The Study On Prevention And Mechanism Of BDP Against The Ulcerative Colitis On Rats

Object: Given the right amount of BDP,to observe changes on systemic and local conditions of rats and RAW264.7 cells,and to investigate the prevention and and mechanisms of BDP in the party,provide theoretical and experimental basis for the follow-up research,by TNBS/ethanol induced ulcerative colitis on rats,and LPS induced inflammation model on RAW264.7.1? The prevention of BDPagainst the ulcerative colitis on ratsMethod:Sixty Sprague Dawley(SD)rats were randomly divided into six groups with ten in each group,the normal group,model group,low-dose group(0.25g/kg),middle-dosegroup(0.5 g/kg),high-dose group(1.0 g/kg)and positive control group(Guchang Zhixie Pills,1.08 g/kg).After each dose of each group gavage 5 days,thenenema modeling with TNBS 60 mg/kg,intragastrical,administration once a day have been performed for 3 weeks continuously.DAI was evaluated on 1st,2nd,3rd and 21 st day during the experiment.30 min after the last administration,the rats were decapitated after anesthesia with ethy1 ether,the colon of the rats were collected consequently.After CMDI evaluation,half of the colon was performed for histopathological examination,and the other half to save at-80?,used as samples of real-time q PCR.Results: Compared With the normal group,model group rats see ulcer granuloma,lesions in the bowel wall thickening,most of rats with intestinal adhesion,infiltration of inflammatory cells in serious condition,CMDI evaluation and pathological histologyscores were significantly increased,with significant(P < 0.01,P <0.01);Compared with model group,BDP group improved the symptoms of colonic membrane congestion,edema,and ulcer.Histopathological results indicated that inflammatory cell infiltration get improved,CMDI evaluation and pathological tissue score significantly decreased(P<0.01?P<0.01).2? The Mechanism in the prevention of BDPagainst the ulcerative colitis on ratsMethod: The grouping and administration methods are same as the first part.30 min after the last administration,the rats were decapitated after anesthesia with ethy1 ether.Blood of the rats were collected for the analysis of SOD,MDA,GSH-Px levels,the rest half of colon were assayed of TLR4,IL–6,NF-?B,TNF-? and IL-10 m RNA expression by real-time quantitative PCR.Results: Compared with model group,MDA content in the tissue significantly was reduced(P<0.01),SOD and GSH-Px activity was higher,significantly(P < 0.01,P < 0.01).The expression of TLR4,IL-6,NF-?B and TNF-? gene downregulated(P<0.01,P<0.01,P<0.01,P<0.01),while IL-10 m RNA expression upregulated(P<0.05).3? Effect of BDP on the inflammatory response of LPS-induced RAW 264.7Method:At first,determine the effect of the cell toxicity about different concentrations of BDP?LPS and LPS + BDP on RAW 264.7 by MTT assay.Then examine the nitric oxide(NO)content in the cell culture medium,determine the appropriate time and concentration of LPS-induced,and establish the model of inflammation in vitro,and study the inflammatory reaction on different concentrations of BDP(0.4,4,40?g/m L)in LPS induced Raw 264.7 cells.NO production was determined by nitratereductive enzymatic methodto evaluate the anti-inflammatory effect of BDP,Real-timeq PCR method was adopted to research theeffects of BDP on expression level of TLR4,NF?B,TNF-? and IL-6 m RNA in LPS-induced RAW264.7 cells.Results:BDPdon't have cytotoxic with the concentration in the range of 0.4~4000 ?g/m L?72 h of the time on RAW264.7 cells,LPS doesn't have cytotoxic with the concentration in the range of 0.1~10?g/m L?48 h of the time on RAW264.7 cells.Determine the concentration and time of LPS established the model of inflammation that produced by LPS-induced RAW 264.7 cells,itis 1?g/m L for 24 hours.Compared with normal control group,after BDP treat the cells for 24 h alone,NOconcentration in the medium of dose group showed no significant difference(P>0.05),after 1?g/m L LPS-induced and drug intervention for 24 h,Compared with normal control group,the content of NO in LPS groupwas significantly higher(P <0.01),IL-6,TNF-?,NF-? B,TLR4 expression was significantly increased(all P<0.01),compared with LPS group,the content of NO in BDP group was significantly lower(P<0.01),the expression of TNF-?,NF-?B,TLR4,IL-6 were significantly lower(both P<0.01).Conclusion:1? BDP showed a positive preventive therapeutic effect on TNBS-induced UC rats,and can significantly improve the damage of colonic mucosa caused by the UC.2? Therapeutic mechanism of BDPcuring UC may be through activation of TLR4-NF-?B path to regulate the secretion of cytokine(IL-6,TNF-?and IL-10)and inflammatory mediators,as well as with the anti-oxidativeand anti-inflammatory effects.