T Cell Immune Globulin Adhesion Molecule-3 Regulates The Production Of Nitric Oxide In Endothelial Cells

BackgroundT cell immunoglobulin and adhesion molecule-3(Tim-3)is an immune molecule widely expressed on various cell surfaces and has multiple regulatory functions.It is expressed in Th1,CD8~+T cells,Th17,Treg,Monocytes,macrophages,dendritic cells,mast cells,glial cells and endothelial cells.Tim-3 is involved in a variety of pathologic processes,including infectious diseases,tumors,autoimmune diseases,transplant rejection and so on.What's more,subsequent research results showed that ischemic postconditioning can reduce the incidence of infarction by suppressing the Tim-3 signaling pathway.Limb remote pretreatment can also affect the transmission of Tim-3 signaling pathway,and play a role in reducing brain damage.Thus,Tim-3 may play an immunoregulatory function in the pathophysiology of a variety of cardiovascular diseases.Cardiovascular and cerebrovascular diseases to ischemic or hemorrhagic heart,brain and systemic tissues caused by hyperlipidemia,blood viscosity,atherosclerosis,hypertension etc.Cardiovascular disease is the most common worldwide disease,a serious threat to human health,it not only led to one-third of the world's death,but also lead to varying degrees of disability in patients.Atherosclerosis(AS)is the main reason of coronary heart disease,cerebral infarction and peripheral vascular disease.The study found that the occurrence of cardiovascular disease is related to endothelial cell dysfunction,and endothelial dysfunction is closely related.to nitric oxide(NO)-mediated vascular dilatation.Nitric oxide synthase(NOS)protein family plays an important role in vascular physiology and is involved in a variety of pathologic processes.NOS has three subtypes:Neurotropic NOS(nNOS),endothelial NOS(eNOS)andinducible NOS(i NOS),in which nNOS and eNOS are expressed consistently.Does Tim-3 regulate the expression of iNOS in endothelial cells and participate in the pathogenesis of atherosclerosis cardiovascular disease?These question and the exact mechanisms remain to be elucidated..ObjectivesTo investigate the effect of Tim-3 on the synthesis of NO in vascular endothelial cell line Ea.hy926,and to explore the role of Tim-3 in the pathogenesis of atherosclerosis.We hope that the results of this study can provide a theoretical basis for the diagnosis and treatment of atherosclerotic disease.MethodsTo construct Tim-3 knockdown and overexpression vascular endothelial cell line Ea.hy926 and determine whether the cell was constructed successfully from the protein level and mRNA level by flow cytometry and RT-qPCR.The Tim-3-Fc was used to block the Tim-3 signaling pathway in the Ea.hy926cell line,and meanwhile the cells were stimulated with LPS.The expression of iNOS mRNA was detected by RT-qPCR.The nitrate in the cell culture supernatant was detected by Griess assay.After the stimulation of LPS,the expression of iNOS mRNA in Tim-3 knockdown and overexpression vascular endothelial cell line Ea.hy926 was detected by RT-qPCR and the nitrite in the culture supernatant was detected by Griess.Results1.Flow cytometry,Western Blot and RT-qPCR results demonstrate the successful construction of stable Tim-3 knockdown vascular endothelial cell line Ea.hy926.2.Flow cytometry,Western Blot and RT-qPCR results demonstrate the successful construction of stable Tim-3 overexpression vascular endothelial cell line Ea.hy926.3.After the simulation of LPS,the expression of iNOS mRNA and the level of nitrite in the cell culture medium in Ea.hy 926 cell line were both decreased when the the Tim-3 signaling pathway was blocked.4.After the simulation of LPS,the level of iNOS mRNA expression in Ea.hy 926 cell line with low expression of Tim-3 was lower than that of control group,and the nitrite level in cell culture medium was also relatively low.The level of i NOS mRNA expression in Ea.hy 926 cell line with high expression of Tim-3 was higher than that of control group,and the level of nitrite in cell culture medium was also relatively high.ConclusionsTim-3 can affect the synthesis of iNOS and release of NO in endothelial cells.