| Purpose Atherosclerosis is a chronic inflammatory disease in artery wall, which involves the comprehensive effect of vascular endothelial cells, smooth muscle cells, monocytes and inflammatory cytokines. Monocytes, macrophages and T lymphocytes are the three predominant cell types in the plaque. Vascular endothelial cells play a crucial role in the development of atherosclerotic plaque. Substances such as ox-LDL exert a direct effect on vascular endothelium which get in straight touch with blood. Stimulated by certain molecules, endothelial cells secrete various cytokines that promote adhesion of leukocytes, and proliferation and migration of smooth muscle cells. Sufficient evidence has proved local activation, recruitment and adhesion of monocytes to be one of the earliest reactions detectable within the lesion of atherosclerosis, which enhances the inflammatory reaction and the development of atherosclerosis.LPS is a pro-inflammatory cytokine which stimulates endothelial cells and leukocytes to release a series of inflammation mediators including NO, chemotactic factors, oxygen derived free radicals, cytokines, prostaglandin and etc. These molecules perform their function of aggravating inflammatory reaction by the autocrine or paracrine pathway. With their existence, intercellular space is expanded and basal lamina of blood vessels is exposed which consequently facilitate neutrophils and monocytes to migrate beneath endothelium and therefore intensify inflammation.Statins are inhibitors of HMG-CoA. They can lower the cholesterol level of blood serum and are used as common medicine against atherosclerosis. Recent researches have demonstrated that statins also perform other functions including ameliorating function of endothelial cells, facilitating fibrinolysis and anti-thrombosis, which markedly ameliorate atherosclerotic lesions and lower the attack rate and death rate of cardiovascular diseases. However, the mechanism of Simvastatin relieving inflammatory reaction of vascular endothelium remains unknown.We used LPS as stimulant and Simvastatin as protector. MCP-1, IL-1 and IL-6 were examined in human umbilical venous endothelial cells on both protein and mRNA level.Method 1. Cell culture:Vascular endothelial cells were collected from umbilical vein of fresh umbilical cord, cultured, identified and passaged. Cells in the 3rd generation were used in the experiment.2. Grouping:We divided the cells into four groups:the control group, the LPS group (100 ng/mL), the Simvastatin group (5μg/mL), and the Simvastatin (5μg/mL) and LPS (100 ng/mL) co-effect group.3. Supernatant was collected and identified for IL-1, IL-6 and MCP-1 by the method of Elisa.4. mRNA was extracted from the cells and real time-PCR was done to examine the expression of IL-1, IL-6 and MCP-1.5. The experiments were repeated 6 times and analyzed.Results 1. Identification of umbilical venous endothelial cells:under a contrast phase microscope, endothelial cells were polygon in shape and arranged in a cobblestones pattern, and formed a single-layer structure that evenly distributed. Stained by SABC method, brown granules were observed in the plasma of the cells, indicating that they were human umbilical venous endothelial cells.2.ELISA: compared with control group, the supernatant of LPS treated group contained markedly increased amount of IL-1, IL-6 and MCP-1 (P<0.01); in contrast to LPS group, the supernatant of Simvastatin and LPS co-effect group contained lower amount of IL-1, IL-6 and MCP-1 (P<0.05).3. Real time-PCR:compared with control group, LPS stimulated cells expressed high level of IL-1, IL-6 and MCP-1 (P<0.01); however, in Simvastatin and LPS co-effect group, the expression of IL-1, IL-6 and MCP-1 was remarkably lower (P<0.05).Conclusion 1. LPS stimulates human umbilical venous endothelial cells to express IL-1, IL-6 and MCP-1.2. Simvastatin inhibits the synthesis and secretion of IL-1, IL-6 and MCP-1 in HUVECs. Simvastatin plays an important role in delaying the process of AS. |
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