Study On The Effects Of Total Flavones Of Ambrette On The Crohn's Disease And Its Mechanisms Based On NF-?B

Crohn's disease(CD)is one of the clinically refractory diseases.Studies have shown that NF-?B signaling pathway regulates the release of inflammatory cytokines and is a key mechanism and important therapeutic target for CD.Autophagy is an important process of cell metabolism,and through the regulation of autophagy pathway to improve process of crohn's disease is the current research hot spot,andNF-KB signaling pathways seem related to autophagy,plays an important role in the process of CD disease pathogenesis,TCM has unique curative effect in the CD treatment,but the foundation and mechanism research has not been confirmed,This study investigated the efficacy and mechanism of TFA in the treatment of CD.Objectives:1.In animal,the effect of TFA on colonic inflammation in CD model mice,the expression of inflammatory factors TNF-?,IFN-?,IL-6,IL-1?,IL-12 and IL-17 was observed.The changes of autophagosomes and the expression of autophagocyte-related proteins LC3-?/LC3-?,Beclin-1,p62,NF-?B pathway marker protein I?B,pIKK,p65,p100,p52 in colon tissues of mice in the model group?SASP and TFA group were observed.2.At the cellular level,TFA was explored to regulate the autophagy of RAW264.7 cells through the regulation of NF-?B autophagy pathway,and to reduce macrophage inflammatory damage.Methods:1.The CD mouse model was established by TNBS.The colonic tissue inflammation level and intestinal fibrosis level of each group were evaluated by the general conditions,DAI score,microscopic pathological score and Masson staining of each group of mice.ELISA was used to detect TNF-?.IFN-?,IL-6,IL-1?,IL-12 and IL-17 levels.2.The changes of autophagosomes in colon tissue were observed by transmission electron microscopy.The autophagy-related proteins LC3-?/LC3-?,Beclin-1,p62 and NF-?B pathway marker proteins expression were observed by Western-Blot and immunofluorescence.3.RAW264.7 cells were cultured in vitro,and different concentrations of TFA were screened by CCK-8 to intervene in LPS-stimulated RAW264.7 cells,and the pathway inhibitor PDTC was added.ELISA was used to detect TNF-?,IFN-?,IL-6,IL-1?,IL-12 and IL-17 in each group of cells in vitro.Western-Blot was used to observe the autophagy-related proteins LC3-?/LC3-?,Beclin-1,P62,NF-?B pathway marker protein I?B,pIKK,p65,p100,p52 protein expression in mouse colon tissues.Results:1 Compared with the normal control group,tnbs-induced CD mice showed increased mortality and colonic weight/length ratio,significantly decreased body weight and DAI score,significantly increased pathological score and fibrosis score,and increased contents of TNF-?,IFN-?,IL-6,IL-1?,IL-12 and IL-17and other pro-inflammatory cytokines in colonic tissues(P<0.05).Compared with the model group,the mortality,colonic weight/length ratio,DAI score,body weight,pathological and fibrosis scores,TNF-?,IFN-?,IL-6,IL-1?,,IL-12 and IL-17 and other pro-inflammatory cytokines were all decreased in the SASP group and TFA group,respectively(P<0.05).Among them,there was a dose-dependent relationship between the expressions of TNF-?,IFN-?,IL-6,IL-1?,IL-12 and IL-17 in colon tissues and TFA,as well as the reduction of mortality,weight gain,colonic weight/length ratio in mice.2.Compared with the normal control group,the contents of I?B,pIKK and p65 in the colon tissue of CD model mice were increased,and the contents of p100 and p52 were decreased.In addition,the contents of autophagosome decreased under transmission electron microscope,LC3-?/LC3-?,Beclin-1 in the colon tissue were decreased and p62 increased(P<0.05).Compared with the model group,the contents of p-IKK?/?/?,I?B? and p-p65 in the colon tissues of the mice in the SASP group and TFA groups were decreased,and the contents of p52 and p100 were increased,and the contents of autophagosome,lc3-?/lc3-? and beclin-1 were increased,and the contents of p62 were decreased.3.To assess the toxicity of TFA using the CCK-8 assay,TFA is not cytotoxic at a dose of 200?g/mL,LPS significantly promotes inflammatory cytokine levels in RAW264.7,including TNF-?,IFN-?,IL-6,IL-1?,IL-12 and IL-17.SASP and TFA reduced the production of inflammatory cytokines in LPS-induced macrophage RAW264.7 cells,and the extent of decline was dose-dependent with TFA.After LPS treatment,the expression of p-IKK?/?/?,I?B? and p-p65 increased significantly,and the expression of p100 and p52 decreased,compared with the normal group(P<0.05).Compared with the LPS group,LPS+TFA group,LPS The expressions of p-IKK?/?/?,I?B? and p-p65 in the PDTC group and LPS+TFA+PDTC group decreased,and the expression levels of p100 and p52 increased(P<0.05).Compared with the LPS+TFA group,The expressions of p-IKK?/p/?,I?B? and p-p65 in LPS+PDTC group and LPS+TFA+PDTC group were increased,p100 and p52 expression levels were decreased(P<0.05);and LPS+PDTC group Compared with the LPS+TFA+PDTC group,the expression levels of p-IKK?/?/?,I?B?,p-p65,p100 and p52 did not change significantly.After LPS stimulation of RAW264.7,LC3II/LC3I and beclin-1 were significantly decreased compared with the normal group,and p62 was significantly increased(P<0.05).On the basis of LPS stimulation,LCAII/LC3I and beclin-1 were significantly increased compared with LPS group.P62 decreased significantly(P<0.05);LC3?/LC3? and beclin-1 in macrophages were significantly decreased compared with LPS+TFA group and LPS group after LPS and PDTC stimulation,and p62 was significantly higher than LPS+TFA group and LPS group.P<0.05);LC3?/LC3? and beclin-1 were significantly decreased compared with LPS group and LPS+TFA group after LPS,TFA and PDTC(P<0.05),and p62 was significantly higher than LPS group and LPS+TFA group.There was no significant difference between the LPS+TFA+PDTC group and the LPS+PDTC group.Conclusion:1.TFA can reduce the levels of TNF-?,IFN-?,IL-6,IL-1?,IL-12 and IL-17 inflammatory factors in colon tissue and serum of CD model mice,alleviate the clinical symptoms of CD mice and improve their the degree of intestinal inflammation.2.TFA can inhibit the expression of NF-?B pathway-related proteins,activate colonic autophagy expression in CD model mice,and increase the expression level of autophagy-associated marker proteins.3.NF-?B pathway has an inhibitory effect on the autophagy level of RAW264.7 cells.TFA may attenuate macrophage inflammation by inhibiting NF-?B signaling pathway and activating autophagy.