| Background and Objective: Athero-inflanimation appears to have a major role in the development and progression of atherosclerotic lesions, which is involved in the full- stage of atherosclerosis process, and provides link between different risk factors, morbidities, and clinical cardiovascular events. Insulin resistance is widely considered equivalence perilous with atherosclerosis, and a low level inflammatory status as well. Inducible cyclooxygenase (COX-2) and its prostaglandin productions play a central role in inflammation, and have been implicated in the pathophysiology of cardiovascular disease, thus might be a potential target to prevent and cure atherosclerosis and acute coronary syndrome. The distribution of COX-2 expression in human coronary arteries specimens from autopsy cases with different atherosclerotic lesions was evaluated to investigate the contribution of COX-2 to the development of atherosclerosis and subsequent risk of plaque instability. The rat vaascular smooth cell (VSMC) and human endothelial cell (EC) were isolated and cultured respectively, with insulin pre-treatment at specifically dose and time interval to investigate the effects induced by INS. NS-398, a selective COX-2 inhibitor was utilized to observe its influence to insulin intervention. In VSMC, the expression of COX-2 mRNA and protein induced by insulin, the activity of COX-2, and the role of involved mitogen-activated protein kinase (MAPK) pathway were detected respectively. EC were transfected with human COX-2 cNDA. After that, pre-treating with aspirin or NS-398, intercellular adhesion molecule-1 (ICAM-1) was measured to identify the anti-atherosclerosis potential of COX-2 inhibitor.Methods: 1, Human coronary arteries specimens (45) were divided into three groups according to pathologic features of HE staining, inflammatory cell containingof, and AHA definition. Among them were 3 healthy coronary arteries as control (no inflammatory cell), 22 fibrotic plaques (inflammatory cell<25%), and 20 complicated plaques (inflammatory cell = 25%). As of complicated plaques, middle (inflammatory cell between 25% and 50%) and severe inflammation (inflammatory cell =50%) groups were subdivided. Immunohistochemical staining was used for location of protein in different type of atherosclerosis lesion. COX-2 positive cells exceed 50% was definited as (+++), 6%-50% as (++), l%-5% as( +), non-staining as (-).2, The rat vascular smooth muscle cells isolated and cultured in 96 wells plate. Insulin (10, 100, 500, 1000U/L), NS-398 (1, 5, 10, 50, lOOuM), and both of them were added for 24, 48, and 72 hours. The proliferation of cells were evaluated by MTT method. Endothelial cells were cultured in 6 wells plate, with insulin (10, 100, 500, 1000U/L) co-incubation for 6 hours, 100U/L insulin for 1, 3, 6, 12 hours, insulin and NS-398 (lOuM) for 6 hours. The expression of intercellular adhesion molecular-1 (ICAM-1) in endothelial cells induced by insulin were measured by RT-PCR.3, Pretreated VSMC with insulin of special concentration and time interval, COX-2 mRNA and protein expression were observed respectively. As to the COX-2 enzyme activity, PGE2 released by VSMC were measured with an EIA kit. The effects of aspirin and NS-398 on insulin induced COX-2 production were analysis by Western-blot. PD98059(p42/44MAPK inhibitor), SB203580 (p38MAPK inhibitor) , and JUK/SAPK antibody assay kit were utilized to identified the intracellular signal pathway that implicated in COX-2 expression.4, After amplification and identification, pCMV-COX-2, a plasmid containing human COX-2 cDNA, were transfected into EC using Lipofectin transfection reagent. The expression of target gene and protein were measured by RT-PCR and Western-blot. The effects of aspirin and NS-398 on ICAM-1 mRNA expression were evaluated after transfection.Result: 1 COX-2 was found in the areas of plaques but not in healthy ornonatherosclerotic vascular walls, which were (-+) in fibrotic plaques, (+ ~ ++) inmilddle inflammation subgroup and (++~+++) in severe inflam... |
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