The Anti-inflammatory Effect Of Kaempferol On Ulcerative Colitis Mice And Its Gut Micro-ecological Mechanism

Objective:Ulcerative colitis(UC)is the highest incidence in inflammatory bowel disease(IBD),and the most significantly factor that causes abnormal activation of UC inflammatory pathways is the imbalance of gut microbiota.Kaempferol is type of flavonoid derived from a variety of medicinal and edible homologous plants.It has anti-inflammatory and antibacterial biological activities.Whether kaempferol can alleviate UC by regulating the gut microbiota has unknown.Therefore,we tried to evaluate the anti-inflammatory effect of kaempferol on UC model mice and explore its specific anti-inflammatory mechanism with the participation of intestinal flora.Method:94 female C57BL/6J mice were randomly divided into negative control group(NC),kaempferol group(Kae),UC model group(DSS),kaempferol treatment group(DSS-Kae),fecal bacteria transplantation negative control group(F-NC)),fecal bacteria transplantation kaempferol group(F-Kae),fecal bacteria transplantation UC model group(F-DSS),fecal bacteria transplantation kaempferol treatment group(F-DSS-Kae),each group of 10 mice;the Co-housing UC model group(Ch-DSS)and Co-housing kaempferol treatment group(Ch-DSS-Kae)had 7 mice in each group,feeding in the same cage.The NC group,DSS group,and Ch-DSS group were given1% sodium carboxymethylcellulose 10ml/kg/d;Kae group,DSS-Kae group and Ch-DSS-Kae group were given kaempferol 50mg/kg/d,dissolved in 1% sodium carboxymethylcellulose.F-NC group,F-Kae group,F-DSS group,F-DSS-Kae group were given 10ml/kg/d of fresh fecal supermatant from NC group,Kae group,DSS group and DSS-Kae group respectively.Gavage was administered for 14 days.Except for the NC group and Kae group,on the 8th day,each group was induced to establish UC model with 3.5% dextran sulfate sodium(DSS)for a total of 7 days.After 7 days,the following indicators were analyzed to explore the therapeutic effect of kaempferol on UC model mice and the role of intestinal flora and its metabolites in this process:1.Recorded the weight of the mice daily,observed the mice’s mental state and feces morphology,calculated the disease activity index(DAI),and detected the pathological state of the colon by HE staining.2.Real-time quantitative PCR method was used to detect the mRNA levels of colitis related factors and tight junction proteins ZO-1,Occludin and Claudin-1.3.ELISA method was used to detect the content of LPS,IL-1β,IL-6 and TNF-α in serum.The chemical method was used to detect the activity of SOD and GSH-PX and the content of MDA in the serum.4.Immunohistochemical method was used to detect the expression levels of TL4 related pathways and tight junction proteins ZO-1,Occludin and Claudin-1 in the colon.5.The 16 Sr RNA high-throughput sequencing method analyzed the changes of the gut microbiota,and the GC-MS method was used detect the changes of the gut microbiota metabolites.6.F-NC group,F-Kae group,F-DSS group,F-DSS-Kae group,Ch-DSS group,Ch-DSS-Kae group mice were recorded the weight of mice daily,and observed the mental state and feces morphology of mice,Calculated the DAI index,HE staining was used to detect the pathological state of the colon,and ELISA was used to detect the levels of LPS,IL-1β,IL-6 and TNF-α in the serum.Result:1.The administration of kaempferol significantly reduced the DAI index of UC model mice,increased the length of the colon,improved pathological damage,reduced pathological scores,and regulated the transcription of inflammatory-related factors in the colon.It reduces the content of inflammatory factors in the serum,regulates the indicators of oxidative stress in the serum,and increases the expression of tight junction proteins.2.After administration of kaempferol,the diversity and abundance of gut microbiota is increased,the ratio of Firmicutes to Bacteroides is increased,and the relative abundance of pathogenic bacteria under Proteobacteria and its phylum is reduced.Increased the abundance of probiotics such as Ruminococcaceae and Prevotellaceae.By changing the metabolism of galactose,phenylalanine,arginine and proline,the metabolic profile of the gut microbiota is changed.3.Kaempferol can significantly inhibit the level of LPS in the serum of UC model mice,and reduce the protein expression of TLR4,MyD88,NF-κB and NLRP3 in colon tissue.4.By observing the basic disease indicators of the fecal bacteria transplantation experiment,it was verified that kaempferol played an anti-inflammatory effect by regulating the gut microbiota.Conclusion:1.Kaempferol can significantly improve the symptoms of UC mice.2.Kaempferol can increase the abundance and diversity of the intestinal flora of UC model mice,reduce the abundance of pathogenic bacteria Proteobacteria,and increase the abundance of probiotics such as Ruminococcaceae and Prevotellaceae.3.Kaempferol can reduce the level of LPS in the serum and thereby down-regulate the activation of TLR4 related inflammatory pathways.