Role Of NF-κB Signaling Pathway In H₂O₂-induced Endothelial Apoptosis And Effects Of Simvastatin

Background: Atherosclerosis is a key pathophysiology of a variety of diseases. Present studies have found a series of factors that lead to it. There are a lot of hypotheses about atherosclerosis as follows: lipid infiltration hypothesis, platelet aggregation (PA) thrombosis hypothesis, vascular smooth muscle cell (VSMCs) clone hypothesis and response-to-injury hypothesis. On the one hand artery intima is a barrier between blood and the vessel wall, on the other hand it is an endocrine organ. In 1973 Ross and Glomseti raise response-to-injury hypothesis for the first time. More and more scholars dedicated to the study of vascular endothelial function who think that the destruction of the intimal integrity is the final results that are caused by atherosclerosis factors. Failure of tunica intima's integrity, endothelial permeability increasing, the dysfunction of the endothelial and atherosclerosis starts. Studies have confirmed that the ultrastructure has changed before intimal thickening and endothelial dysfunction occurred. Imbalance of vasodilator substance such as carbon monoxide (NO) and endothelin-derived shrinkage factor like endothelin-1 (ET-1) vasodilation weakened. Endothelial dysfunction promots the lipid and inflammatory cell infiltration, lipid oxidation, inflammation and smooth muscle cells (SMC) proliferation, extracellular matrix deposit or dissolved, platelet activation and thrombosis, then atherosclerosis begins.Apoptosis is a course of programmed cell death and the intima keep normal by balancing apoptosis and proliferation. Too many endothelial cells apoptosis may undermine the integrity of the intima, reflect effects of endothelial function. NF-κB is a transcription factor, generally exists in multicellular organisms, which is a variety of biological phenomena of core link. It plays an important role in cell proliferation, differentiation, apoptosis, cancer and many other biological events. Though there are more and more studies about mechanisms of NF-κB signal transduction, many mysteries need to be solved. PKC is one of the important cell signaling molecules, which plays important roles in apoptosis, proliferation, differentiation, and other biological events. PKC signal pathway plays opposite biological activity rely on the basis of the stimulus, different cells and different signal transduction pathways. As a complex mechanism, it increases difficulty about the study. Syk is a non-receptor tyrosine kinase, main features in participation and regulation of cell activation, proliferation-related gene expression. Existing literature reports that Syk plays an important role in the process of endothelial cells proliferation,which has a close relationship with NF-κB signal pathway.Simvastatin is 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors reductase inhibitor,which is clinically used as one of the lipid-lowering drugs. Simvastatin also acts against arteriosclerosis, stable atherosclerotic plaque and protect vascular endothelial, anti-inflammatory, but its specific mechanism need further research.Objective: To study the role of NF-κB signal transduction in H₂O₂-induced human umbilical vein endothelial cells apoptosis and the mechanisms of H₂O₂-induced apoptosis. Observe influences of PKC inhibitor and Syk inhibitor in H₂O₂-induced HUVECs apoptosis. Discuss the mechanism of endothelial dysfunction and search for the theoretical basis for atherosclerosis. Observate the role of simvastatin on H₂O₂-induced HUVECs apoptosis and perfect mechanisms of statins in atherosclerosis.Methods: Establish the H₂O₂-induced HUVECs apoptosis model. Observe the role of NF-κB, PKC and Syk signal transduction in the endothelial cell apoptosis interventing by different blockers. Adding different concentrations of simvastatin, explore the effects and to investigate the function of ralative signal transduction pathway. Do as follows:(1)Test the cell proliferation by MTT methods.(2)Detect the cells apoptosis rate by TUNEL.(3)Test NF-κB p65 protein content of all groups by Western blotting. All the data was analysed by the statistical processing of SPSS 13.0.Results: Part one: Draw the growth curve of human umbilical vein endothelial cells cultured in vitro. It is a logarithmic growth stage of HUVECs during the first 4 days cultured in vitro and then the platform stage comes. Part two: Eestablish H₂O₂-induced apoptosis model of HUVECs and the results reflect that H₂O₂ at a concentration of 200umol/L lasting for 6 hours is reasonable. Part three: Test the cell proliferation after joining different inhibitors using MTT methods. The results reflect that cell proliferation noticeable decline interventing by PKC inhibitor while the proportion of positive apoptotic cells has markedly increased comparison with the model group. The NF-κB p65 protein expression are clearly down-regulated in apoptotic cells, comparison with the model group and there are significant differences. Part four: Simvastatin significantly increases H₂O₂-induced endothelial cell apoptosis comparison with the model group. As the concentration of simvastatin grow the apoptosis rate increase. Group of high level of simvastatin expresses less NF-κB p65 conparing with the model group.Conclusion: (1) NF-κB signaling pathway plays a key role in HUVECs proliferation and apoptosis. (2)H₂O₂ inducts apoptosis of HUVECs throuh NF-κB signaling pathway while down-regulates expression of NF-κB p65 protein. (3)PKC and Syk signaling pathway participate in endothelial cell apoptosis process while PKC blockers and Syk blockers promote HUVECs apoptosis. They both suppress NF-κB signaling pathway. (4)Simvastatin increases H₂O₂-induced HUVECs apoptosis and suppresses NF-κB p65 expression.