Screening And Verification Of Anti-obesity Targets Of Chrysin

ObjectiveObesity is a globally prevalent disease,and no safe and effective drug has been found to cure it.According to literature reports,chrysin extracted from traditional Chinese medicine oroxylin has good anti-obesity effect,but the target and mechanism are not clear.In this study,proteomics and gene silencing were used to select and verify the anti-obesity target of chrysin,and the interaction between chrysin and target protein was analyzed to clarify the anti-obesity mechanism of chrysin.MethodsInsulin induced 3T3-L1 preadipocytes into mature adipocytes,and 0.5mmol/L palmitic acid established an obesity model,which was intervened by 50umol/L chrysin.Two-dimensional electrophoresis analysis of differential proteins in control group,model group and chrysin group,and identification of differential proteins by liquid chromatography-mass spectrometry combined with malde tof mass spectrometry to obtain candidate target proteins;The anti-obesity target protein of chrysin was confirmed by gene silencing,ultraviolet spectrum and fluorescence spectrum analysis.ResultsLipid droplet formation from adipocytes in palmitic acid group(model group)was significantly higher than control group and chrysin group.After analyzing and comparing the protein spectrums of each group obtained by two-dimensional electrophoresis,protein spots with twice the gray value difference are obtained by analysis and comparison,and in the model group,the difference is obviously higher or lower than that of the control group(p<0.05).there are eight spots in the chrysin group with no obvious difference from the control group,and eight proteins are obtained by LC-MS identification.The results showed that the 3 proteins,14-3-3 protein epsilon,Heterogeneous nuclear ribonucleoprotein F,T-complex protein 1 subunit beta,were down-regulated in model group,and 5 proteins,T-complex protein 1 subunit epsilon,annexin a2,26S protease regulatory subunit 8,Succinyl-CoA:3-ketoacid coenzyme A transferase 1,60 KDa heat shock protein and heterogeneous nuclear ribonucleoprotein were up-regulated in model group.Among the 8 proteins,annexin a2,26S protease regulatory subunit 8,Succinyl-CoA:3-ketoacid coenzyme A transferase 1 and 60 KDa heat shock protein were related to energy metabolism and inflammation.Gene silencing and triglyceride assay results showed that after down-regulated the gens of annexin a2(down-regulated by 80%,P<0.05),60 KDa heat shock protein(down-regulated by 40%,P<0.05),26S protease regulatory subunit 8(down-regulated by 40%,P<0.05)and Succinyl-CoA:3-ketoacid coenzyme A transferase 1(down-regulated by 40%,P<0.05),the triglyceride in 3T3-L1 induce by palmitic acid is reduced by 30%,45%and 40%respectively,compared with non-silencing group(P<0.05),except for 26S protease regulatory subunit 8 down-regulated group.And UV spectra showed that chrysin could combined with peptide bonds and peptides of annexin a2 and 60 KDa heat shock protein through interaction but Succinyl-CoA:3-ketoacid coenzyme A transferase 1 was not changed.Further more,the fluorescence spectra showed that chrysin could interacted with annexin a2 and 60 KDa heat shock protein in static quenching,and interaction force could be electrostatic force.ConclusionAfter screening and verification,the anti-obesity target of chrysin is annexin a2 and 60 kda heat shock protein,its mechanism is mainly through the influence of protein mechanics and structure,inhibition of annexin a2 and 60 kda heat shock protein in obesity-induced inflammatory signal pathway regulation function,so as to play an anti-obesity role.