| Objective:The mitigative effect of the ethanol extract of Tacca chantrieri André(TCE)on Dextran sulfate sodium salt(DSS)induced ulcerative colitis(UC)was studied,and the effect and mechanism were explored by the lipopolysaccharide(LPS)induced mouse RAW264.7 cell inflammation model,which provided scientific basis for the clinical application of TCE.Methods:1、In this study,the UC mouse model was established by 3.5% DSS aqueous solution,and the effects of low(200 mg/kg)and high(600 mg/kg)doses of ethanol extract from Tacca chantrieri André on UC mice were observed.Its pharmacodynamic effects was evaluated by body weight,diet,disease activity index(DAI)and colonic histopathological score.The levels of related inflammatory factors in the supernatant of colon tissues were detected by ELISA and RT-q PCR,and the expression levels of related proteins in colon tissues were detected by WB technique to explore their mechanism.2、The survival rate of TCE on RAW264.7 cells and the dosage of TCE was determined by MTT method.The contents of IL-6,IL-1β and TNF-α levels were used to screen and determine the optimal concentration and time of LPS stimulated RAW264.7 cell inflammation model.The expression of related inflammatory factors in the cell supernatant and cells was detected by ELISA and RT-q PCR and the expression of related proteins in cells was detected by WB.The anti-inflammatory effect and mechanism of the ethanol extract from Tacca chantrieri André were analyzed.Results:1、Study on the alleviation of TCE on UC mice induced by DSS Compared with the model group,the mice gained weight after the TCE was administered,the DAI score was decreased,and the HE results showed that the symptoms of colitis in mice was decreased,and the difference was statistical significance(P<0.05).The results of ELISA showed that the expression of NO,i NOS and MPO(P<0.01)in the mice colon tissue can be significantly reduced,the expression of pro-inflammatory factors IL-6,IL-8,IL-1β and TNF-α was reduced(P<0.01)and the expression of anti-inflammatory factor IL-10 was increased(P<0.05).The RT-q PCR results showed that TCE could significantly inhibit the m RNA expression of IL-6 and IL-1β in mice colon tissues(P<0.05)and the WB results showed that TCE could reduced the expression of NF-κB and MAPK signaling pathways related proteins in mice colon tissue(P<0.05).2、Study on the mechanism of TCE on LPS-induced RAW264.7 cell inflammation model The results of MTT showed that the survival rate of TCE on cell proliferation starting from40 μg/m L,and there was no obvious inhibition on cell growth.Therefore,the drug doses were determined as 40,20 and 10 μg/m L for the experiment.After the 1000,500,100ng/m L of LPS stimulated the cells for 4,8,16 and 24 h,the levels of IL-6 and IL-1β were detected respectively.Combined with the results of the inflammatory factors,and finally the 1000 ng/m L of LPS was used to stimulate the cells for 24 h to establish the inflammation model.The results of ELISA showed that after LPS(1 μg/m L)stimulated RAW264.7 cells,TCE could significantly reduce the content of inflammatory factors NO,i NOS,IL-6,IL-1β and MCP-1(P<0.01).From the results of RT-q PCR,the m RNA expression of i NOS,COX-2,IL-6,IL-1β and MCP-1 were significantly decreased after the treatment of TCE compared with model group(P<0.05).In the protein results,the related proteins of NF-κB and MAPK signaling pathways were significantly down-regulated after treatment with TCE(P<0.05).Conclusion:The alcohol extract of Tacca chantrieri André of Dai medicine has significant alleviating effect on UC mice induced by DSS,the mechanism of which may be related to the regulation of NF-κB and MAPK pathways. |
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