Effect Of Clostridium Butyricum On DSS-induced Colitis In Mice After Antibiotic Cocktails Pretreatment And Its Preliminary Mechanism

Objective: Using different combination of antibiotic cocktail pretreatment to build a pseudoaseptic mouse model,to observe the influence of ABx on the physiological function,intestinal inflammation and gut microbiota of mice.To observe the therapeutic effect of Clostridium butyricum MIYAIRI 588 on mice with colitis,and to explore its effects on intestinal barrier,inflammation,immune cells in mice.To observe the effect of Clostridium butyricum MIYAIRI588 on gut microbiota construction and short chain fatty acid in mice with colitis.To explore the mechanism of Clostridium butyricum MIYAIRI 588 and its supernatant regulating inflammatory homeostasis in mice with colitis.Methods:(1)Mice were treated with 3ABx(Ampicillin(1g/L),Vancomycin(0.5g/L),Metronidazole(1g/L))and 4ABx(Ampicillin(1g/L),Vancomycin(0.5g/L),Metronidazole(1g/L),Neomycin(1g/L))for 4 weeks,respectively.The body weight,water intake and feed intake of mice were detected.The levels of albumin,alanine aminotransferase,glutamic oxalate aminotransferase,serum creatinine,urea,triglyceride,total cholesterol,blood glucose were detected to evaluate the liver function,renal function,blood lipid,blood glucose of mice.Colon inflammation of mice was evaluated by colonic histology.16 S r RNA sequencing was used to evaluate the structural changes of intestinal microbiota in mice.(2)The mice were pretreated with 4ABx and fed with drinking water containing 3%DSS for 1week to construct DSS colitis mouse model.At the same time,Clostridium butyricum MIYAIRI 588 and its supernatant were given to mice by gavage once a day for 1 week,respectively.The body weight,disease activity index,colon length,histological inflammatory score of colitis mice were observed and recorded.The changes of barrier in mice were detected by RT-q PCR and immunohistochemistry to observe tight junction proteins and mucus-associated proteins expression.The expression of inflammatory factors in colon tissue and serum was observed by RT-q PCR and ELISA.The changes of immune cells in mucosal lamina propria of mice were further observed by RT-q PCR,flow cytometry,immunohistochemistry.(3)RT-q PCR was used to detect the quantitative expression of Clostridium butyricum MIYAIRI 588 in feces,and 16 S r RNA sequencing was used to detect the changes of fecal microbiota in mice.And the changes of short chain fatty acid were detected by GC-MS methods.(4)The RNA-Seq of colon tissue of mice was detected,and the effect of Clostridium butyricum MIYAIRI 588 on gene transcription level of mice was explored through the differentially expressed genes.Furthermore,GO enrichment analysis and KEGG enrichment analysis was conducted to explore the possible signal pathway mechanism of Clostridium butyricum MIYAIRI 588 intervention.Results:(1)From the first week,the body weight,water intake,food intake of mice in ABx group were lower than those in the control group.From the second week,it gradually recovered to the level of the control group.The levels of serum creatinine and urea were increased after ABx intervention,and the degree of increasing was greater in 4ABx group.The levels of serum albumin,glutamic pyruvic transaminase,glutamic pyruvic transaminase,blood glucose,triglyceride decreased in ABx intervention group,among which the contents of serum albumin and glutamic pyruvic transaminase in 3ABx group were the lowest among the three groups,while the contents of glutamic oxalacetic transaminase,blood glucose and triglyceride in 4ABx group were the lowest among the three groups.The total cholesterol content of 3ABx group was lower than that of blank control group,while that of 4ABx group was higher than that of blank control group.There was mild inflammation in the colon of mice in 3ABx and 4ABx group,while mice in 4ABx group had more mild inflammation.In addition,ABx could significantly remove most of the bacteria in the intestinal tract,and the diversity of 4ABX group decreased more than that of 3ABX group.But the genus of Kosakonia still existed in the 3ABx group,while the relative abundance of Bacteroides in the4 ABx group did not significantly decrease.(2)The group treated by Clostridium butyricum MIYAIRI 588 had lower body weight,lower disease activity index,shorter colon length,less inflammatory damage,and the expressions of mucosal tight junction proteins ZO-1,occludin,mucus protein MUC2,as well as mucus related proteins TFF3 and Relmβ were down-regulated.In addition,the expression of pro-inflammatory cytokines TNF-α,IL-6,IL-1β,IL-17 A and anti-inflammatory cytokines TGF-β,IL-10,IL-4,IL-5,IL-13 were down-regulated in the treatment group with Clostridium butyrate MIYAIRI 588.The number of proinflammatory immune cells,neutrophils and macrophages,adaptive immune cells Th1 and Th17 were down-regulated,and the number of anti-inflammatory Th2 and Treg cells were up-regulated in the intestinal mucosa lamina lamina in the treatment group with Clostridium butyric MIYAIRI 588.(3)The mice treated with Clostridium butyricum MIYAIRI 588 had an increasing α diversity of microbiota and the its abundance of short chain fatty acids-producing genera such as Lachnospiraceae,Ruminiclostridium,Ruminococcaceae and anti-inflammatory bacteria such as Alistipes and Rikenellaceae were increased,while its abundance of pro-inflammatory bacteria such as Bacteroides and Alloprevotella were decreased.In addition,the mice treated with Clostridium butyricum MIYAIRI 588 had an increasing content of short-chain fatty acids in feces,especially acetic acid,propionic acid,butyric acid.(4)The group treated with Clostridium butyricum MIYAIRI 588 intervention showed down-regulated of proinflammation-related pathways such as IL-17 signal pathway and TNF signal pathway,as well as cytokine and cytokine receptor interaction pathway and ECM signal pathway.The group treated with supernatant of Clostridium butyricum MIYAIRI 588 mainly showed up-regulated of the pathway of mucin biosynthesis like mucin-type o-glycans and other types of o-glycans,while downregulates PI3K-Akt signal pathway and pro-inflammatory NF-kappa B signal pathway and IL-17 signal pathway.Conclusion: ABx had little effect on body weight,physiological function and colon inflammation in mice.And ABx can remove the gut microbiota of mice effectively,and 4ABx has a more significant removement effect on bacterial diversity than 3ABx.But 3ABx is not effective in inhibiting the genus of Kosakonia and 4ABx is not effective in inhibitingthe genus of Bacteroides.Overall,these results suggest that 4ABx pretreatment may be more effective in simulating germ-free mice.Clostridium butyricum MIYAIRI 588 can relieve the symptoms of colitis mice after 4ABx pretreatment and improve the intestinal barrier and cytokines expression.Meanwhile,it can exerting an anti-inflammatory effect,which is related to regulating the balance between proinflammatory and anti-inflammatory immune cells and its cytokines in mucosa lamina propria.Clostridium butyricum MIYAIRI 588 can regulate the intestinal microbiota and short-chain fatty acid metabolism of mice which tend to normal.The anti-inflammatory effect of Clostridium butyricum MIYAIRI 588 on colitis mice may be related to regulating intestinal microbiota and the metabolism of short-chain fatty acid.The effect of Clostridium butyricum MIYAIRI 588 on inflammation may be related to that promoting mucus production pathway and regulating inflammation pathway.