Study Of Anti-inflammatory Targets And Mechanisms Of Caffeic Acid By Activity-based Chemical Proteomics

BackgroudInflammation is a common pathological process in the body,which is associated with various diseases,such as cancer,diabetes,atherosclerosis and obesity.At present,the common anti-inflammatory drugs in clinic have various adverse reactions,so it is particularly important to find new anti-inflammatory drugs,and the natural compounds with high safety have become the focus of research.We focused on caffeic acid(3,4dihydroxycinnamic acid,CA),a phenolic compound molecule found in many plants,also found in everyday drinks such as coffee and tea.CA has a variety of biological activities,such as antioxidant,anti-inflammatory,anti-tumor,immunomodulatory,and cardiovascular protection.At present,few studies have revealed the specific antiinflammatory target proteins of C A and their molecular mechanisms,and the traditional methods have not been studied in depth.Activity-based protein profiling(ABBP)is a chemical proteomics approach to elucidate the mechanism of interaction between bioactive small molecules and protein targets,which has the advantage of highthroughput identification and quantification of targets at the proteomics level.ObjectivesTo elucidate the anti-inflammatory mechanism of CA,we used the ABPP method to analyze the anti-inflammatory target of CA and its pathway mechanism,and verified the credibility of the target through other pharmacological experiments.Methods1.The mice model of acute pneumonia induced by lipopolysaccharide(LPS)was established,and the positive drug dexamethasone(DEX)was used as control.After 14 days of continuous administration,the pathological sections of the lungs were observed,and the inflammatory factors,tumor necrosis factor alpha(TNF-α)and interleukin-6(IL-6)in the serum were detected.Furthermore,the CA probe(CA-P)was synthesized and its chemical structure was analyzed by 1H NMR and MS.The inflammatory model of RAW264.7 cells stimulated by LPS was established,and the expression of TNF-αand IL-6 in the supernatant of RAW264.7 cells was detected.2.The probe was incubated with RAW264.7 cells stimulated by LPS.Labeling group,competition group and control group were set to enrich the probe labeled protein by click chemistry reaction.The probe labeling and competition effect were observed by SDS-PAGE fluorescence signal and LC-MS/MS and bioinformatics analysis were carried out.3.According to the ABPP results,disulfide isomerase(Protein disulfide isomerase,PDI)was selected for validation.Pulldown method,probe-labeled purified protein,fluorescence co-localization and cellular thermal shift assay(CESTA)were used to verify the results.4.The effects of CA and oxidized CA on the activity and redox state of PDI were analyzed,and the molecular mechanism was further studied,that is,the binding sites between CA and PDI were studied by Iodoacetamide-Probe(IAA-Probe)and LCMS/MS,and verified by mutant.5.Finally,the effects of CA on PDI/PERK(Protein kinase R-like endoplasmic reticulum kinase)/NF-κB(Nuclear factor kappa-B)pathway were studied by PDI inhibitors and knockdown experiments.Results1.The anti-inflammatory effect of CA was verified by in vivo experiments.CA could relieve the pathological symptoms of pneumonia and significantly remove serum inflammatory factors.The structure of CA probe was determined,and the antiinflammatory effect of CA and CA probe was verified in vitro.CA could significantly remove inflammatory factors in cell supernatant and inhibit their expression.2.The labeling concentration of CA probe was 50 μM,and 10 times the concentration of caffeic acid could compete the fluorescence signal.The results of LCMS/MS and bioinformatics analysis showed that PDI was the most reliable protein.3.Traditional molecular biology experiments have proved that there is a direct binding between CA and PDI,which is the binding target of CA anti-inflammatory.4.Oxidative CA can inhibit the activity of PDI.The mechanism is to inhibit the enzyme activity pocket of PDI by covalently binding to the enzyme activity sites Cys53,Cys55,Cys399,and Cys401 of PDI.5.CA targets PDI to play an anti-inflammatory role,which is mediated by PDI/PERK/NF-κB pathway.ConclusionsThese results suggest that CA has a good anti-inflammatory effect both in vivo and in vitro.CA is oxidized after entering cells and covalently binds to PDI to inhibit its reduction activity,thus inhibiting the expression of PDI/PERK/NF-κB pathway proteins and playing an anti-inflammatory effect.