Regulatory Mechanism Of AEE On Lipid Metabolism In Hyperlipidemia Mice

Hyperlipidemia caused by abnormal lipid metabolism is the main cause of the formation of a variety of cardiovascular diseases.At present,the incidence of cardiovascular disease in companion animals also shows a trend of increasing year by year.Previous studies have shown that aspirin eugenol ester（AEE）has a definite therapeutic and preventive effect on hyperlipidemia in rats.In this study,Apo E^-/-mice were used as animal models of hyperlipidemia,and the metagenomic changes in the protein group,transcriptome,lipid metabolome of the mouse liver and cecal contents were analyzed using system biology methods,in order to clarify the regulatory mechanism of AEE in improving hyperlipidemia in mice.Furthermore,using HepG2 cells as the in vitro model of hyperlipidemia,and using molecular biology techniques such as protein western blot,RT-q PCR and gene knockout,the molecular mechanism and key way of AEE in improving lipid metabolism in hyperlipidemia mice were further explored and verified.1.AEE significantly reduced serum TC and LDL-C levels in hyperlipidemia mice.AEE can significantly up-regulate the enzyme activity of CYP7A1 in the liver and regulate the activities of APOA4,PLTP,AOPP and other enzymes,thereby promoting the metabolism of cholesterol in vivo and relieving the inflammatory state of hyperlipidemia mice,and improving the lipid metabolism disorder of hyperlipidemia mice.2.Compared with the HFD group,AEE significantly regulated the lipid content of LPC,PEs,SMs and TAGs.The accumulation of differential lipids suggested that AEE might play a role in regulating glycerophospholipid metabolism,fat digestion and absorption,sphingolipid signaling pathway,c AMP signaling pathway,NF-κB signaling pathway,and Th1/Th2 cell differentiation.3.Compared with the HFD group,AEE significantly improved the intestinal flora disorders in mice.Enrichment results of differential genes showed that the effect of AEE was mainly related to its regulation of lipid transport and metabolism,cardiovascular disease,immune disease,energy metabolism,unsaturated fatty acid biosynthesis,glycosphingolipid biosynthesis,insulin signaling pathway,and secondary bile acid biosynthesis.The results of a combination of lipidomics and metagenomics analyses showed that in the AEE group,the abundance of Verrucomicrobia,Verrucomicrobiales,Candidatus＿Gastranaerophilales,and Candidatus＿Melainabacteria was significantly positively correlated with the concentrations of SM（d18:1/18:0）and PE（16:0/18:1）.4.Transcriptomics research results have shown that compared with the HFD group,AEE can significantly regulate lipid and atherosclerosis pathways,bile acid efflux pathway,energy metabolism and other related pathways.Further verification results showed that AEE could significantly regulate the m RNA expressions of genes related to lipid metabolism and inflammation,such as FXR,CYP7A1,slco1a1,pla2g4a,SQLE,IL18B and IL18R3P in liver tissue,and finally achieve the purpose of relieving hyperlipidemia in mice.5.The results of protein omics showed that compared with the HFD group,AEE significantly regulated bile acid efflux pathway,lipid and atherosclerosis pathway,and immune-related pathway.Further validation showed that AEE significantly increased the protein level of CYP7A1 in liver tissue to promote cholesterol metabolism.6.The results in vitro showed that AEE could significantly reduce the levels of TC and TG in high-fat HepG2 cells and reduce lipid deposition.Molecular biology results showed that AEE could regulate the expression of related genes in FXR-SHP-CYP7A1 and FGF19-TFEB-CYP7A1 pathways and significantly up-regulate the protein expression of CYP7A1 to promote cholesterol metabolism,and finally achieve the purpose of improving the high-fat state of HepG2 cells.In summary,the effect of AEE in improving hyperlipidemia is related to its efficacy in regulating glycerophospholipid metabolism,lipid and atherosclerosis pathways,and bile acid metabolism-related pathways,as well as up-regulating the activity of CYP7A1,the rate-limiting enzyme of cholesterol metabolism.It was further speculated that CYP7A1-related lipid pathways were the key pathways for AEE to play a role in reducing blood lipid.