| Cardiovascular disease has surpassed cancer and other diseases as the top cause of death worldwide.The damage of vascular endothelial cell barrier induced by chronic low-grade inflammation is a major cause of cardiovascular disease.Thus,inhibition of vascular endothelial cell inflammation is a key strategy for the prevention and treatment of cardiovascular diseases.Vitexin,a small active molecule found in a variety of fruits,vegetables,grains,legumes,and Chinese herbal medicines,has functional activities including cardiovascular disease prevention and treatment,anti-tumor and fat reduction.Previous laboratory research and literature survey indicated that vitexin’s exceptional anti-inflammatory function is responsible for its preventative and therapy effect on cardiovascular diseases,but the direct molecular target of action remains unknown.The combination of drug affinity responsive target stability(DARTS)and mass spectrometry is a new approach based on the reduced sensitivity of target proteins to protease degradation after small molecule ligand binding,and can be used to quickly and directly identify potential target proteins for active small molecule.Therefore,based on our previous findings,this paper used DARTS combined with mass spectrometry to screen and identify potential target proteins of vitexin,as well as unlabeled biomolecular interaction and molecular docking techniques to determine the interaction between vitexin and candidate target proteins and their binding sites.Furthermore,molecular biology techniques such as western blot,transcriptome sequencing,immunofluorescence staining,fluorescence labeling,and Me RIP-q PCR were used to investigate the role of candidate target proteins in mediating vitexin’s anti-inflammation to improve barrier function and its molecular mechanism in a model of low-level vascular endothelial cell inflammation induced by IL-17 combined with TNFα.The main results are as follows:1.DARTS analysis showed that vitexin increased the abundance of a 58 k Da protein band on SDS-PAGE,and vitexin was detected in this protein band.The protein represented by this band was most likely FTO(m RNA demethylase),which was a candidate target of vitexin,based on the distinctive peptide,sequence coverage,molecular weight,score and abundance of mass spectrometry.Immunoprecipitation was further used to specifically enrich FTO protein,and vitexin was also detected.Meanwhile,molecular interaction analysis revealed that vitexin significantly quench the intrinsic fluorescence intensity of FTO protein and increased its protease hydrolysis resistance and thermal stability,suggesting that vitexin directly binds to FTO and has a stronger binding ability than the other three common flavonoids galangin,fisetin and curcumin.Molecular docking results showed that,unlike most small molecule FTO inhibitors or agonists,vitexin is bound to the catalytic activity region rather than the substrate binding region,and the hydroxyl and benzene rings on vitexin form hydrogen bonds,hydrophobic interactions and cation-π interactions with FTO protein,which are the major driving forces for the FTO-vitexin complex.The binding of vitexin to FTO further inhibited the intracellular m6 A level increase induced by low-level inflammation,suggesting that FTO-dependent m6 A demethylation is likely to mediate vitexin anti-inflammatory effect.2.Vitexin was absorbed by vascular endothelial cells in a dose-dependent manner,and reduced the gene and protein expression of ICAM and IL-6 in an inflammatory state.Western blot,immunofluorescence and fluorescence labeling experiments showed that vitexin inhibited the down-regulation of the expression of tight junction proteins including Claudin-1,Occludin and ZO-1,as well as the increase in monolayer membrane permeability caused by inflammation,thereby improving endothelial cell barrier function.By blocking the m6 A demethylation activity of FTO with the specific inhibitor FB23,vitexin lost its ability to reduce expression of ICAM and IL-6 and improve endothelial barrier function.These findings suggested that vitexin inhibits inflammation and protects cellular barrier through targeting FTO-mediated m6 A demethylation.3.Vitexin decreased the level of m6 A on the m RNA of the inflammatory factors ICAM and IL-6 and lowered their stability,whereas FB23 treatment reduced the stability of ICAM and IL-6 m RNA,suggesting that ICAM and IL-6 were the direct m RNA targets for demethylation after vitexin activated FTO activity.In addition,transcriptomic sequencing identified mi R-4786 as another most plausible FTO direct m6 A demethylation target,and SRAMP online software predicted a conserved m6 A methylation site GGACU on mi R-4786’s nucleic acid sequence.The expression of mi R-4786 was considerably down-regulated during inflammation,but vitexin significantly up-regulated its expression.It was discovered that after subsequent treatment with specific inhibitors targeting the 5′ and 3′ ends of mi R-4786,vitexin lost its ability to reduce the gene and protein expression of IL-6 and ICAM,suggesting that in addition to directly affecting m6 A on IL-6 and ICAM m RNA,vitexin could also lower inflammatory factors by indirectly affecting mi R-4786 m6 A to increase its expression.In conclusion,the molecular mechanism of vitexin against vascular endothelial cell inflammation is as follows: vitexin directly binds to FTO and activates its demethylase activity,which on the one hand reduces the m6 A level on IL-6 and ICAM m RNA,on the other hand,demethylation of mi R-4786up-regulates its expression,and together reduces the m RNA stability of IL-6 and ICAM,down-regulating the protein expression of inflammatory factors,and finally,playing an anti-inflammatory role. |
