The Structure-activity Relationship Of New Type Of Pectin On Alleviating Colitis Based On Mucous Barrier And Gut Microbiota

Inflammatory bowel diseases(IBDs),including ulcerative colitis(UC)and Crohn’s disease(CD),are chronic relapsing and idiopathic immune disorders in GI tract,marked by a dysbalanced immune system and excessive production of proinflammatory cytokines.So far,the occurrence of these diseases represents a widespread condition with possible mortality and the burden of IBD is constantly rising globally.The medical treatments for IBD are ineffective and many drugs for IBD have been reported with drug resistance and severe adverse effects.However,dietary pectin intervention can effectively and safely alleviate IBD symptom.Pectins,as one of the most important water-soluble dietary fiber,are able to relieve gut inflammation and enhance gut health.The traditional pectin is regarded as anionic polysaccharide containing more than 65%of galacturonic acid.However,as the sources of pectin are becoming more and more,new type of pectins with more complex structural domains have earned more and more focus.The novel pectins mainly contain four domains:HG,RG,arabinan,and galactan,among which neutral sugar accounts for more than 50%.Currently,many studies have reported that novel pectins can regulate colitis.However,due to the various food sources and complex structure of novel pectins,some issues still remain vague,such as the differences among various source,the potential mechanism of action of different pectic domains.Collectively,the structure-activity relationship and mechanism study of pectin in regulating IBD is still in its infancy.Therefore,in this study,novel pectins from different sources or with different domain component were prepared.The stepwise ethanol precipitation and Q-Sepharose Fast Flow(QFF)anion chromatography,atomic force microscopy(AFM),dynamic light scattering,and two-dimensional nuclear magnetic resonance(2D NMR)were used to isolate and structurally characterize pure pectins.At first,the lipopolysaccharide-induced macrophage cell model and In vitro fermentation model were used to determine the anti-inflammatory properties of different pectins.Then,the dextran sodium sulfate(DSS)induced colitis mouse model were applied to evaluate the efficacy of different pectins from disease symptom,pathological features of the colon,the index of immune organs,fecal biomarkers and so on.Finally,the mechanism of different pectic domains were discussed based on the SCFAs absorption and transportation in colon,the colonic barrier and the composition of gut microbiota and spleen germinal center response.The main research results obtained are as follows:(1)The selecting of superior source of anti-colitis crude pectin based on basic disease parametersSix pectins from Goji berry,dragonfruit peel,lime peel,red pomelo peel,potato and raspberry were subjected to dietary intervention experiment of DSS-induced colitis mice.The results of structural analysis showed that these six pectins had different domain components:Goji pectin(G)contained 21%RG,36%arabinose(Ara)sidechain,17%galactose(Gal)sidechain and 23%HG;dragonfruit pectin(D)mainly contained 59%RG;lime pectin(L)mainly contained 49%arabinose sidechain;red pomelo pectin(P)was mainly composed of 78%HG;potato pectin mainly contained51%galactose sidechain;raspberry pectin(R)mainly consisted of 15%RG,19%arabinose sidechain,28%galactose sidechain and 22%HG.The molecular weights of the six samples were all in the range of 3×10~4～2×10~5 g/mol.Except that P is high-methoxylated,all of the others were low-methoxylated.The animal experiment results showed that raspberry pectin,which contained multiple domains,were most conducive to relieving the symptoms of colitis,which could significantly improve the index of immune organs,inhibit colon shortening,decrease the level of pro-inflammatory cytokines and promote SCFAs production.The In vivo antioxidant activity of pectins might be related to its structural composition:pectins containing about 20%HG with Gal A/(Ara+Gal)value of 0.4～0.6 might be best for alleviating oxidative stress in colitis mice.The higher SCFAs production led to better protective effect.Pectins alleviated colitis by promoting the total amount of SCFAs,not the proportion of butyrate.(2)Isolation,purification,and structural characterization of two raspberry pectins with different RGⅠsidechain contentAccording to previous study,raspberry was used to extract pectins by hot acid method and biological enzyme method,respectively.Then,the crude polysaccharides were purified by stepwise ethanol precipitation and QFF gradient elution,and two pure pectins(RPA50%-3 and RPE50%-3)were obtained.The SEC-MALLs,HPAEC,FTIR,AFM,and 2D NMR were used to analyze fine structure of purified pectins.The results showed that RPA50%-3 and RPE50%-3 showed similar M_w(7.7×10~4 g/mol and 5.1×10~4 g/mol)and DM value(36%and 39%),and both consisted of→4)-α-Galp A-(1→,→2)-α-Rhap-(1→,→2,4)-α-Rhap-(1→,→5)-α-Araf-(1→,→4)-β-Galp-(1→and→6)-β-Galp-(1→linkages.RPA50%-3 mainly contained 50%HG,12%RG,and 14%β-1,4-Gal sidechain,while RPE50%-3 mainly contained 59%HG,10%RG,19%β-1,4-Gal sidechain,and 16%α-1,5-Ara sidechain.Both were RGI pectins,and RPE50%-3 contained more RGI sidechains.(3)Comparing the immunomodulatory activities of the two pure raspberry pectins based on in vitro cell culture and anaerobic fermentation systemLipopolysaccharide(LPS)was used to stimuli RAW264.7 cells to establish the inflammatory cell model.Based on LPS-induced macrophage cell culture assays,both of RPA50%-3 and RPE50%-3 could regulate cell immune response through inhibiting production of NO and ROS,reducing the level of pro-inflammatory cytokines(IL-6,TNF-αand IL-1β)in macrophage,and RPE50%-3 showed better effects and acted in a dose-dependent manner,suggesting RGⅠsidechains could enhance the immunomodulatory effect of pectin.RPE50%-3 and RPA50%-3 were subjected to the in vitro fermentation with fecal microbiota from six healthy people,in order to compare the fermentation characteristics of the two pectins.The results showed that compared to inulin,RPE50%-3 and RPA50%-3 were fast-fermented fiber.Within the 48 h period,the carbohydrate consumption amount of RPE50%-3 was larger.And RPE50%-3 with more RGⅠsidechains could produce more SCFAs.Pectins with different sidechain content showed different regulation on microbiota composition:RPE50%-3 could significantly increase the abundance of Bacteroides,Dorea,Blautia,Phascolarctobacterium and Bifidobacterium,while RPA50%-3 mainly promoted the growth of Prevotalla,Bacteroides,Phascolarctobacterium and Lachnoclostridium.Acid monosaccharide Gal A and neutral monosaccharide Ara and Gal could apparently regulate different bacteria.RPE50%-3 could improve theαdiversity of microbiota more remarkably,and had better effects on the growth of some anti-inflammatory bacteria like Eubacterium eligens,Fusobacterium,and RGⅠpectin rich in sidechains had a slight advantage in promoting the proliferation of anti-inflammatory bacteria,such as Faecalobacterium prausnitzii,Eubacterium eligens,Peptostreptococcus and Christensenellaceae,enhancing the immune regulation capacity of intestinal bacteria.(4)The establishment of chronic colitis mouse model and the studies of protective effects of the two pure raspberry pectins on chronic colitis miceDifferent doses of DSS(2%,2.5%,and 3%)were used to construct chronic colitis mouse model,and the results indicated that 2.5%DSS could successfully establish the chronic colitis model with a lower fatality rate(10%).RPE50%-3 and RPA50%-3 were subjected to the dietary intervention experiment on 2.5%DSS-induced chronic colitis mice,in order to explore the protective effects of the two pectins.The results showed that RPE50%-3 and RPA50%-3 could apparently alleviate colonic damage and colon shortening,improve the homeostasis of inflammatory cytokines,relieve oxidative stress and inhibit spleen enlargement,among which RPE50%-3 showed the better effect.Moreover,RPE50%-3 could significantly improve the expression level of TLR4receptors,SCFAs transportation proteins(SLC26A3 and SLC5A1/2)and SCFAs receptors(FFAR2 and FFAR3)in colon tissue,promote the balance of water transportation,and promote the serum level of SCFAs,and thus alleviating colitis,indicate the importance of RGⅠsidechains in anti-colitis function of pectin.(5)The studies of the potential mechanism of different pectic domains on alleviating colitis based on mucous barrier and gut microbiotaBased on the conclusions of previous study,the AG(Gal A content 0%,RGⅠsidechain content 100%),P37(Gal A content 37%,RGⅠsidechain content 54%),P55(Gal A content 55%,RGⅠsidechain content 35%),P85(Gal A content 85%,RGⅠsidechain content 5%)were subject to dietary intervention experiment on chronic colitis mice,in order to discuss whether more RGⅠsidechains lead to better protective effect and explore the potential mechanism of different pectic domains on regulating colitis.The results showed that P37 and P55 had the best protective effects against colitis.Given the suppression of colon shortening and promotion of immune organs,P37 was more effective than P55,which verified the previous results that pectin with multiple domain component had the best effect on alleviating colitis.P37 could alleviate colitis through enhancing colonic barrier,reducing the bacterial burden and inhibiting germinal center response and production of pro-inflammatory cytokines in spleen,suggesting the importance of gut-spleen axis.In the microenvironment of colitis gut,HG domain could promote the expression of tight junction proteins in the colonic epithelium,and the RGⅠsidechains mainly improve the expression of MUC2 and strengthen the mucous layer barrier.In addition,RGⅠsidechains and HG had different regulation trends on the composition of gut microbiota in colitis mice:HG mainly promote the growth of Akkermansia＿muciniphila,Bacteroides,Bacteroides＿thetaiotaomicron,and Odoribacter,thus increasing the ability of microbiota to degrade the mucus layer;100%sidechains structure could promote the proliferation of some pro-inflammatory bacteria like Turicibacter,Alistipes,and Pseudomonas.Pectin with multiple domain component could inhibit the abundance of pathogens such as Erysipelotrichales,Peptostreptococcus and Acinetobacter,promote the proliferation of anti-inflammatory bacteria such as Rikenellaceae＿RC9,Romboutsia,Clostridia,Dorea,Faecalibaculum and Ruminococcus.Thus,new type of pectin with multiple domain component had the best effects on alleviating colitis.