HLP decreased the m RNA expression level,but there was no significant difference with DSS group,while high dose of p-HLP(50 mg/kg)decreased the m RNA expression level of the above two pro-inflammatory cytokines to near the normal level,and there was a significant difference(P<0.01).The expression levels of IL-1βand IL-6 m RNA in p-HLP positive control group were consistent with those in NC group.DSS treatment increased the m RNA expression levels of pro-inflammatory mediators(i NOS and COX-2).The m RNA levels of i NOS and COX-2in colon tissues with different doses(10 and 50 mg/kg)of p-HLP groups were significantly decreased(P<0.01).The m RNA expression levels of proinflammatory/antiinflammatory cytokines and pro-inflammatory mediators in p-HLP-only group(100mg/kg)were consistent with those in NC group.5)The results of 16S r RNA high-throughput sequencing and bioinformatics analysis showed that the top five intestinal dominant species in the colonic contents of the experimental mice were Firmicutes,epsilon bacterium eota,Bacteroidetes,verrucomicrobia and proteobacteria.Compared with NC group,the intestinal microflora of mice with ulcerative colitis changed significantly.Moreover,p-HLP supplementation could reverse the composition and the alteration of gut microbiota in the colitic mice by increasing the abundances of potential beneficial bacteria,including Faecalibacterium,Eubacterium,Allobaculum,Akkermansia,Lactobacillus and Bacteroidetes,and decreasing the abundances of potential harmful bacteria,including Alloprevotella、muribaculacae＿norank、Dubosiella,Lachnospiraceae and Lachnospiraceae NK4A136 in a dose-dependent manner.Among them,high dose of p-HLP(50 mg/kg)showed the obvious effect on maintaining the balance of intestinal microecology.Conclusion:p-HLP is a novel polysaccharide.Different doses of p-HLP could alleviate the ulcerative colitis induced by DSS in mice.Administration of p-HLP could alleviate typical symptoms of IBD including weight loss,colon shortening,pathological tissue damage,decreases in the colitis disease activity index,inhibit the expression of pro-inflammatory factors,promote the expression of anti-inflammatory cytokines,and regulate the composition of gut microbiota,especially the effect of high-dose p-HLP(50 mg/kg)is obvious and unique,and it needs to be studied deeply and systematically.At the same time,p-HLP could be developed as a clinically effective,low toxicity drug for the treatment of inflammatory bowel disease and colitis. |
