Study On Mechanism Of Bai Shao Qi Wu Granule Against Dss-induced Mice Colitis Based On Tlr4/nf-?b/nlrp3 Inflammasome Pathway

The traditional Chinese medicine Baishaoqiwu granule(BSQW)has been previously used to clinically treat ulcerative colitis(UC).Preliminary basic research showed that it had therapeutic effect on TNBS-induced colitis in rats.However,the effect of BSQW on mice colitis induced by dextran sodium sulfate(DSS)has not been reported.Abnormal activation of NLRP3 inflammatory signal plays a vital role in promoting the development of UC.But,it has not been elucidated that wether BSQW treat on UC by regulating NLRP3 inflammatory pathway.The aim of this study was to examine the efficacy of BSQW on UC and TLR4/NF-k B/ NLRP3 pathway both in a mouse model of colitis induced by DSS and in macrophage inflammatory model induced by lipopolysaccharide(LPS)and triphosadenine(ATP).Part 1 : Objective: The effect of BSQWon mice UC induced by DSS and TLR4/ NF-?B/ NLRP3 pathway.Methods: Mice were divided into five groups.3.5% DSS was drunk by animals for 7 days to induce colitis.BSQW treatments were carried out via oral gavage daily for 7 days.All animals were sacrificed after treatment,and colons were collected for detections.The changes of bodyweight,the length and histopathological changes of colon were used to evaluate treatment efficacy.Immunohistochemistry was used to detect the expression of NLRP3 and CD68.RT-PCR and Western blot were used to detect the m RNA and protein expressions of TLR4/ NF-?B/ NLRP3 pathway in colon.Results: BSQW alleviated weight loss,colon shortening and colon tissue damage in mice colitis.Compared with the model group,BSQW down-regulated the m RNA and protein expressions of TLR4/ NF-?B/ NLRP3signal(P< 0.05).Part 2 : Objective : To investigate the regulatory effect of BSQW-containing serum the activation of NLRP3 inflammasome pathway induced by both LPS and ATP in RAW264.7 macrophages.Methods : The macrophages were stimulated by LPS and ATP to activate NLRP3 inflammasome pathway.CCK8 was used to detect the effect of BSQW-containing serum on cell proliferation after the stimulation and select optimal concentration of serum.After treated with the serum,the cells was collected.NLRP3 was detected by immunofluorescence test,and IL-1b in the culture medium was detected by ELISA.The m RNA and protein expressions of TLR4/ NF-k B/ NLRP3 pathway were exaimed by RT-PCR and Western blot.Results: BSQW-containing serum can antagonize the suppression of cells' proliferation and activation induced by LPS and ATP(P< 0.05).Compared with model group,BSQW-containing serum decreased IL-1 b in culture medium(P< 0.05)and down-regulated the m RNA and protein expressions of NLRP3,Caspase1,IL-1 b,TLR4,NF-?B induced by LPS and ATP(P <0.05).Conclusion: 1).Suppression of the TLR4/NF-?B/NLRP3 signaling pathway may be one of the mechanisms involved in the therapeutic efficacy of BSQW against UC in mice induced by DSS.2).BSQW-containing serum can down-regulate TLR4/NF-?B/NLRP3 signaling pathway in mouse macrophage induced by LPS and ATP.