Chitooligosaccharides Promotes The Growth Of Intestinal Lactobacillus Spp.and T Cell Proliferation

Background:Chitooligosaccharides are 2 to 8 glucosamines by?-1,4 glycosidic bonds extracted from shells of marine shells and the like.It has a variety of biological activities,such as anti-infective,anti-tumor,anti-viral,immune regulation and the like.It is generally believed that only monosaccharides and disaccharides in the oligosaccharides can be absorbed through the digestive tract.Since there is no hydrolase in the human body which can hydrolyze the?-1,4 glycosidic bond,the oligosaccharides having a degree of polymerization greater than 2 can't be absorbed.A wide variety of microorganisms are colonized in the human gastrointestinal tract,and these microorganisms are collectively referred to as intestinal microbiota.The intestinal microbiota is combined in a certain proportion,and each intestinal bacteria is mutually restricted and interdependent,forming an ecological balance in quality and quantity.The intestinal microbiota inhibits the growth of pathogenic bacteria through the activity of the dominant commensal bacteria,and prevents pathogenic bacteria from entering the body through this barrier.The disorder of the intestinal microbiota causes a series of immune-related diseases.Normal intestinal microbiota can decompose substances that can't be degraded by the body,and regulate the body's immunity through the influence of lymphocytes.Studies on the use of chitooligosaccharides by the intestinal microbiota and further affecting immune function haven't been reported.Objective:To study the regulation of chitooligosaccharides on T cell proliferation,and to explore its mechanism of action by identifying the intestinal microbiota that specifically utilizes chitooligosaccharides and superior growth,providing a theoretical basis for future development.Method:1.Effects of chitooligosaccharides on intestinal microbiotaSixteen healthy male Babl/c mice were randomly divided into two groups:normal control group?100?l/day H₂O?and chitosan oligosaccharide group?administered with100?l/day 0.1g/ml of chitooligosaccharides solution?,and collected mouse feces for chitooligosaccharides effect on intestinal microbiota.The distribution of intestinal microbiota in the two groups was initially detected by Denaturing gradient gel electrophoresis?DGGE?.Further,16s high-throughput sequencing was used to detect specific bacteria.Volunteers were recruited to collect fecal samples before and after the test of chitooligosaccharides,and RT-PCR was used to detect changes in specific bacteria.2.The regulation of chitooligosaccharides on immune function in miceSixteen healthy male Babl/c mice were randomly divided into two groups:normal control group?100?l/day H₂O?and chitooligosaccharides group?administered with100?l/day 0.1g/ml of chitooligosaccharides solution?to study the immunomodulatory effects of chitooligosaccharides on mice.The proportion of each immune cell in the spleen of mice was detected by flow cytometry,and its statistical significance was analyzed by statistical analysis.The mRNA expression levels of immune-related cytokines in spleen were detected by RT-PCR.3.The growth state of Lactobacillus by chitooligosaccharides and T cell proliferationLactobacillus was cultured in a glycose-free medium,and different concentrations of chitooligosaccharides?0?g/ml,1?g/ml,10?g/ml,100?g/ml and 1000?g/ml?were added to observe the effect on the growth state of Lactobacillus.The OD value at 595nm was measured by a microplate reader,and recorded continuously for 50 hours,and the growth curve of Lactobacillus was plotted.The supernatant of the chitooligosaccharides solution,the Lactobacillus supernatant and the Lactobacillus+chitooligosaccharides co-culture was used to stimulate T cells,and the proliferation was detected by MTT colorimetry and CFSE flow fluorescent labeling.4.Protective effect of chitooligosaccharides on hyperglycemia and blood lipid levels induced by high fat diet in miceFifteen healthy male ICR mice were randomly divided into 3 groups:normal control group?normal feed?,high-fat diet group?high-fat diet?,high-fat diet+chitooligosaccharides group?high-fat diet and administered with 100?l/day 0.1 g/ml of chitooligosaccharides solution?.According to the corresponding grouping,serum samples were taken for ELISA detection of serum blood glucose and lipid levels.Results;1.Effects of chitooligosaccharides on intestinal microbiotaThe DGGE test showed that the intestinal microbiota of the control group and the chitooligosaccharides group were significantly different,and each group was clustered into one group,indicating that chitooligosaccharides changed the intestinal microbiota distribution of mice;Sequencing further revealed a relatively increased number of bacteria in the chitooligosaccharides group:Lactobacillus and Akkermansia muciniphila.16s high-throughput sequencing revealed a specific change and significant difference in the chitooligosaccharides after intragastric administration-Lactobacillus.We recruited volunteers who tried the chitooligosaccharides capsules,collected the feces before and after taking the chitooligosaccharides capsules,extracted the DNA,and detected the changes of the lactobacillus in the feces by RT-PCR.We found that taking the chitooligosaccharides capsules Afterwards,the relative amount of Lactobacillus in the volunteer feces increased significantly.2.The regulation of chitooligosaccharides on immune function in miceThe distribution of each immune cell was detected by flow cytometry by fluorescent antibody staining of mice whole spleen cells.The proportion of CD3⁺T cells in the spleen of mice in chitooligosaccharides group was significantly higher than that of control group;The mRNA expression levels of cytokines IL-2 and IL-4 in the spleen of mice has no significance between the two groups by RT-PCR.3.Chitooligosaccharides can promote the growth of Lactobacillus and T cell proliferationBy plotting Lactobacillus growth curves,we found that chitooligosaccharides can significantly promote the growth of L.gasseri JCM 1131 and L.brevis DM9218.When the concentration of chitooligosaccharides in the medium was 0?g/ml¹00?g/ml,the growth trend of L.gasseri JCM 1131 was positively correlated with the concentration of chitooligosaccharides;when the concentration of chitooligosaccharides was0?g/ml¹000?g/ml,the growth trend of L.brevis DM9218 was positively correlated with the concentration of chitooligosaccharides;whereas for L.casei CGMCC1.2435?ATCC 334?,the concentration of chitooligosaccharides involved in this experiment could not change its growth state.Detecting T cell proliferation by MTT and CFSE,we found that T cells treated with Lactobacillus+chitooligosaccharides co-culture supernatant showed significant proliferation,while a single supernatant of chitooligosaccharides solution or Lactobacillus?the supernatant of Lactobacillus from completed culture medium or glucose-free culture medium?,none of them can cause the proliferation of T cells.4.Protective effect of chitooligosaccharides on hyperglycemia and blood lipid levels induced by high fat diet in miceBy ELISA,in the serum triglyceride,cholesterol and very low-density lipoprotein,the high-fat diet group treated with chitooligosaccharides significantly improved the blood glucose and blood lipid levels,and the food intake decreased,the water intake increased,and the body weight decreased significantly.Conclusion:This experimental study found that chitooligosaccharides can promote the growth of Lactobacillus and T cell proliferation.