Mechanism Of Jiaoqi Powder In The Treatment Of Ulcerative Colitis Based On Gut Microbiota And Treg/Th17 Balance

ObjectiveUlcerative colitis(UC)is a chronic inflammatory disease characterized by gut microbiota dysbiosis and regulatory T cell(Treg)/The helper 17 cells(Th17)immune imbalance.The incidence of UC is increasing gradually worldwide.Therefore,exploring novel therapeutic strategies for UC treatment represent a great clinical importance.Traditional Chinese medicine(TCM)plays an important role in the prevention and treatment of UC,and it is of great significance to use the theory of TCM to treat UC."Blood deficiency and blood stasis" is the key pathogenesis in the occurrence and development of UC.Jiaoqi Powder(JQP)has been shown to have efficacy of nourishing blood,promoting blood circulation,stopping bleeding,removing stasis and regenerating.Our previous study found that Jiaoqi Powder has protective effects against colonic mucosal injury to improve the symptoms in UC patients.The present study aimed to determine the protective effect of JQP and its active ingredient stigmasterol on UC and further explore its underlying mechanisms of regulating intestinal flora and Treg/Th17 balance.Our results provide a scientific basis for the usage of JQP in the treatment of UC.Methods1.The mechanism of JQP regulating gut microbiota and Treg/Th17 balance to inhibit the progression of UC(1)The acute and chronic experimental colitis were induced using dextran sodium sulfate(DSS).At the same time,the treatment of JQP was administrated.The protective effect of JQP against UC was further clarified by comparing the body weights,DAI scores,colon lengths and histopathologic scores of mice.(2)Ten mRNA expression datasets were obtained from the Gene Expression Omnibus(GEO)database.Multichip analysis was performed to obtain differentially expressed genes(DEGs)of UC,and further obtain the DEGs of JQP in the treatment of UC.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed to predict the potential mechanism of JQP against UC.(3)The mRNA expression of the primary moleculars in colon tissues were detected by real-time fluorescence quantitative PCR(qPCR).Enzyme linked immunosorbent assay(ELISA)was used to detect inflammatory factors interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)in colon tissues,and the expression of p65 protein was detected by immunohistochemistry.(4)The proportions of Treg cells(CD4+CD25+Foxp3+)and Th17 cells(CD4+IL17A+)in the spleen and mesenteric lymph nodes(MLN)of mice in each group were determined by flow cytometry.The mRNA expression of transforming growth factor-β1(TGF-β1),interleukin-10(IL-10),forkhead transcription factor(Foxp3),interleukin-17(IL-17),interleukin-23(IL-23)and retinoid-related orphan nuclear receptor yt(RORyt)were detected by qPCR.(5)Faeces of mice were collected and genome DNA was extracted.The 16S rRNA sequencing was performed to evaluate the intestinal flora in each group.2.The mechanism of stigmasterol restoring Treg/Th17 balance by regulating gut microbiota and its metabolites(1)The acute experimental colitis was induced using DSS.Meanwhile,the treatment of stigmasterol was administrated.The body weights,DAI scores,colon lengths and histopathologic scores and the expression of IL-6,IL-1β and TNF-α in colon tissues were detected to clarify the protective effect of stigmasterol against UC.By utilizing flow cytometry,the proportions of Treg cells and Th17 cells in spleen,MLN and colon were determined.The expression of IL-10,TGF-β and IL-17A in the serum of mice were detected by ELISA.(2)The fecal microbiota transplantation(FMT)experiment was performed.First,the mice were administered an antibiotic cocktail(ABX)orally by gavage to deplete the gut microbiota.Then,fresh faeces were collected from donor mice(from the DSS and stigma+DSS groups)and resuspended in PBS.The supernatant was intragastrically administered to antibiotic treated mice.Next,DSS-induced acute colitis model was established.Finally,the therapeutic effect of stigmasterol on the treatment of UC was investigated by body weights,DAI scores,colon lengths and histopathologic scores,flow cytoretry and ELISA.(3)The 16S rRNA sequencing was performed to analyze gut microbiota in the faeces of mice in each group.The contents of faecal Short Chain Fatty Acids(SCFAs)were assessed using gaschromatography coupled with mass spectrometry.Pearson correlation-based network analysis was performed to determine the relationship of gut microbiota and SCFAs.(4)The molecular docking calculations were performed to predict the direct interaction between butyrate and peroxisome proliferator-activated receptor γ(PPARy).The expression of PPARγ in colon tissue was detected by immunofluorescence.Blood from UC patients was collected and peripheral blood mononuclear cells(PBMCs)were isolated from diluted blood.Naive CD4+T cells were then isolated by magnetic bead cell sorting and cultivated to induce Treg and Th11 cell differentiation in vitiro.Meanwhile,naive CD4+T cells were treated with either butyrate alone or both butyrate and PPARy inhibitors.Next,the cells were used for flow cytometry analysis,western blotting,glycolytic rate analysis and a mitochondrial stress assay.(5)To further observe the role of the gut microbiota in the regulation of Treg/Th17 balance by stigmasterol,the mice were pretreated with an ABX cocktail to deplete the intestinal flora and then treated with stigmasterol.Results1.The mechanism of JQP regulating gut microbiota and Treg/Th17 balance to inhibit the progression of UC.(1)JQP administration ameliorated the symptoms of DSS colitis.This effect was shown by the significantly reduced weight loss and DAI scores of mice in the JQP group.Furthermore.JQP significantly mitigated colon shortening and histology scores in DSS-induced colitis.Taken together,these results demonstrated that JQP treatment significantly protected mice against DSS-induced colitis.(2)Based on the GEO database,479 active UC patients and 185 healthy controls were included for analysis.A total of 224 DEGs were obtained,and 37 DEGs of JQP in the treatment of UC were further screened.GO analysis revealed that the core targets were mainly related to inflammation,microbiota and immune response.In parallel,the core targets were primarily enriched in immune response,inflammatory and metabolic signalling pathways,such as NF-κB signaling pathway,TNF signaling pathway,IL-17 signaling pathway,Th17 cell differentiation signaling pathway as well as PPAR signaling pathway,etc.(3)Subsequent animal-based in vivo experiments revealed that JQP significantly reducing the expression levels of NF-κB/p65,IL-1β,IL-6,TNF-α,COX-2,CCL2 and CXCL2 in colon tissue in DSS colitis,exerting anti-inflammatory effect.JQP also reduced the levels of HIF-1,MMP3 and MMP9 to maintain intestinal barrier integrity.(4)JQP administration increased the percentage of Treg cells.Further analysis revealed that JQP treatment increased the mRNA expression of TGF-β1,IL-10,and Foxp3 in the colon tissues of DSS-induced colitis mice.Similarly,JQP administration decreased the percentage of Th17 cells.Moreover,JQP administration significantly reduced the mRNA expression levels of IL-17,IL-23,and RORγt in the colon tissues of DSS-induced mice.This indicated that JQP could regulate Treg/Th17 immune balance.(5)At the genus level,JQP increased the composition of Bacteroides.Lachnoclostridium,Blautia,Lactobacillus,etc.Additionally,JQP decreased the abundances of Colidextribacter,Desulfovibrio and Oscillibacter.This indicated that JQP can regulate gut microbiota,which in turn regulating mucosal immunity,thereby regulating Treg/Th17 balance.2.The mechanism of stigmasterol restoring Treg/Th17 balance by regulating gut microbiota and its metabolites(1)Stigmasterol administration significantly reduced weight loss,DAI scores and colon shortening of mice.Furthermore,stigmasterol appreciably reduced the pathohistological score in colon and downregulated the expression of inflammatory factors,including IL-6,IL-1β and TNF-α,in DSS-treated mice-Stigmasterol increased the Treg proportion in the spleen,MLNs and colon.Similarly,stigmasterol reduced the percentage of Th17 cells.Additionally,stigmasterol increased the expression of IL-10 and TGF-β but downregulated the expression of IL-17A in serum.(2)Stigmasterol FMT treatment alleviated DSS-induced experimental colitis.Furthermore,stigmasterol FMT treatment increased the Treg proportion and reduced the percentage of Th17 cells in the spleen and MLNs.Similarly,stigmasterol FMT treatment increased the expression of IL-10 and TGF-β but downregulated the expression of IL-17A in serum,indicating that the gut microbiota which affected by stigmasterol restored the Treg/Th17 balance.(3)At the genus level,stigmasterol increased the composition of Ruminococcus,Prevotella,Paraprevotella,Helicobacter,Odoribacter,Clostridium_IV,and Clostridium_XIVa.Additionally,stigmasterol decreased the abundances of Streptococcus,Escherichia,Enterococcus and Allobaculum.Stigmasterol also improved the diversity of intestinal microflora.Stigmasterol treatment markedly increased the acetate,propionate,butyrate,isobutyrate and valerate levels in the faeces of DSS-induced colitis mice.Pearson correlation-based network analysis revealed that the abundance of Clostridia in the gut was positively correlated with the butyrate concentrations in faeces,and the butyrate level was most strongly correlated with the abundance of gut microbiota.(4)Molecular docking analysis suggested that butyrate directly interacted with PPARγ,a key target for IBD therapy which was upregulated by stigmasterol treatment.Butyrate might bind to the active sites of PPARy at Arg-288.Additionally,the expression of PPARy in Treg and Th17 cells was upregulated after butyrate intervention.Further analysis indicated that butyrate supplementation increased the differentiation of Treg and decreased Th17 cell differentiation from naive CD4+T cell.Mechanistically,butyrate promotes the metabolic transition of glycolysis to OXPHOS by activating PPARy,thereby inducing T-cell differentiation into Treg cells but not Th17 cells.(5)When the gut microbiota was suppressed,the therapeutic effects of stigmasterol were abolished.Similarly,no significant differences were found in Treg cells or Th17 cells.Overall,these results indicated that the gut flora is important in the therapeutic efficacy of stigmasterol.ConclusionThe present study demonstrated that JQP and its active ingredient stigmasterol could treat UC by inhibiting inflammation,repairing intestinal barriers,and modulating gut microbiota as well as Treg/Th17 balance.Furthermore,the gut microbiota and its metabolites played significant roles in the mechanism of JQP and stigmasterol in regulating Treg/Th17 balance.The results also suggest that gut microbiota-derived butyrate-mediated PPARy activation restored the balance of Treg/Th17 cells and improved the colon mucosa barrier,and this may be a possible mechanism,by which JQP attenuates UC.These findings provide the scientific basis for the use of JQP for the treatment of UC.