Protective Effect Of Polysaccharide From Enteromorpha Prolifera On Intestinal Barrier In Mice With Inflammatory Bowel Disease

Inflammatory bowel disease(IBD)is a kind of digestive tract disease,which seriously affects the physical and mental health of human beings all over the world.Crohn’s disease(CD)and ulcerative colitis(UC)are the two main manifestations of IBD.The severity,mortality and complication rate of IBD are among the highest in gastrointestinal diseases.The severity,mortality and complication rate of IBD are among the highest in gastrointestinal diseases,and it is still difficult to cure.Therefore,the use of natural dietary substances to prevent IBD has practical significance,so it has gradually attracted the attention of researchers and become a research hotspot at home and abroad.Prebiotics is a group of dietary polysaccharides which can not be digested and absorbed by the body,but it can be selectively fermented and utilized by beneficial bacteria in the intestinal tract of the human body.Prebiotics can promote the growth and metabolism of beneficial bacteria in the intestines,but does not stimulate the development and reproduction of harmful bacteria in the body to improve health.There is already evidence of the positive effects of probiotics on experimental colitis and human inflammatory bowel disease.Enteromorpha prolifera belongs to marine algae with abundant sources,low cost and high yield and could be used in food and medicine.Studies have shown that polysaccharides from Enteromorpha prolifera have the functions of regulating immunity,anti-inflammation,anti-tumor and anti-oxidation.In order to test the protective effect of polysaccharides from Enteromorpha prolifera prebiotics on IBD,this study aims to verify that polysaccharides from Enteromorpha prolifera can alleviate the intestinal inflammation in mice with inflammatory bowel disease by regulating the intestinal flora and intestinal immunity of mice,and protecting the intestinal barrier.It provides new ideas for preventing inflammatory bowel disease and promoting the clinical application of polysaccharides from Enteromorpha prolifera.Objective:Aiming at the mouse model of inflammatory bowel disease induced by sodium dextran sulfate(DSS),to explore whether polysaccharides from Enteromorpha prolifera regulates intestinal immunity by regulating intestinal flora and repairing intestinal barrier,thereby reducing the degree of intestinal inflammation in mice.Methods:1.The polysaccharides from Enteromorpha prolifera were extracted by water extractionalcohol precipitation method,the polysaccharides were precipitated by the principle that the polysaccharides were insoluble in the alcohol of 60%-80%.2.The C57BL/6J mice were arranged in three groups(5 mice in each group): Normal control group(NC),treatment group [i.e.on polysaccharide(DSS+PEP)] and the model group(DSS).The normal control group did not administer any treatment.During the whole experiment,the polysaccharide was administered intragastric dosage of 200mg/kg/day in DSS+PEP group while the same volume of normal saline was administered intragastrical in model group for 21 days.On the 21 st day of the experiment,the drinking water of DSS+PEP group and the model group was replaced with 3% DSS aqueous solution with every second day replacement for 7 days.Then,only used pure water for mice to drink.On the 29 th day of the experiment,the mice were sacrificed.Blood was obtained from the eye orbital.Mice feces,colon,thymus,and spleen were obtained and preserved for subsequent analysis.3.The disease activity index of inflammatory bowel disease was evaluated according to the characteristics of feces,bloody stool,and body weight of mice.Histopathological analysis and histopathological damage assessment were performed on the colonic tissue of mice after H&E staining.Myeloperoxidase kit was used to detect the activity of MPO in mouse colon.4.For the detection of intestinal biological barrier,extract the total DNA of mouse fecal bacteria,perform PCR-DGGE amplification and electrophoresis separation of 16 S r RNA gene to initially detect the changes in the structure of fecal flora.Then the total DNA of fecal bacteria was subjected to 16 S r RNA gene high-throughput sequencing to accurately detect changes of the intestinal flora in mice.5.For the detection of intestinal mechanical barrier,IHC and Western blotting were used to detect the expression of tight junction proteins CLDN1 and ZO-1.6.For the detection of intestinal chemical barrier and immune barrier,the content of mucin MUC2 was detected by immunohistochemical,while the content of inflammatory factors in mice was detected by enzyme linked immunosorbent assay(ELISA)and RT-q PCR.Results:1.The polysaccharide of E.prolifera protect intestinal microbiome diversity and their proportion in mice with inflammatory bowel disease.Polysaccharide treated group showed selectively increase in growth of beneficial bacteria such as Ruminococcus,Prevotella,Oscillospira and Odoribacter while decrease in growth in pathogenic bacteria: Proteobacteria and Actinobacteria.These results confirmed that the polysaccharide obtained from E.prolifera would enhance the growth of beneficial bacteria and reduce the potential bacteria in intestine of mice.The polysaccharide decreased the disease activity index,degree of inflammatory injury,MPO activity,and relieved the symptoms of Inflammatory bowel disease in mice.2.The polysaccharide Increased MUC2 secretion was observed.ZO-1 and CLDN1 expression was also increased which indicate that it could protect the chemical as well as mechanical barrier of colon.3.Pro-inflammatory cytokines;IL-1 β,IL-6,TNF-α and LPS were noted to be decrease as compare with DSS model group,while anti-inflammatory cytokines IL-10 in mice was increased.It showed that the polysaccharides could regulate the intestinal and systemic immunity of mice and hence,inhibit the development of inflammation.Conclusions:1.The polysaccharide from E.prolifera can reduce the degree of intestinal injury due to inflammation in mice with Inflammatory bowel disease.2.The polysaccharide regulates intestinal microbial diversity and increase the relative abundance of beneficial bacteria in DSS-induced Inflammatory bowel disease mouse model.Polysaccharide from E.prolifera also have protective effects on intestinal chemical,mechanical and immune barriers in mice with inflammatory bowel disease.