| BackgroundCoronary atherosclerotic heart disease is one of the main causes of human death.Proliferation of vascular endothelial cells and Infiltration of inflammatory cells,which is related to the atherosclerosis of coronary vascular, resulted in myocardial ischemia and anoxia or necrosis caused by the stenosis or obstruction of vascular.The clinical symptoms are significantly correlated with the degree of disease, and the patients could show precordial discomfort, palpitation, angina, even heart failure and sudden death. Interleukin 8 (IL-8) is a kind of cell derived cytokines secreted by infiltrating mononuclear cells, macrophages and activated endothelial cells, which further causing neutrophil aggregation and inducing the expression of adhesion molecules and chemokine to defend pathogens and tissue injury.Studies showed that the level of IL-8 in peripheral blood of patients with coronary heart disease was significantly higher than that of healthy controls, and the patients with acute phase were higher than those in remission period. The expression level of IL-8 was positively correlated with the degree of coronary artery stenosis. Vascular cell adhesion molecule -1 (VCAM-1) and intercellular adhesion molecule -1 (ICAM-1) is an important member of the family of cell adhesion molecules, which mediate interactions between cells, cells and extracellular matrix, regulation of cell proliferation, migration and participation function. The results showed that the expression of cell adhesion molecules increased in CHD patients, and the follow-up mortality was significantly higher than that of normal adhesion molecules. Our experiment using human umbilical vein endothelial cell line (HUVEC) as a model to analyze the effect of IL-8 on the secretion of inflammatory cytokines of vascular endothelial cells, such as IL-2, IL-4,IL-6, IFN-y and TNF- a. At the same time, we analyzed the effect of IL-8 on HUVEC to express cell adhesion molecules. By blocking IL-8R, we further explored the mechanism of IL-8 affecting the function of vascular endothelial cells, which may provide a theoretical basis for the pathogenesis of coronary heart disease and blocking the IL-8/IL-8R pathway may be an usefull treatment to coronary heart disease.ObjectiveHuman umbilical vein endothelial cell line (HUVEC) was used in this experiment as a model to analyze the effect of IL-8 on a variety of vascular endothelial cells and the secretion of inflammatory cytokines, then we analysis of the influence of IL-8 on HUVEC expression of cell adhesion molecules. And by blocking IL-8R, we further explore the mechanism of effect of IL-8 on the function of vascular endothelial cells. This will provide a theoretical basis for understanding the pathogenesis of coronary heart disease and for the clinical application of IL-8 antibody or IL-8R antibody to treat coronary heart disease. Methods1. Detect the proliferation of HUVEC cells stimulated by IL-8: HUVEC cells were cultured with 1 ng/ml?2ng/ml?4ng/ml?8ng/ml?16ng/ml IL-8, and after 24h,the proliferation of HUVEC cells was detected by MTT.2. Detection of the inflammatory cytokine secretion in HUVEC cells by ELISA:HUVEC cells were stimulated by 4ng/ml IL-8, and and after 24h, the inflammatory cytokine secretion, such as IL-2?IL-4?IL-6?IFN-yand TNF-a, were detected by ELISA.3. Detect the expression of adhesion molecule in HUVEC cells: The expression of VCAM-1 and ICAM-1 protein in HUVEC cells dealt with IL-8 was detected by immunofluorescence confocal microscopy.4. IL-8R antibody inhibits inflammatory cytokine secretion in HUVEC cells:After adding IL-8 into the culture system, IL-8R antibody was added to block IL-8R and block the binding with IL-8. The concentration of IL-2, IL-4, IL-6, IFN- and TNF-were detected by ELISA after stimulation.5. IL-8R antibody inhibited the expression of adhesion molecule in HUVEC cell:after adding IL-8 into the culture system, IL-8R antibody was added to block IL-8R and block the binding with IL-8. The expression of VCAM-1 and ICAM-1 in HUVEC cells was detected by immunofluorescence confocal microscopy.6. Statistical methods: The measurement data to the number of addition and subtraction standard deviation (means±s), the difference between the two groups using t test. P<0.05 means statistically significant.Results1.IL-8 stimulated proliferation of HUVEC cells: HUVEC cells were cultured with different concentrations of IL-8, such as 1ng/ml, 2ng/ml, 4ng/ml, 8ng/ml,16ng/ml. When the concentration of IL-8 was less than 8 ng/ml, the proliferation of HUVEC cells was rise along with the increase of IL-8 concentration.2. IL-8 stimulated cytokine secretion of HUVEC cells: After stimulated by IL-8,IL-4 and IL-6 concentration in the culture supernatant were (1.62±0.33) ng/ml and(4.23±1.03) ng/ml, which were higher than the control group and there was significant difference. While the concentration of IL-2, IFN-?, TNF-a was(0.54±0.16), ng/ml (0.88±0.22) ng/ml and (0.36 ±0.10) ng/ml, respectively, and there was no significant difference compared with the control group.3. IL-8 stimulated the expression of adhesion molecules of HUVEC cells: The expression of VCAM-1 and ICAM-1 on the surface of HUVEC cells increased after IL-8 stimulation.4. IL-8R antibody inhibits the inflammatory cytokine secretion of HUVEC cells:After being blocked by IL-8R antibody, the supernatant of IL-4 and IL-6 were(0.79±0.10) ng/ml and (1.52±0.12) ng/ml, which were lower than those without IL-8R antibody group with the concentration of IL-4 and IL-6 were (1.62±0.33)ng/ml and (4.23±1.03) ng/ml, and there was significant difference. The concentrations of IL-2, IFN-? and TNF-? after the addition of IL-8R antibody were(0.36±0.06) ng/ml, (0.81 ±0.12) ng/ml and (0.31 ±0.14) ng/ml, and there was no significant difference between the two groups.5. IL-8R antibody inhibits the expression of cell adhesion molecules HUVEC cells: After being blocked by IL-8R antibody, the relative fluorescence intensity of VCAM and ICAM were (15.71 ±5.17) and (34.26±5.34), respectively. In the group without IL-8R antibody, the relative fluorescence intensity of VCAM and ICAM were(103.11 ±11.53) and (61.99±9.36), respectively. The expression of adhesion molecules was increased in the latter group compared to the before group, and there were significant difference.Conclusions1.1. IL-8 can stimulate the proliferation of human umbilical vein endothelial cell line (HUVEC), and promote the secretion of inflammatory factors IL-4 and IL-6,and promote the expression of adhesion molecules VCAM-1 and ICAM-1.2. IL-8 mainly affects the function of HUVEC cells by binding to IL-8R receptor, which can inhibit the secretion of IL-4, IL-6 and the expression of VCAM-1 and ICAM-1 in HUVEC cells by blocking IL-8R antibody. |
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