Regulate The Expression Of Intestinal Epithelium Cytokines By The Integral Membrane Protein Of Lactobacillus Plantarum Through Modulation Of Epigenetics

Objective To observe the effect of MIMP on mice with inflammatory bowel disease in intestinal inflammation and intestinal microbiota.To investigate the effect of MIMP on inflammatory cytokines and the way of regulation,and to explore the related mechanism.Methods(1)The mice were divided into 3 groups: DSS+MIMP group,DSS group and healthy control group.During the intervention,the body weights,incidences of diarrhea,bloody stools,mortalities and the intestinal tract of mice were observed,H&E staining was used to observe the pathological changes of intestinal epithelium,dextran fluorescein isothiocyanate(fluorescein isothiocyanate-dextran,FITC-Dextran)by gavage were used to detect the vivo intestinal permeability of mice,and Western blot and real-time quantitative polymerase chain reaction was used to detect the expression of intestinal epithelial Tight junction protein in mice;q RT-PCR was used to detect the inflammatory cytokines(IFN-?,IL-17,IL-23,IL-4,IL-10)in intestinal epithelium of mice;16Sr RNA sequencing technique was used to detect the changes of intestinal microbiota in mice(2)Caco-2 cells and peripheral blood mononuclear cells(PBMCs)were established by coculture of Tranwells.Lipopolysaccharide as an inflammatory stimulus,Enzyme linked immunosorbent assay(ELISA)was used to detect the effect of MIMP on inflammatory cytokines(IFN-?,IL-17,IL-4,IL-10,IL-23)in the intestinal microenvironment at different concentrations and incubation time.The effects of MIMP and TLR4 antagonist on the secretion of inflammatory cytokines secreted by LPS were detected by ELISA and q RT-PCR.Detection of MIMP and TLR4 antagonist on MAPK and NF-kappa B signaling pathway by Western blot.Dual luciferase reporter gene assay was used to detect effects of MIMP on the activity of NF-kappa B at different concentrations and incubation time.(3)Western blot was used to detect effects of MIMP and TLR4 antagonist on the overall level of Histone(H3,H4)acetylation in Caco-2 cells induced by LPS.Chromatin immunoprecipitation(Ch IP)was used to detect effects of MIMP on the specific level of Histone(H3,H4)acetylation in promoter of IL17 and IFN-? at different concentrations and incubation time.q RT-PCR was used to dectect effects of MIMP on the expression level of(histone deaceylase,HDAC)induced by LPS.Results(1)the overall situation of MIMP+DSS group is better,the degree of light weight reduce,diarrhea,hematochezia and low incidence of death,disease activity index decreased significantly when compared with DSS mice;MIMP not only can improve the overall intestinal condition,improve the intestinal length(p<0.05),but also can effectively improve the histological score(p<0.001);The plasma content of FITC-Dextran of MIMP+DSS group was significantly lower than that of DSS group(p<0.01),however,the expression level of tight junction protein(ZO-1,JAM-1 and Occludin)in intestinal epithelium was significantly higher in MIMP+DSS group than that in DSS group(p<0.01).In addition,MIMP reduced the levels of proinflammatory cytokines(IFN-,IL-17,IL-23)in the intestinal epithelium of mice,and increased the levels of inflammatory cytokines(IL-4,IL-10).16 Sr RNA sequencing showed that the MIMP+DSS group had significantly higher intestinal microbiota diversity than the DSS group,the Shannon index was significantly lower(p<0.05),the Simpson index showed an upward trend(p=0.055);Principal component analysis and principal coordinate analysis showed that there were significant differences among the three groups in intestinal microecology;The sample clustering tree and histogram analysis showed that the MIMP+DSS group and healthy control group have higher intestinal microbita similarity;Phylogenetic tree analysis and LEf Se showed that Firmicute is the dominant bacteria in DSS group,Leuconostocaceae is the dominant bacteria in DSS+MIMP group and Bacteroides is the dominant bacteria in the healthy control group.(2)In the coculture model,MIMP can regulate the inflammatory cytokines induced by LPS,reduce the level of proinflammatory cytokines(IFN-?,IL-17,IL-23),and increased the level of antiinflammatory cytokines(IL-4,IL-10);The regulation of MIMP was dose dependent and time dependent,and the effect of MIMP was the most significant at specific concentration and time(1ng/ml,12h);inhibition experiment demonstrated that MIMP and TLR4 antagonist have the same ability to regulate inflammatory cytokine secretion;In addition,MIMP and TLR4 antagonist can effectively block the activation of MAPK and NF-kappa B pathway,and the activity of NF-kappa B also showed a downward trend with the increase of MIMP dose and the extension of incubation time.(3)The LPS improve histone(H3,H4)acetylation at the overall level,while MIMP and TLR4 antagonist intervention can effectively reduce the degree of acetylation at dose-dependent manner;in the specific level,MIMP can effectively reduce degree of histone(H3,H4)acetylation in the promoter region of IL-17 and IFN-gamma with increasing dose of MIMP and incubation time;at the same time,MIMP also can upregulate the expression of HDAC.Conclusion MIMP can effectively improve the inflammatory state and intestinal barrier function in IBD mice,significantly down regulate the expression of proinflammatory cytokines,up regulate the expression of inflammatory cytokines,and reverses the intestinal microbiota dysbiosis in IBD mice;MIMP can regulate the secretion of inflammatory cytokines in a dose and time dependent manner,and can effectively block the activation of MAPK and NF-kappa B signaling pathway;MIMP can effectively reduce the overall and specific levels of histone acetylation and increase the expression of HDAC.The study found that MIMP improved intestinal inflammation and clarify its mechanism,expanded knowledge about the treatment of inflammation,has certain theoretical innovation,in the future,MIMP should continue to promote the study as a new generation of anti-inflammatory drugs.