The Ameliorative Effects Of Inosine-Producing Gut Bacteria On Colitis And The Mechanism

Inosine is a purine nucleoside widely present in the body,which not only provides energy for cells in the hypoxic environment,but also maintains the integrity of the intestinal barrier.It is reported that the intestinal barrier of colitis mice is disrupted,and the levels of inosine in the feces and serum are significantly lower than those of healthy mice.Therefore,exogenous supplementation of inosine may become a strategy for alleviating colitis.However,as a small molecule substance,inosine is easily absorbed in the small intestine after oral administration,making it difficult to calculate the amount it reaches the colon.The intestinal level at which it alleviates colitis is not clear.Some gut bacteria,such as Bifidobacterium pseudolongum,Lactobacillus johnsonii,and Akkermansia muciniphila,have the ability to produce inosine.Whether oral administration of these inosine-producing bacteria can alleviate colitis,their dose-effect relationship,and mechanism of action have not been studied clearly.Therefore,we adopted two intervention methods,rectal delivery and oral administration,to study the ameliorative effect of inosine on colitis and clarify the effective intestinal level.Gut bacteria with the ability to produce inosine were screened to study the relationship between their ameliorative effect on colitis and inosine-producing ability.The strain with strongest ability to produce inosine was selected to study its dose-effect relationship in alleviating colitis,and further analyze its mechanism of alleviating colitis through inosine production.The main results are shown as follows.A mouse model of colitis was constructed using dextran sulfate sodium（DSS）,and the dose-response relationship of inosine in alleviating colitis was explored using rectal delivery and oral administration.The results showed that rectal delivery of 50 mg/kg inosine effectively inhibited the loss of body weight,increases of disease activity index（DAI）score,shortening of colon,decreased the levels of colonic inflammatory cytokines（TNF-α,IL-1β,and IL-6）,decreased the level of anti-inflammatory cytokine（IL-10）,deduced the accumulation of malondialdehyde（MDA）,increased the activities of antioxidant enzymes（SOD,CAT,and T-AOC）,decreased the colon tissue lesions and the loss of tight junction protein in colitis mice.The ameliorative effect of rectal delivery of 10 and 100 mg/kg inosine on colitis was relatively poor.Oral administration of high-dose（800 mg/kg）inosine can effectively alleviate symptoms of colitis mice,but low-dose（100 mg/kg）inosine intervention has no significant ameliorative effect on colitis.Gut bacteria with the ability to produce inosine was screened in vitro.The results showed that there were strains differences among B.pseudolongum within Bifidobacterium genus,with strains CCFM1253 and Bp7 having the higher inosine-producing ability.Lactobacillus has a weaker ability to produce inosine than Bifidobacterium,with L.paracasei CCFM1222 having a higher ability.Six strains of gut bacteria with different abilities of inosine production were screened to study their relieving effects on colitis,including B.pseudolongum CCFM1253,Bp7,Bp8,and FJLHD9M1,L.paracasei CCFM1222,and FQHXN96L1.The results showed that both B.pseudolongum CCFM1253 and Bp7 with higher inosine-producing abilities significantly inhibited the increases in DAI score,prevented colon shortening,improved colon tissue structure,regulated inflammatory response,maintained redox stability,and prevented the loss of tight junction proteins in colon tissue in colitis mice.However,B.pseudolongum FJLHD9M1 and Bp8 with lower inosine-producing abilities did not significantly improve the colitis symptoms.In addition,both B.pseudolongum CCFM1253 and Bp7 significantly increased intestinal levels of inosine in colitis mice,while B.pseudolongum FJLHD9M1 and Bp8 had no significant effect on the intestinal levels of inosine.Heat-deactivated B.pseudolongum CCFM1253 failed to improve the colitis of mice.B.pseudolongum CCFM1253,which has the strongest ability to produce inosine,was selected to analyze the relationships among inosine-producing gut bacteria,intestinal inosine,and colitis.Different doses(10⁷,10⁸,10⁹,and 10¹⁰ CFU)of B.pseudolongum CCFM1253 were administered by gavage to colitis mice.The results showed that the intestinal levels of inosine increased with the increase of dose of B.pseudolongum CCFM1253,and there was a significant linear positive correlation.10⁸,10⁹,and 10¹⁰ CFU of B.pseudolongum CCFM1253significantly increased intestinal levels of inosine,but 10⁷ CFU of B.pseudolongum CCFM1253 had no significant effect on intestinal levels of inosine.Daily administration of 10⁹or 10¹⁰ CFU of B.pseudolongum CCFM1253 significantly regulated the levels of inflammatory factors,increased the activities of antioxidant enzymes,and prevented the loss of tight junction proteins in the colon.However,10⁷ or 10⁸ CFU of B.pseudolongum CCFM1253 had no significant effect on the colitis symptoms.Correlation analysis revealed a significant linear correlation between intestinal levels of inosine and inflammatory response and oxidative stress-related parameters.Thus,B.pseudolongum CCFM1253 can regulate the intestinal levels of inosine and alleviate colitis in mice at a dose-dependent manner.The adenosine receptor(A_2AR)antagonist was used to treat colitis mice,and the ameliorative effects of B.pseudolongum CCFM1253 and inosine on colitis symptoms in mice disappeared,indicating that the key metabolite B.pseudolongum CCFM1253 to ameliorate colitis was inosine.Through transcriptomics,RT-qPCR,and immunofluorescence analysis,it was found that both B.pseudolongum CCFM1253 and inosine significantly increased the expression of PPARγin colon tissue of colitis mice.Using intraperitoneal injection of PPARγinhibitors to block PPARγexpression,the result showed that PPARγinhibitors can prevent B.pseudolongum CCFM1253 from affecting body weight,DAI score,PPARγexpression,colon length,tight junction protein,inflammatory factors,oxidative stress,and related gene expressions,which can no longer alleviate colitis.These results indicated that PPARγis a key target for the ameliorating colitis by B.pseudolongum CCFM1253.Therefore,B.pseudolongum CCFM1253 produces inosine in the intestine,which binds to receptor A_2AR and activates PPARγRelated pathways to alleviate colitis.