Molecular Mechanism Of AEE Against Oxidative Damage Of Vascular Endothelial Cells Based On Multi-omics

The oxidative damage model of human umbilical vein endothelial cells(HUVECs)induced by H₂O₂ was used to reveal the molecular mechanism of aspirin eugenol ester(AEE)against endothelial cell injury at the cellular level via transcriptome,proteomics and acetylation modification techniques.The HUVECs were randomly divided into three groups,the control group(normal group),H₂O₂ group(model group),and AEE group.Western blotting,cellular immunofluorescence and gene knockout techniques were used to verify the regulatory mechanism of AEE against vascular endothelial injury via AMPK/m TOR,PI3K/AKT,and ASK1 signal pathways.1.Compared with H₂O₂ group,there were 9111 differentially expressed genes(DEGs)in AEE group,of which 4532 DEGs were up-regulated and 4579 DEGs down-regulated(p<0.05).Gene ontology(GO)showed that DEGs were related to protein binding,transferase activity,protein phosphorylation,nucleus,mitochondrial membrane,and endoplasmic reticulum.The analysis of Kyoto Encyclopedia of Gene and Genome(KEGG)showed that DEGs are mainly involved in TCA cycle,MAPK/m TOR signal pathway and so on.201 DEGs(p<0.01)were mainly involved in lipid synthesis and degradation,stress response,and autophagy.2.Compared with H₂O₂ group,there were 355 differentially expressed proteins(DEPs)in AEE group DEPs(p<0.05),of which 205 DEPs were up-regulated and 150 DEPs down-regulated.GO enrichment showed that the DEPs were related to antioxidant activity,transcription factor activity,and signal transduction.KEGG analysis showed that the DEPs were mainly involved in apoptosis,AMPK signal pathway and so on.154 DEPs(p<0.01)were mainly involved in the process of apoptosis,stress,and atherosclerosis.3.Compared with H₂O₂ group,there were 383 in the AEE group,including 154 differentially expressed acetylated sites,including 329 up-regulated and 190 down-regulated(p<0.05).GO enrichment showed that the differentially expressed acetylated proteins were mainly related to molecular transduction and antioxidant activity.KEGG analysis showed that the differentially expressed acetylated proteins were mainly involved in cellular gluconeogenesis,metabolic pathway,and oxidative phosphorylation.109 differentially expressed acetylated proteins and 123 acetylated sites(p<0.01)were mainly involved in the process of oxidative phosphorylation and autophagy.4.Compared with H₂O₂ group,AEE pretreatment could significantly increase the expression of p-AMPK,p-ULK1 and Bcl2,and significantly inhibit the expression of p-m TOR,p-p70S6K and Bax.N-acetyl-L-cysteine(NAC),chloroquine(CQ),rapamycin(RAPA),AMPK inhibitor and AMPK sh RNA were used to interfere with HUVECs.The results showed that AEE could effectively prevent H₂O₂-induced HUVECs oxidative damage via AMPK/m TOR signal pathway.5.Compared with H₂O₂ group,the expression of p-PI3K,PI3K,p-AKT and AKT increased significantly after HUVECs were treated with different concentrations of AEE(0.5,1.0,2.0 and 4.0?M)at different time(6,12,24,36 h).PI3K inhibitor and PI3K sh RNA technique were used to knock down PI3K,respectively.The results showed that AEE could effectively prevent HUVECs oxidative damage induced by H₂O₂ via PI3K/AKT signal pathway.6.Compared with control group,H₂O₂ could significantly increase the expression of ASK1,p-p38MAPK,p-SAPK/JNK and inhibit the expression of p-ERK1/2 at different time(8,16 h).Compared with H₂O₂ group,p-p38MAPK,p-SAPK/JNK,ASK1 decreased significantly,and p-ERK1/2increased significantly after HUVECs were treated by AEE(1.0?M)at different time(8,16 h).The inhibitors of ASK1,ERK1,ERK2 and sh RNA were used to knock down ASK1 and ERK1/2,respectively.The results showed that AEE could effectively prevent H₂O₂-induced HUVECs oxidative damage via ASK1 signal pathway.In conclusion,the pharmacological activity of AEE against vascular endothelial oxidative injury may be related to AMPK/m TOR,PI3K/AKT,and ASK1 signal pathways based on the combined analysis of transcriptome,proteomics,and acetylation modification.