| Aim:In this study,the DSS method was used to replicate the recurrent colitis model in mice.First,the curcumin treatment of colitis was evaluated and the abundance and composition of the intestinal flora were observed.At the same time,detect the TLR/My D88 signaling pathway related proteins,regulatory B cell subtypes and subgroups and the secretion of related cytokines in mouse colon tissue.And further clarified that curcumin regulates the intestinal flora,regulatory B cells and cytokines through the TLR/My D88 pathway to alleviate the possible mechanism of recurrent colitis in mice.Methods:1.Animal grouping,model replication and drug administration methods:Forty SPF grade male BALB/c mice,18～22 g,were used for the test after 7 days of acclimatization feeding.The mice were divided into the following four groups by random number method:normal group（Normal）,DSS group（DSS）,recurrent colitis model+curcumin treatment group（DSS+Cur）,recurrent colitis model+mesalazine control group（DSS+5-ASA）.Ten animals in each group were grouped with picric acid markers.Normal drinking water was prepared with 3%（w/v）DSS（35 000-50000 k D）solution and all 30 mice were given 3%DSS solution ad libitum for 5 days,except for the normal group of mice.After 1 week of normal drinking,the mice were then given 2%DSS solution for 5 days.The normal group was given normal drinking water.The curcumin treatment group was given 200 mgkg-1 curcumin suspension by gavage;the mesalazine control group was given 300 mgkg-1 mesalazine suspension by gavage;the normal group and the DSS group were given equal volume of saline by gavage for 10 consecutive days.2.Evaluation of the efficacy of curcumin in the treatment of DSS-induced recurrent colitis:The mice in each group were observed daily for changes in diet,hair status,activity frequency,colorectal bleeding and stool sticking,and other physical signs,and changes in body weight were recorded.On the day of disposal,the body weight of the mice was weighed,the mice were anesthetized,the eyes were extracted and blood was stored in the blood collection vessel,the mice were executed by removing the cervical vertebrae,the colon was quickly removed,the colon was naturally laid flat and the length from the ileocecal part to the anal portion was gauged,and the photographs were kept.The weight of the colon was weighed on an electronic balance,the weight index of the colon was calculated,the weight of the colon per unit length was determined,pathological sections of the colon were made,the transverse section of the colon was observed under an electron microscope,and tissue damage was assessed.3.Sequencing of the gut microbe:Samples were collected,six mice were rapidly and randomly selected under sterilized conditions from the ileocecal feces,immediately placed in solidifed carbon dioxide for temporary freezing,and stored in a-80°C refrigerator.Fecal DNA extraction and PCR amplification,high-throughput sequencing was performed to obtain the intestinal flora data,and the data were compared with the gene library for data analysis,to explore the correlation between flora and recurrent colitis,and the effect of curcumin on intestinal flora,and TLR2and TLR5 were correlated with intestinal flora to find the association between them.4.Analysis of regulatory B cell subtypes and subgroups:the ratio of CD25+Breg,Foxp3+Breg,CD1d+Breg,PD-L1+Breg,Tim-1+Breg,CD27+Breg cells using flow cytometry Determine and analyze the correlation between the above-mentioned cells and the intestinal flora to explore whether curcumin can directly regulate the differentiation and function of Breg through the intestinal flora or treat recurrent colitis.5.Analysis of regulatory B cell-related cytokines:ELISA method was used to detect colon tissue IL-1β,IL-4,IL-6,IL-10,IL-13,IL-33,CCL-2,IFN-γ,Ig A,The secretion of TGF-β1 and TNF-αcytokines,and analyze the correlation between IL-1β,IL-4,IL-6,IL-10,IL-33,Ig A and the intestinal flora,and explore the regulation of curcumin the relationship between intestinal flora and cytokines and its mechanism to relieve inflammation of colitis.6.Detection of TLR/My D88 signaling pathway related protein:TLR4 TLR2,TLR5,My D88,IRAK1,IRAK4,NF-κB p65,TRAF6,TAB1,TAB2,TAK1,MKK3,MKK6,p38 MAPK,CREB were detected by Western Blot Assy method to explore the regulatory effects of curcumin on the TLR/My D88 signaling pathway.7.Statistical analysis:SPSS22.0 software was used to conduct statistical tests and analysis,and each group of data was subjected to normal test and independent sample t-test,one-way ANOVA was adopted to determine salience,and correlations were analyzed by linear regression correlation.The measurement data were presented as mean±standard deviation（（?）±SD）,with P<0.05 representing a significant difference and P<0.01 regarding a highly significant difference.Results:1.The body weight growth rate of mice started to diminish on day 5 after the first administration of DSS.On day 9,the body weight growth rate of mice peaked,and then recovered slowly.On day 14,mice consuming 2%DSS started to receive DSS,and the body weight growth rate of mice in the DSS group started to fall,while the body weight growth rate of the curcumin-treated group and the mesalazine control group tended to level off,which had a preventive effect.The body weight gain rate of mice started to decrease on day 5 after the first administration of DSS.On day 9,the body weight production rate of mice reached the lowest point,and then recovered slowly.On day 14,mice consumed 2%DSS could be found that the body weight growth rate of mice in the model group started to decrease,while the body weight growth rate of curcumin and mesalazine groups tended to level off,which had a preventive effect.The growth rate of body weight was significantly better in the normal group compared with the DSS group on the day of execution and after the administration of the treatment.Compared with the normal group,the body weight and colonic length of mice in the DSS group dropped significantly（P<0.01,P<0.05）,and the level rose markedly or tended to raise obviously after treatment（P<0.01）.Compared with the normal group,the DSS group showed remarkable recovery in colonic weight index,colonic weight with colonic weight/colonic length and colonic tissue damage score（P<0.05,P<0.01）,and the level were greatly reduced after treatment（P<0.01）.After treatment with curcumin and mesalazine,the ulcer and erosion area were diminished,inflammatory cell infiltration was reduced,vascular proliferation was decreased,glands were aligned,and crypt and cupped cells were restored.It is suggested that curcumin can effectively treat recurrent colitis in mice.2.Research on the gut microbe:Sequencing of the intestinal flora found t hat the diversity of the model group was reduced compared with the normal g roup,but the unique flora of the model group increased.Uncultured＿bacterium＿g＿Lachnospiraceae＿NK4A136＿group,uncultured＿Bacteroidales＿bacterium＿g＿Allo prevotella,uncultured＿bacterium＿g＿Prevotellaceae＿UCG-001,unclassified＿g＿Oscil libacter are the dominant species in the model group,and their proportions inc rease significantly（P<0.05）.In unclassified＿g＿Alistipes,unclassified＿g＿Roseburi a,unclassified＿g＿Brevibacterium,Lachnospiraceae＿bacterium＿DW52,uncultured＿bacterium＿g＿Sphingobacterium,the abundance hiking prominently in the normal group and the curcumin group（P<0.05,P<0.01）.Staphylococcus＿equorum is a unique dominant flora of the curcumin group,and its abundance increased sig nificantly（P<0.001）.The relationship between TLR2 and TLR5 and the intesti nal flora was analyzed,and it was found that the intestinal flora of the model group was more closely related to TLR2 and TLR5.The normal group had t he farthest relationship with the expression of TLR5,and the relationship betw een the curcumin group and TLR2 Farthest.It shows that the disorder of the i ntestinal flora in mice leads to TLR2,and the overexpression of TLR5 further leads to the occurrence of colitis inflammation.And curcumin could regulate t he abundance and balance of intestinal flora by decreasing TLR2,TLR5 expres sion.3.Regulatory B-cell changes:CD25+Breg,Foxp3+Breg,CD1d+Breg cell ratios were dropped in the DSS group compared with the normal group（P<0.05,P<0.01）,and the above cell ratios were restored markedly in the curcumin group compared with the DSS group（P<0.01）,and the CD25+Breg,Foxp3+Breg cell ratios were greatly raised after mesalazine suspension administration（P<0.05,P<0.01）.CD27+Breg,Tim-1+Breg,PD-L1+Breg cell ratios were dramatically elevated in the DSS group（P<0.05,P<0.01）,and CD27,Tim-1,PD-L1 cell expression was suppressed after drug treatment compared with the DSS group（P<0.05,P<0.01）.It tended to be consistent with the normal group.It demonstrated that curcumin could effectively regulate the expression of Breg cell subtypes.4.Detection of regulatory B cell-related cytokines:Compared with the normal group,the expression of IL-4,IL-10,IL-13,Ig A in the colon tissue of the model group was remarkably reducing.（P<0.01）,TGF-β1 There is a downward trend in the amount of expression.The contents were clearly enhanced in the colonic tissues of mice after treatment.（P<0.05,P<0.01）.The expression of the cytokines was visibly declined after curcumin treatment（P<0.05,P<0.01）,the expression level of IL-6 and IFN-γhas an upward trend.The expression of the cytokines in curcumin group was significantly reduced was brilliant inferior to the model group（P<0.05,P<0.01）.The above results indicate that curcumin can inhibit the other inflammatory factors,and can promoter anti-inflammatory factors.5.TLR/My D88 signaling pathway-related protein detection:TLR/My D88signaling pathway important node proteins were detected,and it was found that the expression of TLR4,My D88,IRAK1,IRAK4,NF-κB p65,TRAF6,TAB1,TAB2,TAK1,MKK3,MKK6,p38 MAPK,CREB expression was remarkably raised（P<0.05,P<0.01）,and compared with the DSS group,the contents of the above proteins were considerably closer to the normal group after treatment by drug gavage（P<0.05,P<0.01）.It is recommended that curcumin can restrain TLR/My D88signaling pathway.Conclusion:The effective treatment of DSS-induced recurrent colitis may be achieved by curcumin regulating the balance of gut microbe and inhibiting the TLR/My D88 signaling pathway to regulate regulatory B cell activation. |
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