| BackgroundCilostazol as an antiplatelet agents was synthesized by Otsuka Pharmaceutical Co. in1978. It was as the treatment of the chronic arterial occlusive diseases on the market in Japan in1988. With the large-scale evidence-based medicine confirmed that cilostazol can improve the symptoms such as ulcers,pain,chills and intermittent claudication which are caused by chronic lower extremity arterial occlusive disease. This antiplatelet agents has been approved as the first one for use in treating intermittent claudication by the US Food and Drug Administration on the market. It was also used in the clinical treatment of diabetes-induced peripheral vascular disease.Cilostazol is a specific inhibitor of phosphodiesterase III. Its principal actions are associated with its ability to elevate intrecellular adenosine3’,5’-cyclic monophosphate (3’,5’-cAMP) levels, and include the inhibiton of platelet aggregation and enhancement of the microcirculation via vasodilation. In recent years, it was found that cilostazol has the effects of anti-inflammatory and anti-proliferation in the smooth muscle cells. The application of cilostazol after stent implantation reduces neointimal hyperplasia and remodeling, has an advanced effect of anti-restenosis.We believe that these results complement the antiplatelet effect and make this agent unique in the management of ischemic disease.Effect on atherosclerosis in the vascular endothelial cellsAtherosclerosis is a common cause of ischemic heart and cerebravascular diseases,has been serious harm to human health.It was thought that AS is the result of dyslipidosis. However, with the further research,most scholars consider that AS is the performance of vascular imflammatory. They believe that AS is the response of inflammatory after dysfunction of vascular endothelial cells and the process of the damage-repair.Vascular endothelial cells as a mechanical barrier to the blood vessel wall, is a highly active and the largest endocrine organ between the blood and tissue boundaries, which with the effects of cell permeability, selective barrier, hemostasia, anticoagulation, fibrinolysis, blood transport and metabolism of vasoactive substances and regulation of vascular tone, producing the growth factors, hyperplasia of fier matrix, etc; take part in inflammation, effecting angiogenesis, vascular permeability and fluid balance. In the early stages of atherosclerosis, the site of injury activated endothelial cells secrete adhesion molecules and chemokines, such as vascular cell adhesion molecule-1(VCAM-1) and monocyte chemoattractant protein(MCP-1), leading to immune cells and mononuclear cell aggregation and migration to the vessel wall intima. VCAM-1in the early atherosclerotic plaque formation plays a major role, and MCP-1is a chronic inflammatory response of the media, the two lead to monocyte adhesion. In addition, reactive oxygen species and TNF-a are also involved in the formation of atherosclerosis. Some studies showed that the expression of the nuclear factor--κB (NF-κB) activation of genes in human atherosclerosis increased.As a new phosphodiesterase3inhibitor, the fundamental role of cilostazol is anti-platelet aggregation. Its effects of antiflammatory and against atherosclerosis are via many additional targets:inhibiting the activity of PDE and restraining hydrolysis of cAMP, preventing atherosclerosis, thrombosis and vascular occlusion, reducing the expression of adhesion molecules(such as VCAM-1), inhibiting cytokine release and onset(eg,MCP-l,PDGF and TNF-α,etc). Furthermore, it increases intracellular cAMP levels for vasodilation, promotes endothelial recovery and inhibition of angiogenesis, increases perfusion, play a protective effect for ischemic brain tissue. Studies indicated that cilostazol has-a significant protective effect on endothelial cells. MATSUMOTO et al showed that cilostazol improve the mesenteric artery endothelial cell function of rats with type2diabetes via animal experiments. Further studies showed that cilostazol resistance to endothelial cell apoptosis induced by LPS via the ERK1/2and p38MAPK pathway.In addition, cilostazol also inhibit inflammatory cell adhesion to endothelial cells and inhibit the expression of endothelial cell, MORI et al found that cilostazol upregulates monocyte cAMP concentration to inbibit its effect of adhesion to endothelial cells.Effect on vascular smooth muscle cellsVascular smooth muscle cells is a major component of the vessel wall, which is the important factor to determine its activity and configurations. The proliferation of VSMC play an important role in atherosclerosis, restenosis, hypertension and other vascular diseases. It is the most active cells in the process of atherosclerosis. Clinical and experimental studies have confirmed that cilostazol can inhibit VSMC proliferation and prevent coronary restenosis after interventional treatment. Cilostazol can inhibit different growth factors, including platelet-derived factor insulin and insulin-like growth factor-1induced rat aortic vascular smooth cell proliferation, and also inhibit the in vitro induced by PI3GF cultured human vascular smooth muscle cell proliferation. Recent studies also showed that plasma PAI-1level is related to coronary heart disease, intimal hyperplasia and restenosis. Cilostazol inhibit the vascular smooth cell proliferation via downregulating various mechanisms to inhibiting the expression of cytokines, including TGF-B, JNK and P38MAPK signal pathways.三、Effect on Mitogen-activated protein kinase (MAPK)Mitogen-activated protein kinase (MAPK) family are higly conserved serine/threonine protein kinase, is widely distributed in eukaryotic cytoplasm. MAPK signal pathway is a crucial way when extracellular-signal triggered cell nucleus reactions, it plays a significant role in the cell formation, differentiation, growth, proliferation and apoptosis. There are four pathways in these family:the extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38MAPK and ERK5/BMK1. The classic MAPK cascade consists of three active steps:MAPK kinase kinase (mitogen-activated protein kinase ki-nase kinase, MAPKKK) activiated and phosphorylation, it activated MAPK kinase(mitogen-activated protein kinase kinase, MAP-KK), then the dual phosphorylation of MAPKK activated by neighboring threonine and tyrosine. The activation of MAPK can be exported from cytoplasm to nucleus and acting on the target. Substantial evidence indicates that mitogen-activated protein kinase are also targets of cilostazol.PurposeAt present, Cilostazol is widely used as primary and secondary prevention in clinical vascular disease, including ischemic heart diseases and strokes, but also the basis for anti-platelet agents therapy. This agent has been shown to reduce atherosclerosis-related events in a multitude of clinical studies. Vascular endothelial dysfunction and abnormal proliferation and migration of vascular smooth muscle cells(VSMCs) play important roles in the physipathology of atherosclerotic diseases. The present study undertaken try to observe the effects of cilostazol on vascular endo-thelial cell and vascular smooth muscle cell proliferation in vitro and on the activity of P38Mito-gen-activated protein kinase(MAPK) phosphorylation, respectively, in order to reveal the possible protective mechanism of cilostazol on atherosclerosis.Methods1. EA.hy926cells line were passed on96well assay in vitro and stimulated by different level of cilostazol(10、30、100、300umol/l) for24hours, and were observed in comparison with the control group. The ratio of cell proliferation was determined by non-radioactive MTT assay.2. EA.hy926cells line were cultured in vitro, and treated with variosed and concentrations of cilostazol(10、30、100、300umol/l) for24hours, then EA.hy926cells were lysed and the concentration of protein was measured using Bradford method. The expression of p38MAPK phosphorylation protein was evaluated by the immunoblotting technique using anti-p38phospho-MAPK antibody.3. Rat aortic VSMCs were cultured in vitro and treated with various concentrations cilostazol(10、30、100、300umol/l) for24hours, and were observed in comparison with the control group. The ratio of cell proliferation was determined by non-radioactive CCK-8assay.4. Rat aortic VSMCs were cultured in vitro and stimulated by different level of cilostazol(10、30、100.300umol/l) for24hours, the Rat aortic VSMCs were lysed and the concentration of cilostazol of protein was measured using Bradford method. The expression of P38MAPK phosphorylation protein was evaluated by the immunobloting technique using anti-p38phospho-MAPK antibody.Results1. The ratio of endothelial cells proliferation was0.909±0.013in the control group, and was0.903±0.026,0.851±0.023,0.699±0.013and0.651±0.036in cilostazol groups corresponding to cilostazol concentrations of10,30,100and300umol/L respectively. It was shown that cilostazol significantly inhibits the vascular endotheliao cell proliferation when the concentration used was above30umol/L(P<0.05). Cilostazol produced a dose-dependent inhibition of vascular endothelial cell proliferation compared with the control group(P<0.05).2. Compared with the control group(OD100), there was no effection of10umol/L cilostazol on the expression of P38MAPK phosphorylation protein(OD96.3±5.9, P>0.05), the expression of P38MAPK phosphorylation protein was significantly inhibited by cilostazol in30、100、300umol/L, respectively(92.5±2.4,72.6±5.2,70.6±3.2, P<0.05).3. The ratio of VSMCs proliferation was0.835±0.022in the control group, and was0.829±0.019,0.706±0.106,0.605±0.003and0.581±0.032in cilostazol groups corresponding to cilostazol concentrations of10,30,100and300umol/L respectively. It was shown that cilostazol significantly inhibits the VSMCs proliferation when the concentration used was above100umol/L(P<0.05).4. Compared with the control group(OD100), There was not effection of10and30umol/L cilostazol on the expression of P38MAPK phosphorylation protein (OD99.7±2.3,99.8±3.7, P>0.05), the expression of P38MAPK phosphorylation protein was significantly inhibited by cilostazol in100and300umol/L,respectively (86.7±1.6,77.9±2.3, P<0.05). ConclusionOur study suggests that decreased p38MAPK activation contributes to inhibit VEC and VSMC proliferation by cilostazol,respectively. Arrest of endothelial cell and VSMC proliferation may be an important mechanism of the beneficial effect of cilostazol in ischemic heart and cerebravascular diseases. These observations indicate that P38MAPK inhibition can represent a novel therapy against atherosclerotic diseases. |
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