The Effect Of Simvastatin On The Expression Of Tribbles In Human Umbilical Vein Endothelial Cells With LPS Treatment

ObjectiveAtherosclerosis is a kind of chronic inflammatory disease,lt is resulted from the effect of vascular endothelial cells,monocytes,vascular smooth muscle cells and inflammatory factors.There are monocytes,macrophages and T lymphocytes in the plaque.Vascular endothelial cells play an important role in the process of atherosclerosis development.Abnormal substances in the blood may be a direct impact on vascular endothelial cells,because vascular endothelial cells directly contact with blood. When vascular endothelial cells are stimulated,they can secret some factors that can promote the adhesion of leukocytes,the proliferation and migration of vascular smooth muscle cells.Tribbles,which were first found in Drosophila melanogaster,are kinds of protein kinase that can block cell mitosis,and also can be called SINK.There are three members in mammalian,including Trb1,Trb2, Trb3.Tribbles have a same central domain,70-100 amino acids sequences in N-segment,25 amino acids in C-segment.The central domain is like serine/threonine kinase but lacks lysine active site,so they can regulate the protein kinases.Tribbles can regulate the expression of MAPKK,and then regulate the phosphorylation of MAPK.The signal pathway of MAPK is related with transcription factors NF-κB and AP-1.LPS is a kind of classic inflammatory stimulus.LPS can activate vascular endothelial cells and promote the expression of NO,oxygen free radical,chemokine,cytokine,prostaglandin and so on.The inflammatory factors can damage cells,increase the gap of endothelial cells,expose the vascular basement membrane and promote the migration of neutrophil and monocyte.LPS activates the signal transduction pathways of MAPK and NF-κB in vascular endothelial cells through TLR-4.Statins are potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase.This kind of drugs is capable of lowering the serum levels of cholesterol and is successfully used to treat hypercholesterolemia and atherosclerosis.In addition,the anti-inflammatory effect of statins has been reported.This effect can be reached through the signal pathway of MAPK and NF-κB.This research used LPS as a inflammatory treatment and simvastatin as a protect drug,then observed the expression of Tribbles mRNA in HUVEC and the effect of simvastatin on them.Method1.Cell culture:Collect the fresh umbilical core,cultivate HUVEC, identified,passage,the cells of 2-3 era could be used.2.Cell division:The cells are divided into 12h,24h groups in random,and then are divided into four groups respectively:①cntrol group;②LPS group(100ng/mL);③simvastatin group(5μg/mL);④LPS and simvastatin group.The supernatant is poured and the cells are preserved in -80℃.3.The expression of IL-6,TNFαand Tribbles mRAN of HUVECs are detected by the real-time quantitative PCR(RT-PCR).4.The experiment above are repeated six times,then use the SPSS statistics software to do the statistical anlysis.Result 1.The identification of HUVEC:The morphology identification in the microscope,the endothelial cells distribute equably,grow in monolayer, polygon and cobblestone-appearance.FactorⅧantigen SABC detection,in the microscope,there are brown granules in the cytoplasm.2.The result of real time quantitative PCR:Compared with control group,the expression of IL-6,TNFα,Trb1 and Trb3 mRNA in HUVEC are significantly increased in LPS group(P＜0.05).Compared with LPS group, the expression of IL-6,TNFα,Trb1 and Trb3 mRNA in HUVEC are reduced significantly in simvastatin and LPS group(P＜0.05).Conclusion1.The expression of IL-6,TNF-α,Trb1 and Trb3 mRNA in HUVEC can be increased by LPS.2.Simvastatin can inhibit the expression of IL-6,TNF-α,Trb1 and Trb3 mRNA in HUVEC with LPS treatment.3.Trb1 and Trb3 can promote the apoptosis of HUVEC with LPS treatment.