In Vitro Human Intestinal Microbiota Culture For Treatment Of Ulcerative Colitis In Mice

Ulcerative colitis(UC)is a non-specific chronic inflammatory bowel disease.The incidence of global ulcerative colitis has increased year by year,and because there is no good therapeutic drug,it has a great burden on patients and society.Microbiota in the human gut have an important role in human health.Studies have shown that intestinal mucosal immune abnormalities caused by dysbacteriosis in the intestinal tract are one of the important mechanisms of the pathogenesis of ulcerative colitis.Clinical trials have shown that fecal microbiota transplantation(FMT)has a good effect on the treatment of ulcerative colitis,but the fecal microbiota transplantation technology has shortcomings such as shortage of donor intestinal microbiota and unstable treatment effects.Therefore,development of donor fecal bacteria substitutes is of great significance to the development of fecal microbiota transplantation technology.In this study,in vitro culture approach was used to culture the human intestinal microbiota from fecal microbiota transplantation donors,studied the effects of prebiotics,berberine and medium composition on human intestinal microbiota during in vitro culture,and investigated the efficacy of in vitro cultured intestinal microbiota FMT in the treatment of ulcerative colitis mice.It is of a great significance to develop donor intestinal microbiota substitutes for clinical fecal microbiota transplantation.The main work of this thesis includes the following four parts:First,investigated the effects of prebiotics on human intestinal microbiota composition and metabolism during in vitro culture.Five kinds of prebiotics,oligoisomaltose,arabinogalactan,pectin,inulin and stachyose,were selected as the research objects.16S rRNA high-throughput sequencing and liquid chromatography techniques were used to investigate the intestinal microbiota diversity,composition and SCFA metabolism changes during in vitro culture.The results showed that oligoisomaltose promoted the growth of Lachnospiraceae during in vitro culture.Arabinogalactan increased the level of intestinal microbiota metabolism relative gene during in vitro culture.Pectin facilitated the maintenance of intestinal microbiota diversity and promoted of the metabolism of propionic acid and butyric acid.Inulin and stachyose promote the growth of the Veillonellaceae during in vitro culture.The above results indicated that the selection of oligoisomaltose,arabinogalactan and pectin as the medium components for in vitro intestinal microbiota culture could improve intestinal microbiota,promote the growth of beneficial bacteria and improve intestinal microbiota metabolism.Second,evaluated the effects of berberine on human intestinal microbiota composition and metabolism during in vitro culture.The results showed berberine reduced intestinal microbiota diversity during in vitro culture.Increased the relative abundance of Faecalibacterium and decreased the relative abundance of Bifidobacterium,Streptococcus and Enterococcus during in vitro culture.Berberine significantly inhibited the intestinal microbiota acetic acid and propionic acid metabolism,but did not inhibit the metabolism of butyric acid intestinal microbiota.Metabolomics results showed that berberine impacted the intestinal microbiota amino acid metabolism,which can promote the metabolism of glycine,serine and threonine metabolism,and inhibit the metabolism of arginine and proline.The above results indicated that berberine inhibits the metabolism of acetic acid and propionic acid during in vitro culture,reduced the intestinal microbiota diversity and inhibits the growth of beneficial bacteria Bifidobacterium,indicating that berberine is not suitable for in vitro intestinal microbiota culture.Third,the effects of different nutrient composition media on in vitro culture human intestinal microbiota were studied.Investigated the effects of four media,including brain heart infusion medium(BHI),gut microbial culture medium(GMM),fastidious anaerobe broth(FAB)and bacterial growth medium(BGM)on intestinal microbiota diversity,composition,network and SCFA metabolsim during in vitro culture.The results showed that BHI medium could help maintain the intestinal microbiota diversity and promote the growth of Streptococcus during in vitro culture.GMM medium promoted the growth of Bifidobacterium and metabolism of acetic acid and propionic acid.FAB medium promoted the growth of Clostridium sensu stricto and Enterococcus during in vitro culture.BGM medium promoted the growth of Akkermansia and facilitated the metabolism butyric acid during in vitro culture.The above results indicated that GMM medium could facilitate the growth of Bifidobacterium and promote the metabolism of SCFA.Fourth,GMM medium with oligoisomaltose,arabinogalactan and pectin was selected to culture human intestinal microbiota in vitro.Investigated the therapeutic effects of in vitro cultured human intestinal microbiota on ulcerative colitis mice induced by dextran sulfate sodium(DSS).The results showed that in vitro cultured intestinal microbiota FMT treatment can greatly improve the survival rate of ulcerative colitis model mice,and improved the general symptoms of ulcerative colitis mice(such as recovery of body weight and colon length)and reduce mice Disease Activity Index(DAI).In vitro cultured human intestinal microbiota FMT treatment can improve the ratio of proinflammatory cytokines and anti-inflammatory cytokines in mice with ulcerative colitis.The results of serum metabolomics showed the energy metabolism in mice was restored.In vitro intestines cultured FMT treatment can change the intestinal microbiota of mice with ulcerative colitis,such as restored the diversity of intestinal microbiota,and increased the relative abundance of functional genes related to intestinal microbiota metabolism.Enriched beneficial bacteria,inhibited the growth of harmful bacteria,and improved the dysbacteriosis of intestinal microbiota caused by DSS.The above results indicated that in vitro cultured human intestinal microbiota as the FMT content has a good therapeutic effects on ulcerative colitis in mice.In vitro cultured human intestinal microbiota transplantation could regulate intestinal and immune function in mice.It could reduce the degree of inflammatory reaction in mice,restore the intestinal microbiota diversity and alleviate the symptoms of intestinal microbiota disorder,thereby significantly improved the survival rate of mice and restored the health of mice.