Study On Effect Of COX-2 Inhibitor Celecoxib On Atherosclerosis And Its Inflammatory Mechanism

Objective:To establish the AS lesion model caused by the ApoE^-/-mice associate with a western diet and the mice RAW264.7 macrophage inflammatory model induced by LPS;And then,based on these two AS model,choosing the up-regulation of leukotrienes（LTs）as breakthrought point,to investigate the effect and possible mechanism of celecoxib,a selective inhibitor of COX-2,on AS and its inflammatory response at the whole and cellular level,in order to provid the new theoretical basis and new ideas for the rational use of COX-2 on AS and the treatment and prediction of AS-related cardiovascular risk.Methods:1.After the two weeks of adaptive feeding,twenty-eight6-week-old male ApoE^-/-mice on a C57BL/6 background were used as model group and fed on a western diet（normal chow+21%fat+0.15%cholesterol）,another fourteen 6-week-old male C57BL/6 mice were used as normal control group and fed on a normal chow.The model group was further randomly divided into（1）AS model group;（2）celecoxib group（80mg/kg/d,ig）,fourteen miceineachgroup.At10weeks（18-week-old）,lowdensity lipoprotein-cholesterol（LDL-C）,high density lipoprotein-cholesterol（HDL-C）,total cholesterol（TG）and triglyceride（TC）)in serum and the whole aorta oil red staining were detedted in 4 mice.At the end of 18 weeks（26-week-old）,the remaining mice were sacrificed,the serum levels of LDL-C,HDL-C,TG and TC were measured,and the full-length aorta oil red O staining was made to mesure the positive plaque area ratio（%）by by image analysis.Hematoxylin-eosin（HE）stain of aortic roots were also made to observe the formation of atherosclerotic plaques and the aortic plaque area ratio（%）and aortic intima-media thickness（IMT）were quantitatively analyzed by using computer image analysis software.Contents of prostaglandin E₂（PGE₂）,Tumor necrosis factor-α（TNFα）,LTs,including Cysteinyl leukotrienes（CysLTs）and leukotriene B₄（LTB₄）,in mice aortic homogenate were measured by ELISA method.2.To establish the RAW 264.7 macrophage inflammatory model,mice RAW264.7 macrophages were cultured and stimulated with 1μg/ml LPS for 24hours.Cells were pre-incubated with Celecoxib（40,30,10,8,3,1,0.3,0.1,0.03μmol/L）for 1 hour and then stimulated by 1μg/ml LPS for 24 hours.MTT assay was used to detect the toxicity of celecoxib on mice RAW 264.7macrophage induced by LPS in order to determine the concentration of celecoxib.The mice RAW 264.7 macrophages were then divided into normal control group,LPS model group（DMSO+LPS）and celecoxib pre-treatment group（celecoxib+LPS）.In LPS model group（DMSO+LPS）and celecoxib pre-treatment group（celecoxib+LPS）,the macrophages were pre-treated with celecoxib（8μmol/L）or DMSO for 1 hour respectively,and then further treated with 24-hour 1μg/mL LPS stimulation.The cells in normal control group were treated with serum-free DMEM as the control.Then,the contents of NO（by Griess Reagent）,TNF-α,PGE₂,CysLTs and LTB₄（by Elisa Kit）in the cells and medium were detected by the assay kit.Results:1.Compared with the normal control group,the levels of LDL,HDL-C and TC in the AS model group were significantly higher than those in the control group（P<0.05 or P<0.01）,and there was no significant change in TG（P>0.05）.The aortic plaque area ratio（%）stained by oil red O in aortas of the mice in AS model group was significantly increased（P<0.01）.The results of HE staining showed that aortic plaque area ratio（%）and the IMT in AS model group were significantly higher than those in normal control group（P<0.01）.There is no significant difference in blood lipids between AS model group and celecoxib group at 10-week and 18-week feeding（all P>0.05）.Compared with the AS model group,positive plaque area ratio（%）stained by oil red O in aortas of the mice in celecoxib group was significantly increased（P<0.01）.The results of HE staining showed that aortic plaque area ratio（%）in celecoxib group were significantly higher than those in AS model group（P<0.01）,while the IMT had no difference between celecoxib group and AS model group（P>0.05）.The levels of PGE₂ and TNFαin the aorta homogenate of the celecoxib group were significantly lower than those in the AS model group（P<0.01 and P<0.05）,while the CysLTs and LTB₄ were significantly higher than those in the AS model group（both P<0.01）.2.Compared with the normal control group,obvious cellular morphologic change and the increase of NO,TNF-α,PGE₂ and CysLTs were observed on mice RAW 264.7 macrophages after stimulated by LPS for 24 h,indicating that mice RAW 264.7 macrophage inflammation model induced by LPS was successfully established.Compared with LPS model group,the release of inflammatory cytokines NO,TNF-αand PGE₂ induced by LPS were significantly inhibited in celecoxib pre-treatment group（8μmol/L）（all P<0.01）,while the contents of CysLTs and LTB₄ were up-regulated（both P<0.01）.Conclusions:1.ApoE^-/-mice associate with a western diet for 18 weeks can cause obvious AS lesions.Intragastric administration of celecoxib（80mg/kg/d）for 18 weeks significantly increases ApoE^-/-mice aortic AS lesions,the mechanism is independent of blood lipids,but may be associated with the up-regulation of aortic LTs.2.The mice RAW264.7 macrophage inflammatory model induced by lipopolysaccharide is successfully established.Celecoxib（8μmol/L） significantly inhibits the secretion of inflammatory cytokines NO,TNF-αand PGE₂,but up-regulate the release of CysLTs and LTB₄.