Research On Antidiabetic Activity Of Polysaccharides From Stichopus Japonicus Prepared By Simulated Gastrointestinal Digestion

Type Ⅱ diabetes mellitus is characterized by insulin resistance,accompanied by oxidative stress and inflammation.At present,the drugs for treating diabetes have obvious side effects,so it is of great significance to find green and safe natural drugs.As one of the highest edible value of species,sea cucumber Stichopus japonicus is mainly composed of protein covalently associated with sulfate polysaccharide,which has a high potential of antidiabetic activity.The fine structure of polysaccharide related to antidiabetic activity is still controversial,and its activity and antidiabetic mechanisms are not thoroughly studied.Firstly,an in vitro gastrointestinal digestion model was established to simulate digestion of the defatted body wall of S.japonicus.With the progress of gastrointestinal digestion,the degree of proteolysis increased from 76.5%to 87.3%.The gastric digestion fractions（GDFs）and intestinal digestion fractions（IDFs）were divided into<3,3-10,10-30 and>30 k Da,respectively.All had certain inhibitory effects on DPP-IV.The activities of<3 k Da fractions were the highest,with IC₅₀values of 0.51±0.05 mg/m L and 0.52±0.07 mg/m L in GDFs and IDFs.In the concentration range of 10 to 100μg/m L,the fractions could increase the glucose consumption of 3T3-L1 cells and insulin-resistant Hep G2 cells with no cytotoxicity.For insulin-resistant Hep G2 cells,fractions with high molecular weight,especially fractions>30 k Da,showed the highest activity.The fraction>30 kDa was further digested by papain and separated by column chromatography.Three polysaccharides were obtained,including fucoidan.Besides FCSsj,a neutral polysaccharide NPsj was also obtained.The structures of the two polysaccharides were analyzed by HPLC,NMR,mass spectrometry and methylation,etc.The weight-average（Mw）and number-average（Mn）molecular weight of FCSsj were 60.99 k Da and 57.89 k Da,respectively.The branch chain types of FCS sj were Fuc 2,4S,Fuc 4S and Fuc 3,4S with the molar ratio of 1.00:0.26:0.65.There are four kinds of Gal NAc in the backbone,which are Gal NAc 0S,Gal NAc 4S,Gal NAc 6S and Gal NAc 4,6S.It contains a novel di-fucosyl branch chain Fuc 2,4Sα（1→3）Fuc4S,which is connected to the Glc A of the main chain by 1,3 glycosidic bond.The Mw of NPsj is 301.75 k Da,which belongs to a neutral glucan.The backbone is anα-1,4-glycosidic bond linked glucan with a branch at the O-6 position of the backbone.For every 7～9 glucose residues in the backbone,oneβ-type glucose branch is connected to the backbone by aβ-1,6 glycosidic bond.The cell model and zebrafish model were established to study the antidiabet ic activity of FCSsj.It was found that FCSsj could alleviate the glucosamine induced damage of Hep G2 cells and improve the glucose consumption,glucose uptake and glycogen synthesis.At the same time,the contents of ROS and MDA in the cells were decreased,and the contents of GSH and SOD were increased.The oligosaccharide s of FCSsj were prepared by partial N-deacetylation–deaminative cleavage method,and the 6-mer component d F5 with highest activity was obtained.FCSsj and d F5 coould improve the uptake of 2-NBDG,reverse the decrease of lipid in yolk sac,and reduce the accumulation of ROS in zebrafish embryos caused by high glucose immersion.The activity of d F5 oligosaccharide is higher than that of FCS sj at the same concentration.The mechanism of FCSsj and dF5 against type Ⅱ diabetes mellitus was studied by transcriptomic analysis.Glucosamine induced 3918 genes up-regulated and 3505genes down-regulated in Hep G2 cells.FCSsj treatment reversed the down-regulation of 139 genes and up-regulation of 192 genes,while d F5 reversed the down-regulation of 298 genes and up-regulation of 389 genes.GO enrichment analysis showed that FCSsj and d F5 could conversely up-regulate the expression of genes related to antioxidant activity,carbohydrate metabolism process and oxidative stress response,such as GPX2,GPX3,PRDX2,PFKL,ISYNA1,GAA and HYAL2,meanwhile,down-regulate the expression of guanylate binding,nucleoside binding and guanylate triphosphate binding genes.KEGG enrichment analysis showed that FCS sj and d F5reversely up-regulated the genes in insulin resistance,cholesterol metabolism,PPAR signaling pathway and carbon metabolism,and down-regulated the genes in protein endoplasmic reticulum process,sterol synthesis and JAK-STAT signaling pathway.FCSsj and d F5 increased glucose consumption and decreased lipid accumulation in insulin-resistant Hep G2 cells by down-regulating the lipid synthesis genes IDI1,SQLE,SC5D,HMGCS1,and up-regulating the glucose and lipid metabolism genes G6PD,PFKL,SLC2A1,IDH2,ACADS and ECSH1.At the same time,the inflammatory and apoptotic pathways mediated by node genes JAK1,JAK2,HSP90AA1 and PTPN11 were down-regulated,and the inflammatory response and apoptosis of insulin-resistant Hep G2 cells were decreased.Direct targets of FCSsj and d F5 were studied by pull-down method.The subcellular localization of FCSsj was mitochondria,and both FCSsj and d F5 were significantly aggregated in zebrafish liver.They regulate potential downstream signal pathway by targeting FDXR.