Anti-inflammatory Effect And Mechanism Of Polysaccharides From Sargassum Fusiforme After UV/H₂O₂ Degradation

Ulcerative colitis（UC）is a non-specific chronic inflammatory bowel disease of unknown aetiology.Its incidence and prevalence are mainly in developed countries,and increasing year by year in developing countries.At present,most of the drugs commonly used to treat UC have low efficacy,high price and side effects,etc.Therefore,How to prevent,alleviate and treat UC is of great interest to scholars at home and aboard.Polysaccharides have high safety,low toxicity and diverse biological activities.Polysaccharides are one of the most important bioactive components in Sargassum fusiforme,which have multiple biological activities,including antioxidant,antitumor,anticoagulant and immunomodulatory.However,its dark color and large molecular weight greatly limit the studies of its biological activities and structural properties.Therefore,this study firstly investigated the degradation of polysaccharides from Sargassum fusiforme by Ultraviolet（UV）combined with H₂O₂（UV/H₂O₂）treatment.Next,the structural properties of polysaccharides from Sargassum fusiforme with different degrees of degradation were analyzed,and their in vitro anti-inflammatories were evaluated to screen the degraded polysaccharides with the best activity.Then,the effect and mechanism of the selected degraded polysaccharides on colitis in mice were investigated.Finally,the anti-inflammatory activities of purified fractions from the selected degraded polysaccharides were evaluated,and the structural properties of the fraction with the best activity were analyzed.These studies were conducted with a view to laying a theoretical foundation for the development of low molecular weight polysaccharides from Sargassum fusiforme with colitis-relieving function.The main research methods and results were as follows:（1）UV/H₂O₂ treatment had obvious degradation effect on polysaccharides from Sargassum fusiformeMolecular weight analysis of polysaccharides from Sargassum fusiforme by gel permeation chromatography showed that its molecular weight decreased from 280.73 k Da to13.71 k Da after UV/H₂O₂（150 mmol/L）treatment for 2 h,while there was no significant change in the molecular weight after only UV or H₂O₂（150 mmol/L）treatment for 2 h.Thedegradation of polysaccharides from Sargassum fusiforme by UV/H₂O₂ treatment increased with increasing H₂O₂ concentration and treatment time,while its degradation weakened with increasing polysaccharide concentration.In adition,UV/H₂O₂ treatment had a better degradation when the initial p H value was three.Besides,UV/H₂O₂ treatment had a significant decolorization effect,while only UV or H₂O₂（150 mmol/L）treatment had no significant decolorization effect.（2）Structural properties and in vitro anti-inflammatory activity of polysaccharides from Sargassum fusiforme after UV/H₂O₂ treatmentPolysaccharides from Sargassum fusiforme with different degrees of degradation were obtained by controlling the time of UV/H₂O₂（100 mmo L/L）treatment.Chemical compositions analysis showed that the contents of total sugar,protein and uronic acid in polysaccharides from Sargassum fusiforme with UV/H₂O₂ treatment for 2 h decreased by14.29%,57.11%and 43.18%,respectively,while the contents of reducing sugars and sulfate groups increased by 202.86%and 31.77%,respectively,compared with those in untreated polysaccharides from Sargassum fusiforme.Next,the molecular weight of degraded polysaccharide was obviously reduced,and the morphological characteristics gradually changed from irregular flakes to small uniform particles.In addition,the molar percentages of xylose and glucose in the degraded polysaccharides gradually decreased,but the changes of other constituent monosaccharides showed no obvious pattern.The in vitro anti-inflammatory activity studies showed that the degraded polysaccharide（PSF-T2）,obtained by UV/H₂O₂treatment for 2 h,had the strongest ability to inhibit NO,IL-1β,IL-6 and TNF-αsecretion,and the expression of inflammation-related genes Tlr4,Irak,Il-1β,Il-6,Il-12 and Tnf-α,indicating that PSF-T2 had a good anti-inflammatory activity.（3）PSF-T2 could effectively relieve the symptoms of colitis in miceBased on the dextran sodium sulfate-induced mouse model of colitis,studies showed that PSF-T2 increased body weight and colon length of mice with colitis,and reduced its colonic edema and pathological damage.Secondly,PSF-T2 could inhabit the inflammatory response by regulating the protein expression of IκBαand NF-κB p65 in the NF-κB signaling pathway and the secretion and gene expression of IL-1β,IL-6 and TNF-α.Besides,PSF-T2 could also protect intestinal barrier function of mice with colitis by promoting the secretion of intestinal mucus protein and upregulating the protein expression of MUC-2 and tight junction proteins（ZO-1,Occludin and E-cadherin）.In the PSF-T2 groups with different concentrations,high concentration group of PSF-T2（200 mg/kg·BW）had better effects on alleviating colitis in mice.（4）PSF-T2 could regulate gut microbiota and metabolism in mice with colitisThe results of 16S r DNA high-throughput sequencing showed that PSF-T2 treatment increased the diversity and abundance of gut microbiota in mice with colitis.At the phylum level,the main dominant flora in each group of the experiment were Firmicutes and Bacteroidota,the abundance of Firmicutes increased and that of Bacteroidota decreased in mice with colitis after PSF-T2 treatment.At the family level,PSF-T2 could reduce the abundance of microorganisms associated with pro-inflammatory and causing damage to the intestinal mucosal barrier,such as Bacteroidaceae,Erysipelotrichaceae,Ruminococcaceae and Marinifilaceae,while increasing the abundance of microorganisms associated with anti-inflammatory,promoting the production of short chain fatty acids and the secretion of intestinal mucus protein,such as Lactobacillaceae,Desulfovibrionaceae,Saccharimonadaceae,Oscillospiraceae and Eggerthellacea.In the non-targeted metabolomics analysis,OPLS-DA analysis showed that the serum metabolites in mice with colitis were significantly differences between the Model group and PSF-T2 group.A total of43 metabolites with significant differences were screened by comparing metabolites between the two groups.KEGG pathway enrichment analysis of the above differential metabolites showd that PSF-T2 alleviated colitis mainly by modulating antifolate resistance,mineral absorption,central carbon metabolism,protein digestion and absorption,amido-t RNA biosynthesis,insulin resistance and phenylalanine/tyrosine/tryptophan biosynthesis.The above significant enrichment pathways involved a total of five key differential metabolites,namely acetylcarnitine,L-methionine,L-phenylalanine,deoxyuridine and3-carbamoyl-2-phenylpropionic acid.The correlation analysis of the above five metabolites with gut microbiota at the family level showed that acetylcarnitine was significantly and positively correlated with Pseudomonadaceae,L-methionine and L-phenylalanine weresignificantly and negatively correlated with Helicobacteraceae,DUMP was significantly and positively correlated with Lachnospiraceae,and 3-carbamoyl-2-phenylpropionic acid was significantly and negatively correlated with Pseudomonadaceae,and positively correlated with f＿unclassified＿o＿Oscillospirales.Besides,PSF-T2 could also promote the production of short chain fatty acids,such as acetic acid,propionic acid and n-butyric acid.（5）Structural properties and in vitro anti-inflammatory activity of purified fractions of PSF-T2PSF-T2 was purified using Toypearl-DEAE 650M anion exchange column to obtain two fractions,PT-0.25 and PT-0.5,with yields of 39.50%and 23.90%,respectively.PT-0.5 had the best anti-inflammatory activity,because it could better inhibit the secretion of NO and the secretion and gene expression of IL-6,IL-12 and TNF-α.Chemical compositions analysis of PT-0.5 showed that the contents of total sugar,protein,reducing sugar,uronic acid and sulfate group contents were 50.33±1.40%,1.15±0.03%,11.39±0.38%,8.70±1.02%and 5.00±0.23%,respectively.Its monosaccharide composition was mainly composed of fucose,galactose,mannose and glucuronic acid,and molecular weight was mainly 22.89 k Da.In addition,its morphological characteristic was irregular lamellar structure,and surface was smooth and non-porous.Besides,the results of methylation analysis showed that the structural composition of PT-0.5 was more complex,consisting of three terminal residues,six linear residues and one branching type residue.Among them,the most dominant residue types were4-Fucp、t-Manp、6-Galp、t-Fucp和3,4-Glc Ap.