Investigation Of Preventing Colitis In Mice Via Flora-SCFAs-PPAR-γ Pathway By Ripened Pu-erh Tea

Inflammatory bowel disease(IBD)was a chronic disease affecting the gastrointestinal tract,and its incidence is increasing with the years.Biotherapy had side effects and high cost,resulting in social and economic burden.Therefore,dietary therapy for intervening and improving IBD symptoms need to be strengthened research direction.There are few studies on the preventive effect and mechanism of pu-erh tea,especially ripened pu-erh tea(RPT)and bioactive constituents on colitis.In this study,16S r RNA sequencing,fecal microbiota transplantation and immunofluorescence and so on were used to systematically explore the mechanism of RPT and its active components of theabrownins(TB)in preventing colitis in mice from the perspectives of histopathology,intestinal flora and inflammatory factors and so on.The content and result of each chapter was described below:In chapter 1,classification,active ingredients and functional effects of pu-erh tea,pathogenesis and treatment measures of IBD,and relationship between intestinal flora and IBD were systematically reviewed.In chapter 2,the preventing effect of RPT on mice colitis was investigated.First of all,mice were administered with low,medium and high concentrations of RPT(3%,L-RPT;6%,M-RPT;9%,H-RPT)one week and then induced colitis by dextran sulfate sodium salt(DSS).The results showed that three doses of RPT significantly relieved weight loss and colon shortening,decreased DAI score(p<0.001),and reduced intestinal mucosa ulceration and the disruption of crypt structures.Secondly,the effect of RPT on inflammatory factors in mice was further investigated,the results showed that all RPT could significantly reduce contents of serum inflammatory factors TNF-α(DSS+L-RPT,p<0.01;DSS+M-RPT,p<0.001;DSS+H-RPT,p<0.05)and IL-6(p<0.001),and the m RNA expression levels of TNF-α,IL-6 and IL-1βin colon tissue(p<0.001).Lastly,RPT inhibited the inflammatory cell of macrophage(CD68~+)and neutrophil(Gr-1~+)infiltration into colon by immunofluorescence.In chapter 3,on the basis of the discovery in the above chapter that RPT can improve colitis in mice,the mechanism of RPT in preventing colitis from the perspective of intestinal flora was explored.Firstly,the effect of RPT on barrier function was assessed.The results observed that three doses of RPT group significantly increased the m RNA expression level of zonula occludens-1(ZO-1)in colon(p<0.01),and medium and high concentrations of RPT significantly decreased the serum FITC-Dex concentration(p<0.05),which suggested that RPT benefit enhancing the intestinal barrier integrity.Secondly,analysis of gut microbiota by 16S r RNA and found that RPT obviously changed the intestinal microflora structure of mice.Compared with DSS group,RPT intervention decreased level of potentially harmful bacteria(Enterobacteriaceae,Helicobacter,and Lachnoclostridium)and enriched in the relative abundance of prospectively salutary bacteria(Lactobacillus,Muribaculum,Akkermansia,and Ruminococcaceae UCG-014),and significant stimulation of the production acetate,propionate,and butyrate in cecal contents(p<0.001).Lastly,Western blot in mouse colon showed that RPT increased peroxisome proliferator activated receptor-γ(PPAR-γ)expression in colon,and medium and high concentrations of RPT significantly inhibited the phosphorylation level of NF-κB(p65)(p?0.001).In chapter 4,antibiotics treatment and FMT were further used to verify that RPT prevention of colitis in mice in chapter 3 by regulation of intestinal flora,that is,intestinal flora was a key target for improving colitis.Firstly,the mice were treated with broad-spectrum antibiotics(ABX)showed that compared with the ABX+DSS group,ABX+DSS+H-RPT group did not relieved weight loss and protected colon shortening,and did not decrease TNF-α,IL-1βand IL-6 levels,and did not reduce macrophage aggregation in colon.Secondly,the transplantation of RPT-regulated and control donor intestinal flora to the recipient(FMT group of RPT-regulated:DSS+L-FMT、DSS+M-FMT and DSS+H-FMT group;FMT group of control:DSS+C-FMT group)via FMT technology,the results showed that compared with DSS group,FMT group of RPT-regulated and control all significantly alleviated weight loss(p<0.001),delayed colon shortening and reduced the degree of pathological injury of colon tissue in colitis mice.FMT group of RPT-regulated and control all significantly decreased the m RNA expression levels of TNF-α,IL-6,and IL-1βin the colon tissue(p<0.001),DSS+C-FMT group(p<0.001),DSS+L-RPT(p<0.001),DSS+M-RPT(p<0.01)and DSS+H-RPT(p<0.01)group significantly decreased IL-6 levels in the serum compared with DSS group.However,the contents of TNF-α(p<0.05)and IL-1β(p<0.001)were significantly reduced in the DSS+H-FMT group,while there were no significant differences in the other groups(p?0.05).Meanwhile,FMT group significantly elevated the levels of acetate(p<0.05,DSS+L-FMT;p<0.05,DSS+M-FMT;p<0.01,DSS+H-FMT),propionate(p<0.01,DSS+L-FMT;p<0.05,DSS+M-FMT;p<0.001,DSS+H-FMT),and increased PPAR-γprotein expression and inhibited the phosphorylation of NF-κB(p65)in colon.In chapter 5,on the basis of elucidating the preventive effect and molecular mechanism of RPT on colitis in mice above three chapters,the preventing effect of component theabrownins(TB)on colitis mice was investigated.First of all,mice were administered with TB(7 mg/m L)and TB-r(3 mg/m L,4 mg/m L and 7 mg/m L)one week and then induced colitis by DSS.The results showed that TB and low and medium concentrations of TB-r(3 mg/m L and 4 mg/m L)treatment significantly relieved weight loss(p<0.001),retained a longer colon length(TB and TB-r/4 mg/m L,p<0.01),improved severe crypt damage,significantly decreased the serum levels of TNF-αand IL-1β(p?0.001)and reduced the protein levels of TNF-α(TB-r/3 mg/m L and TB-r/4mg/m L,p?0.01),IL-1β(TB,TB-r/3 mg/m L and TB-r/4 mg/m L,p?0.01)and IL-6(TB,p?0.01)in colon,and reduced macrophage infiltration into colon.However,high concentration of TB-r(7 mg/m L)did not improve colitis.TB treatment significantly increased the contents of acetate(p<0.05),propionate(p<0.001)and butyrate(p<0.001)in cecum.However,TB-r(3 mg/m L)significantly decreased above organic acids(p<0.001),the levels of butyrate in TB-r/4 mg/m L treatment were significantly increased(p<0.001).Western blot showed that TB treatment significantly enhanced PPAR-γexpression(p<0.001)and decreased phosphorylation of NF-κB(p65)(p<0.01).The components of TB-r were analyzed by UHPLC-MS/MS based on untargeted metabolomics and results showed that TB-r including 8 catechins,72 flavonoids and their glycosides,12 phenolic acids and their derivatives,14 alkaloids,18 sugars and 37amino acids and their derivatives,etc.The relative contents of caffeine,ellagic acid,curdione and gallic acid were higher in TB-r.In chapter 6,a brief summary about preventive effect and molecular mechanism of RPT and its active ingredient TB on DSS induced colitis in mice,and the future research direction were prospected.In summary,this paper confirmed that RPT and active components of TB can prevent colitis in mice,and preliminarily explored the mechanism that regulating the structure of gut microbiota and its metabolites short-chain fatty acids(SCFAs),mediating the PPAR-γ/NF-κB pathway in colon,thus improving the symptoms of colitis in mice.