Analysis Of The Anti-inflammatory Products In F.prausnitzi Supernatant And Its Mechanism

Background:Inflammatory bowel disease(IBD)is a disease characterized by chronic and recurrent intestinal inflammation,mainly including ulcerative colitis(UC)and Crohn’s disease(CD).Although the pathogenesis of IBD is not yet fully understood,a loss of probiotics including Faecalibacterium prausnitzii(F.prausnitzi)in the intestinal flora of IBD patients has been widely observed.It has been found that the supernatant of F.prausnitzi can exert an anti-inflammatory effect by reducing T helper 17(Th17)cells and promoting regulatory T(Treg)cells.However,the anti-inflammatory substances in F.prausnitzi supernatant and the mechanism in regulating T cells have not yet been fully investigated.Methods:F.prausnitzi supernatant was separated by macroporous resins.Evaluate the anti-inflammatory effects of different fractions by Th17-and Treg-differentiation models in vitro.Anti-inflammatory substances were identified by gas chromatography-mass spectrometer(GC-MS).Th17-and Treg-differentiation models in vitro and Trinitro-benzene-sulfonic acid,(TNBS)-induced colitis rats were respectively used to validate the anti-inflammatory effects of the identified substance in vitro and in vitro,and the anti-inflammatory effects of other protein substances were excluded.The proportions of T cells were detected by flow cytometry.The levels of cytokines in cell culture supernatants or rats’ plasma were detected by enzyme linked immunosorbent assay(ELISA).Next,immunofluorescence was used to detect the expression of histone deacetylase(HDAC)in normal and colitis rats.Immunohistochemistry was used to detect the expression of HD AC 1 in the guts of normal subjects and IBD patients.The HDAC1 plasmid was transfected into Jurkat cells and the protein levels in the cells were determined by western blotting.Use the differentiation models of Th17 and Treg cells in vitro and detected the protein level in the cells by Western blotting.Finally,the colitis models were induced by dextran sulfate sodium(DSS).The body weight changes,colon length,pathological score and Th17/Treg cell ratio in mice were detected.Results:1.A method for the separation of F.prausnitzi supernatant was established.F.prausnitzi supernatant was separated into five fractions according to polarity.2.Established differentiation models of Th17 and Treg cells in vitro were and the most polar fraction was found to inhibit Thl7 cells and interleukin(IL)-17A,promote Treg cell differentiation.3.GC-MS identified butyrate from that anti-inflammatory fraction.Both the anti-inflammatory fraction and butyrate(the same concentration as in the anti-inflammatory fraction)could inhibit the differentiation of Th17 cells and the secretion of IL-17A and IL-6,and promote the differentiation of Treg cells and the expression of transforming growth factor(TGF)-β in vitro.Both F.prausnitzi supernatant and butyrate could inhibit Th17 cells and IL-17A,IL-6 secretion,promote Treg cell differentiation and TGF-β secretion in TNBS-induced colitis rats.4.Differential expression of HDAC 1 was found in the intestine of control and colitis rats,as well as in healthy people and IBD patients.5.Overexpression of HDAC 1 in Jurkat cells was found to promote the levels of IL-6/signal transducer and activator of transcription(STAT)3/IL-17 pathway-associated proteins and decrease TGF-β.Inhibition of HDAC1 in T cells can inhibit IL-6/STAT3/IL-17 pathway-related proteins in Th17-polarizing condition and promote Forkhead box protein 3(Foxp3)level in Treg-polarizing condition in vitro,respectively.6.At last,animal studies confirmed that both butyrate and HDAC1 inhibitor can ameliorate the inflammation in DSS-induced colitis mice and reduce the proportion of Th17/Treg cells.Conclusions:It is butyrate produced by F.prausnitzi,that maintains Th17/Treg balance and exerts significant anti-inflammatory effects in colorectal colitis rodents,by inhibiting HDAC1 to promote Foxp3 and block the IL-6/STAT3/IL-17 downstream pathway.The present study provides experimental information for the purification experiments and mechanisms studies of other probiotics and provides confirmations for the clinical application of butyrate-producing probiotics including F.prausnitzi.Targeting the butyrate-HDAC1-T cell axis offers an effective novel approach in the treatment of inflammatory diseases including IBD.