The Anti-inflammation Effect Of Phloretin On Ulcerative Colitis Mice And Its Intestinal Micro-ecological Mechanism

BackgroundUlcerative colitis(UC)is one type of inflammatory bowel disease(IBD).UC patients mainly manifest as abdominal pain,diarrhea,and bloody stool,and lesions mainly affect the rectum and the distal colon.Until now,more and more studies have shown that the disorder of gut microbiota plays an increasingly vital role in the development of UC.Phloretin(Ph),a kind of flavonoid,exacted from fruit root bark with various biological activities,for example,anti-inflammatory,antibacterial and anti-oxidation properties.It remains unknown whether phloretin could relieve UC.Whether gut microbiota participated in the anti-UC of phloretin also need further research.Objective1.To evaluate the anti-UC effect of phloretin,and further analyze the intestinal microecological mechanism in phloretin anti-UC.Method1.Sixty male mice were randomly divided into six group: the healthy control group(CK),phloretin control group(Ph),UC mice model group(DSS),phloretin-treated UC group(DSSPh),co-housing UC mice model group(CoDSS)and co-housing phloretin-treated UC group(CoDSSPh).The CoDSS and CoDSSPh group were the UC mice model group and phloretin-treated UC group,respectively,and the two groups were housed in one cage.Taking advantages of coprophagy of mice,gut microbes will exchange between the two groups.The anti-UC effect of phloretin and the influence of gut microbiota on phloretin antiUC were detected by the below indicators:1)Measuring body weight change and DAI,the colon histopathologic structure was examined by H&E stain;3)The mRNA levels of inflammatory cytokines and mucin-2(Muc-2)in colon were examined by RT-qPCR method.2.Forty male mice were randomly divided into five group: CK,Ph,DSS,DSSPh and DSSFMT group(FMT-treated group).Fresh fecal bacteria from DSSPh group mice was administered by gavage to DSSFMT group mice.The intestinal micro-ecological mechanism in phloretin anti-UC was examined by the below indicators:1)Measuring body weight change,DAI and colon length,the colon histopathologic structure was examined by H&E stain;2)The mRNA levels of inflammatory cytokines and Muc-2 in colon were examined by RT-qPCR method.3)The relative levels of Claudin-1?ZO-1?I?B?p-I?B?NLRP3?ASC and IL-1? protein in colon were detected by Western blot analysis.The oxidant stress in colon was measured by biochemical method(including TBA method,WST-1 method and microenzymatic method).4)The immune cells levels in spleen were measured by flow cytometry analysis;5)16S rDNA gene high-throughput sequencing detected the change of gut microbiota.Based on 16 S rDNA high-throughput sequencing results,bioinformatics methods were used to analyze the correlation of gut microbiota with DAI,pathological score,colon length and inflammatory cytokines,and to predict the function of gut micobial genome.Result1.Gut microbiota took part in the process of phloretin anti-UC.1)Phloretin treatment significantly increased UC mice body weight,decreased DAI,improved colon histopathologic structure,decreased colon pathological score,regulated the mRNA levels of inflammatory cytokines and decreased the mRNA level of Muc-2.2)Compared with DSSPh group mice,CoDSSPh group mice were more severe disease sympoms and colon inflammation.Compared with DSS group mice,CoDSS group mice were with less severe disease sympoms and colon inflammation.2.The anti-inflammation effect of phloretin on ulcerative colitis mice and its intestinal micro-ecological mechanism.1)FMT treatment(Fecal microbes were come from DSSPh group mice)significantly increased mice body weight,decreased DAI,increased colon length,improved colon histopathologic structure,decreased colon pathological score and regulated the mRNA levels of inflammatory cytokines in colon tissues.2)FMT treatment significantly improved the intestinal barrier function,inhibited the activation of NF-?B and NLRP3 inflammasomes,decreased the oxidant stress in colon and regulated the content of immune cells in spleen.3)FMT and phloretin treatment both increased the Chao index of gut microbiota and increased the levels of Bacteroidetes,Alistipes and Lactobacillus,and decreased those of Firmicutes and Oscillibacter.Moreover,there was no difference in the composition of gut microbes on every classification level between the treatment of FMT and phloretin.4)The abundance level of Oscillibacter was negatively related with those of Alistipes and Lactobacillus.5)The analysis of the relationship between gut microbes and environmental factors shown that the level of Alistipes was negatively related with that of DAI,TNF-? and IL-6,and the level of Lactobacillus was negatively related with that of DAI,and the level of Oscillibacter was negatively related with that of IL-1?,IL-6 and pathological score.6)The expression levels of genes related to metabolism were significantly different between the treatment groups(including DSSPh group and DSSFMT group)and DSS group.Conclusion1.Phloretin had the anti-UC effect,and gut microbiota participated in the process.2.The gut microbiota intervened by phloretin produced the same anti-UC effect as phloretin.3.Phloretin had the anti-UC effect by regulating the composition of gut microbiota through increasing the levels of Alistipes,Lactobacillus and decreasing that of Oscillibacter.