Mechanism Of AEE Inhibiting Cholesterol Metabolism Of Macrophage Foam Cells Induced By Ox-LDL

In recent years the number of pet owners in China has been increasing year by year.The existence of improper feeding and other wrong feeding methods in the process of pet ownership,coupled with the triggering of chronic diseases in old age,has greatly increased the risk of atherosclerosis in pets.Aspirin eugenol ester（AEE）is a non-steroidal drug synthesized in the laboratory,and preliminary studies have confirmed its good lipid-lowering,anti-inflammatory and other pharmacological effects.AEE has the potential to be developed as an anti-cardiovascular drug for veterinary use to meet clinical drug needs.This study was based on the Apo E^-/-mice atherosclerosis（AS）model.The biochemical detection,tissue Oil-Red O staining and tissue H.E.staining were used for exploring the intervention effect of AEE on AS plaque.The RAW264.7 macrophage-derived foam cell model induced by oxygenized low density lipoprotein（ox-LDL）was used for revealing the mechanism of AEE inhibiting of ox-LDL-induced macrophage foaming at the cellular level by molecular biological techniques such as q RT-PCR,immunofluorescence,ELISA and Western blotting.1.Taking C57BL/6J mice as the control group,Apo E^-/-mice were divided into the model group,the AEE H group,the AEE M group,the AEE L group and the atorvastatin group.The control group was given the standard diet,and the model group and the dosing group were given the high-fat diet.After 10 weeks of free feeding,the blood and heart-aortic samples of mice were collected for blood biochemical detection,tissue Oil-Red O staining and H.E.staining.The results demonstrated that the levels of total cholesterol and low density lipoprotein cholesterol in the blood of the model group were significantly higher than those in the control group.The area of aortic intimal plaque was increased significantly,the aortic arch intima was thickened significantly,and the lipid deposition in the cross-sectional section of the aortic root was manifest,indicating that the AS model in Apo E^-/-mice was established successfully.After AEE treatment,the above blood biochemical indexes and the histopathological alteration were improved significantly,indicating that AEE had a certain therapeutic effect on AS.2.In vitro foam cell models were established by ox-LDL induction of RAW264.7macrophages for 24 h,and the suitable induction concentration was screened by the BODIPY staining and the Oil-Red O staining.The results showed that the ox-LDL of 40～80μg/m L could induce cells to take up a grand amount of lipid and form foam cells.Finally,the 80μg/m L was selected as the induction concentration for subsequent trials.The results of CCK-8assay showed that after induction with ox-LDL for 24 h,the proliferative activity of AEE pretreated cells was up to 115.33%.The results of CCK-8 showed that compared with the control group without any drug treatment,after pre-treatment with different concentrations of AEE（80,160,320μM）for 24 h,the proliferative activity of RAW264.7 cell was 98.67%,100.33%,102%.The proliferation activity of cell pre-treated with AEE was up to 115.33% after being induced by ox-LDL for 24 hours.It was illustrate that AEE and ox-LDL were no toxic to RAW264.7 macrophages.3.After 80μg/m L ox-LDL induced RAW264.7 cells for 24 h,a large number of cells differentiated,the content of lipid droplets increased significantly,the uptake of ox-LDL increased significantly（P<0.01）,the excretion rate of cholesterol was reduced significantly（P<0.01）,and the content of the total cholesterol and free cholesterol in the cell were increased significantly（P<0.01）.CD36 and LDLR expression were increased significantly（P<0.01）,and the expression of PPARγ,LRX and ABCA1/G1 were decreased significantly（P<0.01）.After AEE pre-treatment,it inhibited the uptake of cholesterol by cell and promote cholesterol efflux,diminished the accumulation of intracellular lipids,and reverse the expression of the above genes.After pre-treatment with AEE,it could effectively inhibit the uptake of cholesterol and promote cholesterol efflux,reduce the accumulation of intracellular lipids,and reverse the expression of the above genes.4.The results of RAW264.7 cells and its culture supernatant reveal that AEE inhibited the secretion levels of the tumor necrosis factor-α（TNF-α）,Interleukin-1β（IL-1β）and Interleukin-6（IL-6）,and down-regulated the expression levels of NF-κB,i NOS,TNF-α,IL-1βand IL-6 during the formation of ox-LDL-induced foam cells.It showed that AEE played an anti-inflammatory role in the process of macrophage foam cell formation.5.AEE reduces the expression of NF-κB and the secretion of pro-inflammatory cytokines in cells,mainly by increasing the expression of PPARγin RAW264.7 cell,inhibiting the activation of NF-κB pathway,decreasing the production of pro-inflammatory cytokines TNF-α,IL-1βand IL-6,and reducing the inflammatory response in cells.In short,the effect of AEE in the treatment of AS is obvious,and its therapeutic effect is related to the reduction of total cholesterol and low density lipoprotein-cholesterol.AEE inhibits ox-LDL-induced foamy formation of RAW264.7 macrophage,mainly by down-regulating the expression of low density lipoprotein receptor（LDLR）and CD36 to inhibit macrophage uptake of cholesterol.AEE promotes the reverse cholesterol transport（RCT）through the PPARγ/LXR/ABCA1/G1 pathway,dwindling the accumulation of lipids in cells.And AEE inhibits the NF-κB pathway-mediated cellular inflammatory damage by increasing the expression of PPARγ,thereby inhibiting the foaming process of macrophages.