Screening And Anti-inflammatory Effects Of Unsaturated Fatty Acid Alcohol Amines And Flavonoid Derivatives Targeting Nur77

Current studies have shown that the nuclear receptor Nur77 has great potential in alleviating various inflammatory diseases,and may be a key target for the treatment of inflammatory-related diseases.The purpose of this thesis is to discover new small drugs that can potentially target Nur77 in the treatment of inflammatory diseases.Through in Binding assay,the unsaturated fatty acid alcohol amines and flavonoid derivatives were screened for Nur77 affinity;the ones with better affinity were obtained.Then,We select the candidate compounds B6 and 4k to further study after structure-activity relationship analysis.The research content and results of this thesis are as follows:(1)Preliminary screening of two types of derivatives:Based on SPR experiments,we found that the synthetic unsaturated fatty acid alcohol amine derivatives(PUFAAAs)and flavonoid derivatives(FLs)both showed a certain affinity for Nur77 in vitro.In the LPS-induced inflammatory macrophage model,4a,4f and 4k in PUFA-AAs and A6,B6,B7 and C5 in FLs can significantly inhibit the secretion content and the mRNA expression of pro-inflammatory factors NO,IL-1β,TNF-α and IL-6.However,only some of the compounds 4k and B6 showed a good inhibitory effect on the protein expression of IL-1β and TNF-α.Based on the screening results,we obtained two potential candidates for Nur77,B6 and 4k,with high anti-inflammatory activity.(2)Preliminary study on the targeting Nur77 and anti-inflammatory properties of candidate compounds B6 and 4k:B6 and 4k can exert anti-inflammatory effects in a concentration-dependent manner;B6 and 4k can bind to Nur77-LBD in vitro,with Kd values of 9.95 × 10-6 M and 4.97× 10-8 M,respectively;Molecular docking experiments show that they can work well on the binding pocket of Nur77-LBD.Then,through Nur77-siRNA technology,it was confirmed that B6 and 4k depend on Nur77 to inhibit NF-κB inflammation signaling pathway,and B6 can induce Nur77 to exit the nucleus and localize to mitochondria,suggesting that compound B6 may exert an antiinflammatory effect by affecting the subcellular localization of Nur77.However,4k did not significantly affect the subcellular localization of Nur77.(3)Study on the effect of B6 and 4k in Acute Inflammation Model(Acute Lung Injury,ALI):B6 and 4k can dose-dependently reduce the secretion of TNF-α,IL-1βand IL-6 in alveolar lavage fluid;down-regulate the expression of TNF-α,IL-1β,and up-regulate the expression level of IκBα;improve the infiltration of inflammatory cells and the thickening of alveolar compartments;reduce interstitial edema and lung tissue damage.Overall,these results indicate that B6 and 4k can effectively protect the lung from inflammation.(4)Research on the effect of 4k in Chronic Inflammation Model:4k can reduce palmitic acid(PA)-induced lipid acculation in hepatocytes HepG2.Using lipidomics methods,we found that 4k may improve palmitic acid-induced lipid acculation by regulating the expression of lipid metabolites(such as TGs).In addition,in the mouse obesity model induced by HFD,4k can significantly reduce the weight of the mouse,improve the fat acculation of the mouse,and show a good weight loss effect in the body.All in all,this article obtained two compounds with high anti-inflammatory activity,B6 and 4k,that both rely on Nur77 to exert their anti-inflammatory effects.It is further verified that Nur77 is a key target for the treatment of inflammation,suggesting that we can continue to develop more effective anti-inflammatory drugs based on this target.This is not only conducive to the process of new small molecule targeted drugs with anti-inflammatory effects,but also provides research ideas for the design and synthesis of more novel anti-inflammatory small molecules in the future,at the same time,provides basic support for the development of such anti-inflammatory active compounds.