| Ulcerative colitis is one of the most common inflammatory bowel diseases to emerge since hygiene and urbanization improved in North America and Europe in the early 20 th century.More recently,it has begun to spread to middle-and high-income developing countries such as China.Characterized by persistent abdominal pain,diarrhea,and blood in the stool,the inflammation affects the inner lining of the intestine from the colon to the rectum.Current drug treatments have been successful in alleviating symptoms,but these treatments are limited by high recurrence rates and inevitable side effects.Therefore,there is an urgent need to develop new and effective therapeutic drugs.According to the Chinese Pharmacopoeia,both GG and FG have the effect of stopping diarrhea.At present,flavonoids are the main active components of pueraria and pueraria pueraria,while there are few studies on polysaccharides in this aspect,especially the comparison of the two at the same time.Whether there is a difference in the antidiarrheal effect of the two and whether there is a difference in the mechanism of action is worth further discussion.At present,the existing problems in the study of FG and GG in the treatment of ulcerative colitis are as following: Puerarin and Gegenqinlian decoction have previously been reported to have the efficacy of treating ulcerative colitis,but the anti-diarrhea efficacy of FG and GG has not been studied.Whether the mechanism of action of pueraria polysaccharide in the treatment of ulcerative colitis is the same is worth further exploration.Plant-derived polysaccharides,composed of multiple monosaccharides,complex glycosidic bonds and multiple molecular weights,have been shown to reduce UC discomfort symptoms in multiple modes of action.To heal damage to the intestinal barrier,it can control the quantity and variety of intestinal flora as well as the aberrant expression of the TJ protein.The use of metabolomics and intestinal flora research techniques is now expanding rapidly.Intestinal flora research in conjunction with metabolomics is a crucial research tool for understanding polysaccharide mechanism.si RNA and AAV transfection have been widely used in gene experiments,which can be used to study the mechanism of polysaccharide action and the correlation between genes.Based on this,In order to treat ulcerative colitis,this project focuses on the antidiarrheal activity of GG and FG polysaccharide as well as the mechanism of action.The following are the primary research findings:1.Pharmacodynamics of Pueraria polysaccharide on ulcerative colitis in C57BL/6miceMale C57BL/6 mice were used as research objects to construct the colitis model induced by DSS.After three days of prophylactic administration,the mice in the administration group were fed 2.5%DSS drinking water and intragastric administration of 5-aminosalicylic acid(5-ASA)or different doses of GG and FG polysaccharide(50.0,100.0,200.0 mg/kg).Daily measurements of body weight,blood in the stool,and diarrhea were taken.The disease activity index(DAI),colon length,and the activity of the inflammatory factors IL-6,IL-1β,and TNF-α were also examined.By using H&E staining,pathological abnormalities in colon tissue were found.After 10 days of intervention,GG and FG polysaccharide(50,100,200mg/kg)significantly reduced DAI score,inhibited the shortening of colon length and weight loss,reduced the content of inflammatory factors in colon and improved the pathological injury of colon tissue,and improved the spleen index.It was proved that GG and FG polysaccharide had good anti-ulcerative colitis effect.It was proved that GG and FG polysaccharide both had antidiarrheal effect.There was no statistically significant difference between GG and FG polysaccharide.2.Pharmacodynamics of Pueraria polysaccharide on inflammation in Caco-2 cellsHuman Caco-2 cells were selected as the research object to construct the intestinal injury model induced by DSS in vitro.After 4h of pre-administration,different doses of GG and FG polysaccharide(10.0,20.0,40.0 μg/ml)were continued to be administered for 24 h from the day of modeling.Cell survival rate,apoptosis rate,transmembrane resistance and tight junction protein expression were recorded in each group.After intervention,the administration of polysaccharide(10.0,20.0,40.0 μg/ml)had no significant effect on cell survival rate,but could reduce the apoptosis induced by DSS,and the expression of transmembrane resistance and tight junction protein.It was proved that Pueraria polysaccharide had good anti-inflammatory effect in vitro.From the detection index,GG polysaccharide was better than FG polysaccharide.3.Treatment of ulcerative colitis mice with Pueraria polysaccharide based on metabonomicsIn order to study the mechanism of pueraria polysaccharide in the treatment of ulcerative colitis,the metabolomics of animal plasma was studied.There were 64 different metabolites between normal mice and model mice.After the intervention of Pueraria polysaccharide,the metabolic profile of mice with ulcerative colitis was changed,suggesting that pueraria polysaccharide and pueraria polysaccharide may have therapeutic effects on ulcerative colitis by influencing endogenous components in mice with ulcerative colitis.It was found that GG polysaccharide and FG pueraria polysaccharide had regulatory effects on the above pathways.According to correlation analysis of KEGG and Cytoscape,all four metabolic pathways are directly or indirectly correlated with PI3K/AKT.Therefore,it can be concluded that GG and FG polysaccharide can play a role in regulating the metabolism of ulcerative colitis through PI3K/AKT signaling pathway.4.Effects of Pueraria polysaccharide on intestinal microflora of ulcerative colitis in C57BL/6 miceThe interaction between polysaccharide and intestinal tract plays an important role in its efficacy.Therefore,this study studied the effects of GG polysaccharide and FG pueraria polysaccharide on intestinal flora.In this chapter,the cecum contents of mice were studied based on 16 Sr RNA high-throughput sequencing.The results of Alpha diversity analysis showed that the bacterial diversity of pueraria polysaccharide was close to that of normal group.Coverage index of each group was above 0.99,indicating that the sequencing results could represent the real situation of microorganisms in the samples.There was no significant difference between FG polysaccharide and GG polysaccharide groups.The results of community composition analysis showed that Firmicutes and Bacteroidetes had different action trends after the intervention of pueraria polysaccharide.There was no significant difference between GG polysaccharide and FG pueraria polysaccharide.The intestinal flora of the normal group and the model group differed significantly,according to a beta diversity analysis.The composition of intestinal flora was more similar to the normal group following the administration of p pueraria polysaccharide.These findings suggested that the cecal microflora might be altered by the addition of G pueraria polysaccharides to return it to the normal group.The expression in the route of the model group was lower than that of the normal group,according to KEGG metabolic pathway enrichment analysis,and there was a tendency for correction following polysaccharide intervention.Spearman correlation analysis showed that metabolites were significantly correlated with bacterial flora,but whether there was a direct relationship between bacteria and plasma metabolites still needs to be further verified by experiments.5.Research on the ulcerative colitis-related pathogenic mechanisms of Pueraria polysaccharide in C57BL/6 miceIn order to verify whether GG polysaccharide and FG polysaccharide have regulatory effects on PI3K/AKT signaling pathway and tight junction protein.In this chapter,Western blotting,IHC,and IF studies were used to examine the colonic proteins of mice.The findings demonstrated that the model group’s expression of the proteins pPI3 K and p-AKT was higher than that of the normal group.All proteins expressed less after polysaccharide intervention,suggesting that pueraria polysaccharide functions by preventing PI3K/AKT phosphorylation.ZO-1,claudin-1,and occludin expression considerably decreased in the model group.Pueraria polysaccharide appears to preserve the intestinal barrier through controlling the expression of tight junction protein since the expression of all proteins increased after polysaccharide intervention.Immunohistochemistry and immunofluorescence outcomes matched those of protein.There was no significant difference in protein expression between GG polysaccharide and FG polysaccharide.GG polysaccharide and FG pueraria polysaccharide may reduce inflammatory response in mice with ulcerative colitis by activating PI3K/AKT signaling pathway,thus exerting antidiarrheal effect.6.Study on the pathological mechanism of Pueraria polysaccharide in the treatment of ulcerative colitis in knockdown-PI3 K miceBased on the results of previous studies,to further determine whether PI3 K is a direct pathway for the interaction of GG and FG polysaccharides.PI3 K gene was knocked down in Caoc-2 cells and mice by in vitro transfection of si RNA and in vivo transfection of AVV.The results showed that after PI3 K knockdown,the effect of GG polysaccharide on animals and cells completely disappeared,while that of FG polysaccharide did not completely disappear.Meanwhile,the results of plasma metabolomics were consistent with the results of drug efficacy.Conclusion: In this study,the effects of GG and FG polysaccharides on the metabolomics and intestinal flora of ulcerative colitis were improved.The effect of GG and FG polysaccharides on preventing diarrhea was verified by pharmacodynamic experiments.Metabolomics studies and intestinal flora studies have found that pueraria polysaccharides have effects on plasma metabolites and bacteria,and the regulation of metabolites is different from that of bacteria,but the regulation of metabolic pathway is the same.The correlation analysis suggested that GG and FG polysaccharides might affect metabolites by regulating the flora.Mechanism verification showed that GG and FG polysaccharides may play a role in the treatment of ulcerative colitis through the regulation of PI3K/AKT signaling pathway.Finally,gene knockdown proved that PI3 K was the direct pathway of GG polysaccharide,while FG polysaccharide had multitarget effect.This study provides a new perspective for exploring the effect and mechanism of pueraria polysaccharide in the treatment of ulcerative colitis and provides an important basis for medical research. |
PDF Full Text Request