Advanced Glycation Endproducts Up-regulate Nuclear Factor-κB And Cyclooxygenase-2 In Rat Cardiac Microvascular Endothelial Cells

IntroductionNo enzymatic protein glycation by chronic hyperglycemia is a complex cascade of reactions yielding a heterogeneous class of compounds, collectively termed advanced glycation endproducts ( AGEs). AGEs are associated with, and may be a causal factor, in the development of vascular complications of diabetes. Recent in vitro and animal experiments suggested that AGEs and their receptor (the receptor for AGEs RAGE ) system take a crucial part in development of diabetic vascular complications. The engagement of RAGE by AGEs has been reported to active multiple signaling pathways. In these pathways, NF - κB and the activation of its downstream consequences have an important role in the development of macrovascular and microvascular damage. The mechanisms regulating NF - kB and COX - 2 expression in microvascular endothelial cells exposed to AGEs are unclear. Thus the experiment cultured rat cardiac microvascular endothelial cells (CMECs) in vitro, and investigate the influence brought by BSA - AGE on inducing the expression of NF - kB and COX -2. Further discuss the relationship between AGEs and diabetic vascular complications.MethodsFirstly, CMECs were cultured according to Nishida M. When CMECs reached a sub confluent, the stimulating factors were added. Group one, divided into five groups, normal control (serum - free DMEM cultured liquid with BSA) and BSA - AGE stimulating groups in varying concentrations ( 25 ,50,100,200mg/L) then acting 24h. Group two, divided into five groups, normal control ( serum - free DMEM cultured liquid with BSA) and the same concentration BSA -AGE (lOOmg/L) in varying stimulating time (6,12,24,48h) . The protein expression of NF - kB and COX - 2 were studied by Western Blot.ResultsThe experiment results had shown that, after treatment with BSA - AGE in varying concentrations, the protein expression of NF - kB and COX - 2 increased in a dose - dependent manner. Compared with controlled group, the protein expression of NF - kB was 1.99 - , 2.76 - , 3. 57 - , and 4. 25 - folds respectively (n=5,p<0.05);the protein expression of COX - 2 was 2. 39 - , 3.41 - , 4.49 - , 5. 34 - fold respectively ( n = 5 , p <0. 05). And after treatment with AGEs of the same concentration but at varying time, with the time prolonged, the protein expression of NF - kB and COX - 2 increased in a time- dependent manner. Compared with controlled group, the protein expression of NF - kB was 1. 83 - ,2. 66 - , 3. 52 - , and 4. 14 - folds respectively ( n =5 ,p<0. 05);the protein expression of COX -2 was 2. 18 - , 3. 21 - , 4. 33 - , 4. 99 - folds respectively (n=5,p<0.05).DiscussionAll forms of diabetes are characterized by chronic hyperglycemia and the development of diabetes - specific vascular complications. Morbidity and mortality in diabetes mellitus are caused chiefly by its vascular complications, both in the microcirculation and in large vessels. Reducing sugars such as glucose react non - enzymalically with amino groups in proteins, lipids and nucleic acids through a series of reactions forming Schiff bases and Amadori products to produce AGEs. AGEs may directly impact on the structural integrity of the vessel wall and underlying basement membrane. In addition other studies have shown that AGEs quench nitric oxide ( NO) and induce oxidant stress. The mechanisms that AGEs led to diabetic vascular complications are not fully understood,three pathways have been proposed :(T)the activation of multiple signal transduc-tion pathways in extracellular matrix ( ECM );(2) AGEs may also exert the expression of cytokines by engagement of cellular binding receptors;(3)many early glycosylation products that produced by glucose and fructose in cells could impair the function of proteins in the target tissue. In these mechanisms, the most important is the AGE - RAGE system. It is increasingly postulated that the inflammation may be possible pathogenetic links between diabetes and its vascular complications. The AGE - RAGE interaction induces an oxidant stress activates multiple signaling pathways, inducing the over expression of many proinflamma-tory cytokines, and results in endothelial dysfunction and vascular inflammation. More and more studies have implicated that NF - kB , which is sensitive to oxidant stress, is important in the development of both macrovascular and microvas-cular complications. Recently, many researches focus on the role of COX -2, an inducible enzyme regulated by NF - kB, in diabetic complications.The experiment cultured CMECs in vitro in order to discuss the influence brought by BSA - AGE on the protein expression of NF - kB and COX -2. Result had showed that with the increase of AGEs concentrations and adding time, the protein expression of NF - kB and COX - 2 were on rise, and the expression of COX - 2 was related to the activity of NF - kB. Researches indicated that AGEs stimulate COX - 2 expression through activating NF - kB and promote the development of vascular complications.Conclusions1. BSA - AGE can activate the NF - kB in microvascular endothelial cells in vitro and in certain rages showing a dose - dependent and time - dependeng manner.2. BAS - AGE can up - regulate the COX - 2 in microvascular endothelial cells in vitro and in certain rages showing a dose - dependent and time - dependent manner.