| Objective:Lactobacillus acidophilus and compound Huan Kui Le(HKL)suspension were used to interfere with ulcerative colitis(UC)induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS)in rats.To analyze the effects of combined intervention on intestinal flora,immunity and serum metabolites of UC rats,and to explore the pathogenesis of UC and the molecular mechanism of combined intervention.Method:(1)110 SPF male Wistar rats were randomly divided into 6 groups.Except for the normal group,the other groups used TNBS/ethanol enema to replicate the rat UC model,and then were divided into model group(UC group),HKL intervention group(HKL group),Lactobacillus acidophilus intervention group(Lac group),Lac+HKL combined intervention group(LH group),5-aminosalicylic acid intervention group(5-ASA group).and give intragastric intervention to rats in each group.Observe the general condition of rats every day and two days to record the weight,After 14 days of continuous intervention,the rats were killed and the samples were taken.Gross specimens were observed with naked eyes and colon tissues were taken for histopathological scoring;m RNA and protein expression levels of IL-4,IL-6,IL-10,IL-12,IL-13,IL-17,TGF-βand IFN-γin colon tissues of rats in each group were detected by RT-q PCR and ELISA,and protein expression levels of TLR4 and TLR9 were detected by immunohistochemistry;(2)Total bacterial DNA was extracted from colon tissues of five groups of rats(except 5-ASA group),and primers were designed for amplification of 16Sr RNA V3～V4region The intestinal flora of rats in each group was sequenced and bioinformatics analysis was carried out by using the Illumina miseq platform,and the representative samples(feces)of each group were selected according to the diversity results for metagenomic sequencing;(3)The serum of five groups of rats was detected by Nuclear magnetic resonance hydrogen spectroscopy(~1H-NMR),and the differential metabolites in serum were screened by multivariate statistical analysis of PCA,PLS-DA and OPLS-DA,and their correlation with intestinal flora was analyzed;(4)Flow cytometry was used to detect the changes of CD4~+T lymphocyte subsets Th1,Th2,Th17 and Treg cells in peripheral blood of the five groups of rats,and correlation analysis was conducted between T cell subsets and intestinal flora.Result:(1)General status of rats in each group:After TNBS/ethanol enema,compared with the normal group,rats in the UC group showed weight loss,reduced activity,lethargy,loose stools or mucous pus and blood stools,and their weight was significantly lower than that in the normal group(P<0.01).Symptoms of each intervention group were relieved to varying degrees after gavage,and body weight increased significantly from the sixth day(P<0.05);(2)Colon histopathological score:the infiltration degree,depth and ulcer depth scores of the UC group were all higher than those of the normal group(P<0.01).Compared with the UC group,the infiltration degree and depth score of HKL group,LH group and 5-ASA group were significantly decreased(P<0.05),and the ulcer depth score of rats in each intervention group was significantly decreased(P<0.05 or P<0.01);(3)Detection of inflammatory factors and TLRs levels:Compared with the normal group,m RNA and protein expression levels of IL-12,IFN-γ,IL-6,IL-17 and protein expression levels of IL-10,TLR4 and TLR9 in UC group were significantly up-regulated(P<0.01),and protein expression levels of TGF-βwere significantly down-regulated(P<0.05).Compared with UC group,m RNA and protein expression levels of IL-12,IFN-γ,IL-6 and IL-17 were significantly decreased in each intervention group(P<0.01 or P<0.05).The expression levels of IL-4 m RNA and protein in HKL group were significantly up-regulated(P<0.01).The expression levels of IL-4 and IL-13 in Lac group were down-regulated(P<0.05).The m RNA and protein levels of IL-4,IL-10,IL-13,TGF-βwere significantly up-regulated in THE LH group(P<0.01 or P<0.05).The m RNA expression levels of IL-4,IL-13 and TGF-βin the 5-ASA group were significantly up-regulated(P<0.01 or P<0.05).Except Lac group,protein expression levels of TLR4and TLR9 in other intervention groups were significantly down-regulated(P<0.01 or P<0.05);(4)Intestinal flora gene sequencing results:1)Changes of intestinal flora in rats of the UC group:compared with the normal group,there are more Proteobacteria,Eggerthella and Coprococcus＿1 in rats of the UC group(P<0.05 or P<0.01);Microflora and Romboutsia,Ruminococcaceae＿ucg-014,Prevotellaceae＿NK3B31＿group,Desulfovibrio,norank＿o＿Mollicutes＿RF9,Ruminiclostridium＿6,Ruminococcaceae＿ucg-010,Bifidobacterium,Ralstonia,Pseudomonas,norank＿o＿Gastranaerophilales,norank＿f＿Rhodospirillaceae,Burkholderia-Paraburkholderia,Brevundimonas,Lachnospiraceae＿ucg-006,Streptococcus,Odoribacter were decreased(P<0.05 or P<0.01);2)Changes of intestinal flora in HKL group:compared with UC group,Chryseobacterium,Norank＿f＿clostridipes＿vadinbb60,Ralstonia,Streptococcus,Achromobacter,and Subdoligranulum were increased in HKL group(P<0.05);3)Changes in intestinal flora of rats in the Lac group:Compared with the UC group,Coprococcus＿1,Klebsiella,erysipeloclostridium,Hungatella,Norank＿o＿NB1-n,Acidovorax bacteria in the Lac group decreased(P<0.05 or P<0.01),and Ralstonia increased(P<0.05);4)Changes of intestinal flora of rats in the LH group:Compared with the UC group,Romboutsia and Ruminococcaceae＿Ucg-014 in LH group,[Eubacterium]＿coprostanoligenes＿group,Bifidobacterium,Staphylococcus,ruminococcaceae＿ucg-010,Aerococcus,Jeotgalicoccus,Streptococcus,Ralstonia,Burkholderia-Paraburkholderia,Odoribacter,Lachnos Piraceae＿NK4B4＿group,Ruminiclostridium＿1,Pseudomonas,Norank＿f＿Coriobacteriaceae genera increased(P<0.05 or P<0.01);5)Coprococcus＿1 is remarkably enriched in the UC group.Bifidobacterium,Bifidobacteriales,and Bifidobacteriaceae were significantly enriched in the LH group;6)Compared with the normal group,the abundance of genes related to glycolysis/gluconeogenesis and tricarboxylic acid cycle metabolism pathway in the UC group was decreased.The gene abundance associated with this metabolic pathway increased in different degrees in each intervention group(P<0.05);(5)Results of serum metabolomics:1)Compared with the normal group:UC group has 6 metabolites including lactic acid and creatine were significantly increased,and 8metabolites including alanine and glucose were significantly reduced.2)Compared with the UC group:the 6 metabolites of alanine and lysine in the HKL group were significantly increased,and the 4 metabolites such as glycine and inositol were significantly decreased;the isoleucine of the Lac group was significantly increased and 5 metabolites including glycine and inositol were significantly reduced;6 metabolites including alanine and LDL in the LH group were significantly increased,and 4 metabolites such as glycine and inositol were significantly reduced.3)The contents of lactic acid,creatine and glycine in serum of rats were positively correlated with Klebsiella.The glycine content was negatively correlated with Ruminococcus.Creatine content was negatively correlated with Prevotellacea6A1 and Prevotellaceae NK3B31.Glucose content was positively correlated with Prevotellaceae NK3B31 and Prevotellacea6A1;(6)Detection results of T cell subsets:(1)The percentages of CD3~+T cells,CD4~+T cells and CD8~+T cells in the blood of UC group decreased(P<0.05).Compared with the UC group,the proportion of CD3~+T cells and CD4~+T cells in HKL group and LH group increased(P<0.05).The proportion of CD4~+IL4~+Th2 in UC group was significantly higher than that in normal group(P<0.05).Each intervention group was significantly lower than the UC group(P<0.05);In terms of the proportion of CD4~+CD25~+Foxp3~+Treg,the UC group was significantly lower than the normal group,and each intervention group was significantly higher than the model group(P<0.05).The proportion of CD4~+ILl7~+Thl7 in UC group was significantly higher than that in normal group(P<0.05).Significantly decreased in each intervention group(P<0.05);(2)Th17 cells were positively correlated with Proteobacteria and Shigella,but negatively correlated with Firmicutes and Ruminococcus＿ucg＿014,Treg cells were positively correlated with bifidobacteria and Romboutsia,Th1 and Th2 cells were positively correlated with Klebsiella.Conclusion:(1)The expression of pro-inflammatory factors in the colon tissue of UC rats is up-regulated and the expression of anti-inflammatory factors is down-regulated,so that the balance between pro-inflammatory factors and anti-inflammatory factors is broken,which promotes the occurrence and development of UC.The combined intervention significantly increased the m RNA and protein expression levels of IL-4,IL-13,TGF-βand IL-10 in the colon tissue of UC rats,indicating that the anti-inflammatory effect of the combined intervention was relatively obvious,and the combined intervention inhibited the expression of TLR4 and TLR9 proteins in colon tissue in UC rats.(2)The abundance of intestinal flora in UC group decreased.The abundance of conditionally pathogenic bacteria such as Shigella and Klebsiella increased.All the intervention measures affected the distribution of Firmicutes,Bacteroidetes and Proteobacteria in the intestinal tract of UC rats,among which the distribution of dominant bacteria was closest to that of the normal group after combined intervention,indicating that the regulatory effect of combined intervention was better than that of single drug.Weight loss,physical weakness,diarrhea and other symptoms in UC group rats may be related to the decrease of Romboutsia in Firmicutes.Coprococcus in Firmicutes was enriched significantly in UC group;Bifidobacteria in Actinobacteria were significantly enriched in the LH group,and Bifidobacteria were positively correlated with IL-13 and TGF-β,while IFN-γwere negatively correlated.This suggests that the combined intervention may exert anti-inflammatory effect by affecting the expression of TGF-βand IL-13 by Bifidobacterium.Combined treatment can alleviate the energy deficiency of colon epithelium in UC group rats by increasing Ruminococcae＿ucg＿014,Ruminococcaceae＿UCG＿010,Romboutsia,Norank-f-coriobacteriaceae and other short-chain fatty acid producing bacteria;(3)Rats in the UC group had energy metabolism disorders,which were specifically reflected in the increased contents of lactic acid,glycine and creatine and the decreased contents of glucose,alanine and taurine,which were related to the decreased abundance of short-chain fatty acid-producing bacteria such as Romboursia in the intestinal mucosa.Energy metabolism,amino acid metabolism and lipid metabolism were disturbed in UC group rats.the rise of Klebsiella abundance is important signals of metabolic disorder in rats UC.It’s abundance was positively correlated with the level of lactic acid,creatine,and glycine.It’s abundance was negatively correlated with acetone levels.Combined treatment influence the metabolism of lactic acid,creatine and glycine by inhibit the Klebsiella.Combined intervention can regulate the metabolic changes caused by intestinal flora disturbance in UC rats;(4)The increase of Proteobacteria(especially Shigella coli)and the decrease of Firmicutes induce the differentiation of Th17 cells and promote the secretion of pro-inflammatory factors,thus causing the amplification of local inflammatory response and promoting the occurrence and development of UC.After the combined intervention,the increase in the abundance of Bifidobacteria and other energy producing bacteria is conducive to the production of Treg cells,and inhibits the differentiation of Th0 cells into Th1 cells. |
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