| ObjectiveIn our study,the compounds with high anti-inflammatory activity were screened from many natural compounds and their derivatives,and the anti-inflammatory activity of the compounds was verified by the model of dextran sulfate sodium?DSS?-induced colitis in mice,and then to discuss the anti-inflammatory mechanism of the compounds.Our research is helpful to broaden the application of anti-inflammatory drugs related to autoimmune diseases and provide a new way for the research and treatment of inflammatory diseases.Methods1. By using Th cell differentiation of fluorescent reporter gene mice and drug screening methods,111 samples including 100 natural products and their derivatives,and 11 extracts from medicinal plants were preliminarily screened,from which small molecular compounds with high activity were selected for further dose-dependent screening.The ratio of GFP in CD4cells was analyzed by flow cytometry.2.DSS-induced colitis in mice was selected as the suitable animal model.A 20-deoxyingenol derivative D9(C25H36O5;M=416),which was provided by this research team,was selected as test sample and administered by intraperitoneal injection.The mice were randomly divided into four groups:normal group,DSS model group,the group with high?10mg/kg?and low?5mg/kg?concentration of D9 respectively.The therapeutic effect of D9 on DSS-induced colitis in mice was studied from the aspects of general conditions and body weight,disease activity index,colon length,and histological changes.3.Through the collection of mouse serum and colon tissue from each group,Elisa and PCR were used to detect the effect of D9 on related indicators of Th1,Th2 and Th17 cells in DSS-induced colitis in mice,and to study the immune mechanism of D9.Results1. In the model of T cell differentiation of reporter gene mice in vitro,Compound YY1?Ingenol 3-angelate?and its structural analogue,20-as well as?-?2?inhibited the expression of IL-17 and IFN-?,and promoted the expression of IL-4.A further dose-dependent screening evaluation of them revealed that ingenol 3-angelate was the most active,while the activities of other 20-deoxyingenol derivatives were close,slightly weaker than ingenol 3-angelate,showing a dose-dependent relationship.However,the preliminary animal experiments showed that ingenol 3-angelate was more toxic,while D9,the derivative of ingenol 3-angelate,was safer.So D9 was chosen as the test sample.2. The animal model of colitis mouse induced by DSS verified that D9?10mg/kg?could relieve and cure the colitis induced by DSS,it had good effect in preventing the excessive weight loss of mice,repairing the damage of colonic mucosa,decreasing of the score of DAI and colonic histopathology,slowing down the deterioration of the disease and played an important role in anti-inflammatory repairment.3. The cytokines and genes associated with colitis mice induced by DSS revealed that D9?10mg/kg?suppressed the expression of IL-17?IL-23R?IL-22?associated with IL23/Th17 cell pathway?and ROR?t?Th17 transcription factor?,at the same time,it down-regulated IFN-?while stimulated the expression of IL4 in order to maintain Th1/Th2 balance.This results were consistent with the trend of screening drugs at cell level by and large.Conclusion1.Ingenol 3-angelate and its structural analogue,20-deoxyingenol derivatives D9,D11,D13,as well as?-?2?exhibited high activity in the screening process and were closely related to inflammatory cytokines.They might have good anti-inflammatory effect and development potential.It could be used in the development and research of drugs related to inflammatory diseases.2.Compound D9 had a certain remission effect on DSS induced colitis in mice.This effect might exerted by regulating the cytokines related to T cells,balancing the abnormal Th1/Th2 drift,inhibiting the differentiation of Th17 cells and suppressing the secretion of IL-17,thus played a role in regulation of inflammation and protection of intestinal mucosa. |
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