Study On The Pharmacodynamic Mechanism Of Bletilla Striata Oligosaccharides To Improve Ulcerative Colitis

ObjectivesBletilla striata（Thunb.）Reichb.f.was widely used in China for the treatment of traumatic bleeding and digestive system disorders.Bletilla striata’s main ingredient,Bletilla striata polysaccharide（BP）,is difficult to study in depth due to its complex structure,large molecular weight and high viscosity.Therefore,this study proposed to degrade BP into Bletilla striata oligosaccharide（BO）and analyze the structure of BO.An ulcerative colitis（UC）model constructed with 3%Dextran Sulfate Sodium（DSS）was used to elucidate the mechanism of BO in improving UC,providing a new strategy for the prevention and treatment of UC.Methods1.Structural analysis of BOFirstly,the monosaccharide composition and its corresponding molar ratios were confirmed by high performance liquid chromatography.Secondly,the structure of BO was identified by methylation analysis,Fourier Transform infrared spectroscopy（FT-IR）analysis,and Nuclear Magnetic Resonance（NMR）analysis.2.Construction of an UC model and pharmacodynamic study on the anti-UC of BOSix-week-old male BALB/c mice were selected for the study and randomly divided into 4 groups:normal group,model group,BO group,and BO+model group.A UC model was induced by 3%DSS and the intervention was administered with BO（200 mg/kg/d）for a modelling period of 7 days.Weight change,disease activity index score,colon length,spleen index,pathological sections of colon,and serum Lipocalin-2 level were used as judging indicators to evaluate the pharmacological activity of BO.3.Study on the effect of BO in inhibiting intestinal inflammation and colonic damageMolecular biology techniques such as Real-time PCR,Western blot,and immunofluorescence/histochemistry were used to detect the expression of inflammatory factors such as Tnf-a,Nlrp3 and Il-1β at the mRNA and protein levels in UC mice.In addition,in order to evaluate the inhibitory effect of BO on colonic injury,the expression of water channel protein,mucin and tight junction protein in UC mice were detected by colon tissue section staining.4.Study on the effect of BO on gut microbiota and its metabolitesThe changes in the gut microbiota of mice with UC were detected by 16S rDNA sequencing technology,while bile acids,short fatty acids and tryptophan metabolites were also extracted and the content of the metabolites was determined by LC/GC-MS,which proved the regulatory effect of BO on the gut microbiota and its metabolites in UC mice.In addition,the important role of gut microbiota in the improvement of UC by BO was further verified by antibiotic experiments.5.Study on the effect of BO on the enterohepatic circulation of bile acidsThe expression of bile acid receptor was detected by Real-time PCR and Western blot,and the effect of BO on the regulation of abnormal enterohepatic circulation in UC mice was investigated in depth.Results1.BO mainly contained glucose and mannose.The ratio of mannose:glucose in BO was calculated to be 5.2:1.2.The main backbones of BO contained（1 → 4）-linked-α-D-Man,（1→2）linked-α-D-Man,and（1→2）-linked-α-D-Glc.3.BO improved physiological indicators in UC mice,such as slowing weight loss,increasing colon length and lowering spleen index.4.BO not only significantly inhibited the expression of intestinal inflammatory factors,but also promoted the expression of water channel proteins,mucins,and tight junction proteins in colon tissues.5.BO restored the diversity of gut microbiota in mice with ulcerative colitis,reduced the abundance of bacteria such as Parabacteroides,Bacteroides,Oscillibacter,and Helicobacter,increased the abundance of Ruminococcaceae NK4A214 group,Odoribacter,and Prevotellaceae UCG001,effectively regulating gut microbiota disorders.6.BO partially reversed the levels of bile acids,short-chain fatty acids,and tryptophan metabolite（5-hydroxytryptamine）in UC mice.7.Antibiotic experiment showed that in the absence of intestinal flora,BO was unable to improve symptoms in antibiotic-treated mice,while BO fermentation broth slowed weight loss,alleviated blood in the stool,reduced spleen index and increased colon length.8.BO effectively regulated the expression of bile acid receptors in the intestine and liver,and improved the enterohepatic circulation in UC mice.ConclusionsIn this study,we initially investigated the mechanism of BO in improving UC.The results showed that BO could inhibit intestinal inflammation and colonic injury in UC mice by regulating the composition of gut microbiota and intestinal metabolites,providing a scientific basis for the future treatment of UC.