| Objective:The objectives of the study were to explore the therapeutical effect of Baizhu Huangqi Tang(BZHQT)on dextran sulphate sodium(DSS)induced ulcerative colitis(UC)mice,and to examine BZHQT's regulating effect on NOD-like receptor protein 3(NLRP3)inflammasomes.In this study,we hope to provide experimental evidences for UC treatment.Methods:1.The therapeutical effect of BZHQT was confirmed through animal study.6-8 weeks old C57BL/6 male mice were randomly divided into control group,model group,5-Amino Salicylic Acid group(5-ASA),BZHQT low dose group(BZHQT,3.9 g/kg/d),BZHQT middle dose group(BZHQT,7.8 g/kg/d)and BZHQT high dose group(BZHQT,15.6g/kg/d)with 8 mice in each group.Control group was given distilled water for 7 days.Other groups were given 2.5%DSS solutions for 7 days.BZHQT and 5-ASA were given simultaneously.Control group and model group were given the same volume of distilled water.Body weight,loose stools,bloody diarrhea,and disease activity index(DAI)were recorded during the study.The weight and the length of colon,hematoxylin and eosin(HE)and myeloperoxidase activity(MPO)of colon tissues were measured after the end of treatment.2.In vitro studies to explore BZHQT's effect on NLRP3 inflammasome.Phorbol-12-myristate-13-acetate(PMA)was added into U937 cells for 48 h.Then,U937cells were incubated with different concentration of BZHQT(0?g/m L,3.125?g/mL,6.25?g/m L,12.5?g/mL,25?g/m L,50?g/mL,100?g/m L).And the cell viability was detected by cell counting kit-8(CCK-8).5×10~5 cells/mL of U937 cells were seeded in 6 well plate with 2 mL of each well.Control group,model group(LPS,1?g/ml),BZHQT group(25?g/m L),and positive control group(INF39,10?M)were designed.U937 cells were induced by PMA for 48 h.Except control group,other groups were stimulated with Lipopolysaccharides(LPS)(1?g/m L).Control group was added with same volume of phosphate buffer saline(PBS).After 2 h,corresponding drugs were added into BZHQT group and positive control group.Control group and model group was added with same volume of fresh medium.The cell supernatant and cells were collected after 48 h.Interleukin(IL)-1?levels in cell supernatant were detected by enzyme linked immunosorbent assay(ELISA).And mRNA and protein levels of NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),cysteinyl aspartate-specific protease-1(Caspase-1)and IL-1?were detected by RT-PCR and western blot.3.In vivo studies to explore BZHQT affected NLRP3 inflammasome.2.5%DSS was used to induce UC model.Control group,model group,positive control group(5-ASA,0.52 g/kg/d)and BZHQT(BZHQT,7.8 g/kg/d)group were designed in this study.The molding and administration were performed simultaneously for 7 consecutive days.The mice were sacrificed on the 8th day.The serum was collected to measure IL-1?by ELISA.The colon tissues were collected to detect both mRNA levels and protein levels of NLRP3,ASC,Caspase-1,and IL-1?by RT-PCR and western bolt.Results:1.The therapeutical effect of BZHQT on DSS induced UC mice results:BZHQT is effective in treatment of UC.Compared with control group,model group mice body weights were obviously decreased.DAI increased,the length and weight of colon length were decreased(P<0.01).Inflammatory cell infiltration was seen in colonic mucosal tissue(P<0.01).MPO activity increased(P<0.01).Compared with model group,each treatment group could improve UC clinical symptoms in varying degrees(P<0.05 or P<0.01).Body weights,loose stools,and bloody diarrhea,and DAI were alleviated after treatment(P<0.05 or P<0.01).The colon weight returned to normal.The inflammatory cell infiltration and MPO activity decreased(P<0.05 or P<0.01).The curative effect of BZHQT middle dose group was better than the other two groups.2.BZHQT affected NLRP3 inflammasome in vitro results:Through CCK-8,50?g/m L and 100?g/mL of BZHQT were found had inhibitory effect on cell viability(P<0.05).In this study,we chose maximum non-toxic concentration(25?g/m L)to conduct experiment.ELISA showed that the IL-1?levels in model group mice serum increased compared with control group(P<0.01).And BZHQT group and positive control group could significantly decrease the levels of IL-1?(P<0.01).In RT-PCR and western bolt studies,the NLRP3,ASC,Capsae-1 and IL-1?levels were obviously increased compared with control group(P<0.05 or P<0.01).Compared with model group,both mRNA and protein levels of NLRP3,ASC,Capsae-1 and IL-1?were ameliorated after BZHQT group treatment(P<0.05 or P<0.01).3.BZHQT affected NLRP3 inflammasome in vivo results:ELISA results showed that BZHQT could significantly decrease the IL-1?levels in UC mice serum(P<0.01).And compared with model group,BZHQT could decrease the mRNA and protein levels of NLRP3,ASC,Caspase-1 and IL-1?in colon tissues(P<0.05 or P<0.01).Conclusion:1.BZHQT has a certain therapeutical effect on UC mice.2.BZHQT could ameliorate inflammatory symptoms of UC mice through inhibiting NLRP3 inflammasomes. |
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