Research On The Mechanism Of Qiaosongsu In Reducing Ulcerative Colitis In Mice

Object:To observe and monitor the pharmacodynamics actions of pinocembrin in the treatment of ulcerative colitis in mice,and explore its mechanisms of alleviating UC,so as to provide theoretical basis and reference for the development of UC therapeutic drugs.Method: Pharmacodynamic evaluation in vivo:UC model was induced by drinking dextran sulfate sodium(DSS)freely in mice,which is available to evaluate the pharmacodynamics effect of pinocembrin on UC,with indexes such as diarrhea,blood stool,weight,colonic morphology and length,histopathological section and score.Pharmacodynamic evaluation in vitro:First,CCK-8 kit was used for the detection of the effect of pinocembrin on the viability of RAW264.7 cells.Then,Griess reagent was used for the detection of the effects of the pinocembrin on the secretion of NO(nitric oxide)in LPS-stimulated RAW264.7 cells.Finally,the effect of pinocembrin on the expression of cell factors and inflammatory mediators in LPS-stimulated RAW264.7 cells could be valued through WB,q-PCR and immunofluorescence staining techniques.Research on the pharmacological mechanism:Mice feces was collected for 16 s RNA sequencing to investigate the effects of pinocembrin on intestinal flora(probiotics or pathogenic bacteria)in UC mice.Meanwhile,WB and q-PCR were used to evaluate the effects of pinocembrin on the expression of tight junction protein(ZO-1,Claudin-1,Occludin,JAM-A)in Caco-2 cells.Further,it is possible for the further evaluation of the effects of pinocembrin on TLR4/NF-?B,NLRP3 inflammasome signaling pathway in colonic tissue of UC mice.With the analysis of the underlying structural mechanism of pinocembrin binding to the MD-2 protein by using the molecular docking method,the mechanism of blocking TLR4 signal transduction was further demonstrated by using immunoprecipitation and WB.With the NLRP3 inflammasome activation model in THP-1 cells was established by PMA and LPS,the effect of pinocembrin on NLRP3 signal transduction and ASC speck proteins can be explored by the technologies,such as WB and immunofluorescence.Results:Pharmacodynamic results in vivo:Pinocembrin can significantly ameliorates the disease symptoms such as lassitude?decrease in activity?weight reduction,diarrhea and blood stool in UC mice.The measurement of colo nic tissue and the score of pathologic section showed that the pathological injury were remarkably attenuated by pinocembrin,such as colon shortening,congestion,edema,unclear boundary and inflammatory cell infiltration.Pharmacodynamic results in vitro:The experimental results showed that pinocembrin inhibited the secretion of NO in LPS-stimulated RAW264.7 cells,as well as the expression of inflammatory cytokines(TNF-??IFN-??IL-6)and mediators(COX-2?i NOS)without cytotoxicity,which depends on its dose.Pharmacological mechanism:The analysis of flora sequencing of mice feces showed that the community composition and abundance of microflora appeared significant change compared to control mice,which changes reflected in dysbacteriosis and damagement of microecosystem.Compared with the model group,with pinocembrin treatment,microbiota disturbance was gradually regressed and the diversity of the flora increased,which maintained the intestinal micro ecological balance to some extent.Pinocembrin promoted the expression of tight junction proteins(ZO-1,Claudin-1,Occludin,JAMA)in Caco-2 cells.Furthermore,pinocembrin could significantly inhibit the abnormal activation of TLR4/NF-?B and NLRP3 signal pathway in colon tissues,as well as the expression of inflammatory mediators.Pinocembrin may competitively inhibit the binding of LPS to MD-2 protein,thereby blocking TLR4-mediated signal transduction.However,the aberrant activation of the NLRP3 signaling cascade was suppressed by pinocembrin,which may relate to depressing the abnormal protein expression involving NLRP3 signaling pathway as well as the assembly of NLRP3 inflammasome.Conclusion:Pinocembrin significantly reduced DSS-induced UC in mice.Its mechanism probably related to regulating intestinal microbiota,inhibiting the over-activation of TLR4/ NF-?B and NLRP3 signaling pathways and increasing the barriers of intestine.