Study On The Preventative Effects Of Polysaccharides From Gracilaria Lemaneiformis Against Colitis And Its Mechanism

Gracilaria lemaneiformis is a kind of edible and medicinal seaweed traditionally,which is rich in carbohydrates,proteins,vitamins and other nutrients.In particular,polysaccharide is one of its important bioactives.Previous studies indicated that polysaccharide extracted from G.lemaneiformis by citric acid had higher yield and lower molecular weight,and showed hypoglycemic,anti-photoaging and glucolipid metabolism regulating effects in vivo.In this study,a sulfated polysaccharide(SP)from G.lemaneiformis was prapared by citric acid extraction,and its chemical composition was also determined.The simulated gastric-intestinal digestion and colon fermentation in vitro were carried out to explore how SP might be absorded and utilized in vivo and what effects SP might exert on gut microbiota in vivo.The mice model with colitis induced by sodium glucan sulfate(DSS)were performed to explore the preventive effect of SP.Several techniques including real-time quantitative polymerase chain reaction(RT-q PCR),gas chromatography and high-throughput sequencing were adopted to investigate the possible protective mechanism of SP.These would provide the theoretical foundation for further application of Gracilaria lemaneiformis polysaccharide.Main results were found as follows:(1)SP was extracted by citric acid,in which the contents of total polysaccharide and uronic acid were 59.93 ± 1.31% and 13.32 ± 0.82%,respectively.The contents of 7 types of monosaccharides including galactose,glucose,xylose,mannose,fucose and arabinose were 71.78%,13.48%,7.92%,4.23%,1.45% and 1.14%,respectively.Besides,SP also contained protein(1.72 ± 0.29%)and sulfate(29.82 ± 0.13%).It had compact microstructure and also a few pits on the surface with the average molecular weight of 21.06 Da.In addition to S element(12.32 ± 0.11%),it also possessed other elements such as Na,Ca,K and Mg.Moreover,the contents of(1?3)glycosidic bond was 86%,while the contents of(1?)or(1?6)glycosidic bond and(1?2)or(1?4)glycosidic bond were 11.4% and 2.6%,respectively.Thus,SP was a typical sulfated polysaccharide,which was mainly linked by(1?3)glycosidic bond and mostly composed of galactose and glucose.(2)SP had slight degradation during the simulated digestion in vitro with the changes of reducing molecular weight(from 22.38 ± 0.57 k Da to 21.97 ± 0.01 k Da)and increasing content of reducing sugar(0.027 ± 0.001 to 0.237 ± 0.070 mg/m L).Besides,SP could reduce the p H value and promote the production of SCFA in vitro fermentation.The result showed that SP was utilized by gut microbiota for 53.7% at the end of fermentation.It could promote the growth of beneficial bacteria with the maintenance of microbial richness and diversity,which would be better to keep microbital composition in a steady state during a short time.The results also indicated that SP could be mainly degraded and utilized by Parabacteroides distasonis,Parabacteroides merdae,Sutterella wadsworthensis,Phascolarctobacterium,Lachnospiraceae UCG-004,Desulfovibrio,Bacteroides.Moreover,SP also influenced the metagenomic characteristics of fecal microbiota.(3)SP treatment for four weeks could alleviate the loss of weight and appetite as well as shortened and edema of colon in mice with colitis induced by DSS.And SP could maintain mice colonic permeability via reducing contents of ET and LBP in serum.SP could also attenuate serious inflammatory response and tissue damage via decreasing contents of inflammatory cytokines including TNF-?,IL-6,IL-1? and activities of MPO in colon.Beside,SP could prevent intercellular junctions against lesions and enhance the intestinal barrier function via promoting the expression of tight junction proteins including Claudin-1 and ZO-1 and MUC-2.Thus,SP possessed protective effects on DSS-induced colitis in mice.(4)Combining several methods(including RT-q PCR,GC and high-throughput sequencing)were used to explore the possible mechanism regarding to the positive effects of SP in mice.The results illustrated that SP played a protective role in colitis via several targets.It could down-regulate the relative expression of inflammatory cytokines(TNF-?,IL-6,IL-1?)to alleviate serious inflammatory response,up-regulate the relative expression of tight junction proteins(Claudin-1 and ZO-1)and MUC-2 to enhance intestinal physical barrier,promote the production of SCFA and activate corresponding receptors GPR43,Olfr78 and GPR109 A to play a role in immune regulation,inhibit the turbulence of gut microbiota to normalize microbial composition,increase the relative abundance of Enterorhabdus,Desulfovibrio,Alistipes,Bacteroides acidifaciens to regulate microbital composition and enhance intestinal biological barrier function.All of these approaches were beneficial for preventing mice from DSS-induced colitis.