The Mechanism Of Action Of Angiogenin In Maintaining Gut Microbiota Homeostasis And Inhibiting IBD

Inflammatory bowel disease?IBD?is characterized by chronic inflammation of the gastrointestinal tract,but its pathogenesis has not yet been fully elucidated.It is believed that antimicrobial peptides,secreted by the host,maintain the stability of the gut microbiota,thus participate in the development of IBD.However,the underlying mechanisms of action of antimicrobial peptides are still poorly understood.Angiogenin?ANG?,a secreted ribonuclease,is highly expressed in colorectal tissues and has antibacterial activity.However,it is unclear whether it can regulate the progression of IBD by maintaining gut microbiota.To this end,this dissertation focuses on the role and mechanism of action of ANG in IBD by employing microbiology,molecular and cellular biology,and bioinformatics methods to comprehensively and systematically explore the contribution of ANG in IBD with both mouse colitis models and clinical samples.Firstly,we established the correlation between ANG and IBD.By comparing the ANG concentration in feces from IBD and healthy subjects,we found that ANG level was significantly decreased in the feces of IBD patients.Meanwhile,Ang1 gene deficiency(Ang1^-/-)significantly aggravated DSS-induced colitis symptoms in mice,including more body weight loss,increased disease activity index,shorter intestinal length,more severe intestinal epithelial damage,and increased expression of inflammatory factors.These findings indicate that ANG could inhibit IBD development.Secondly,we uncovered the contribution of ANG-regulated gut microbiota in IBD.We compared the gut microbiota differences between wild-type?WT?mice and Ang1^-/-mice through 16S r DNA amplicon high-throughput sequencing.The results showed that the microbiota composition was significantly altered in Ang1^-/-mice,in which?-Proteobacteria increased while Lachnospiraceae reduced.Furthermore,we found that WT and Ang1^-/-mice displayed indistinguishable colitis symptoms after gut microbiota removal;nevertheless,transfer of Ang1^-/-mice fecal microbiota into WT mice significantly increased their susceptibility to different colitis models.These data indicate that ANG inhibits IBD development by maintaining a healthy gut microbiota.In order to identify specific strains associated with the pathogenesis of colitis,we performed screening and identification experiments.As a result,we found that the abundances of B.diminuta and S.paucimobilis,belonging to?-Proteobacteria,were significantly increased in the feces of Ang1^-/-mice;on the contrary,Anaerostipes sp.and Blautia sp.,belonging to Lachnospiraceae,were significantly reduced in Ang1^-/-mice.Based on this result,we explored the relationship between these strains and mouse colitis by bacteria gavage experiments.As expected,increasing the abundances of two strains of?-Proteobacteria aggravated the colitis severity of WT mice;while supplementing Ang1^-/-mice with strains of Lachnospiraceae could alleviate colitis.In addition,we found that fecal butyrate concentration was significantly reduced in Ang1^-/-mice,while the administration of sodium butyrate significantly ameliorated DSS-induced colitis.The above results indicate that ANG could inhibit IBD development by regulating the abundance of gut?-Proteobacteria and Lachnospiraceae.Thirdly,we explored the mechanism by which ANG regulates gut microbiota.We found that ANG had a significant killing effect on B.diminuta and S.paucimobilis,but much weaker on Anaerostipes sp.and Blautia sp.through in vitro antibacterial experiments.Meanwhile,the propidium iodide staining experiment and scanning electron microscope analysis showed that ANG killed the B.diminuta and S.paucimobilis by destroying the cell membrane in a dose-dependent manner.The above data suggest that ANG differentially inhibits the growth of?-Proteobacteria and Lachnospiraceae bacteria by directly destroying the cell membrane of susceptible bacteria.Finally,we established the clinical significance of ANG and its related bacteria.On one hand,gavage of ANG1 recombinant protein significantly reduced the abundance of?-Proteobacteria and increased the abundance of Lachnospiraceae in Ang1^-/-mice feces;meanwhile,gavage of ANG1 recombinant protein significantly alleviated the DSS-induced colitis symptoms.On other hand,we compared the difference in gut microbiota from IBD patients and healthy volunteers,and found that the abundance of?-Proteobacteria?B.diminuta and S.paucimobilis?significantly increased and Lachnospiraceae?Anaerostipes sp.and Blautia sp.?significantly reduced in the feces from IBD patients.Consistent with this,the fecal ANG concentration in the patients with IBD was also significantly decreased.Together,our data suggest that fecal ANG concentration and ANG-associated bacteria can be used as biomarkers for the clinical diagnosis of IBD.In summary,this dissertation revealed that ANG can suppress IBD progression by balancing the abundance of?-Proteobacteria and Lachnospiraceae in gut microbiota.ANG itself could be a drug for the treatment of IBD,and its related bacteria have the potential to be therapeutic targets and diagnostic markers of IBD.