| Background:The prevalence of diabetes mellitus can cause a variety of vascular complications, such as diabetic nephropathy, atherosclerosis(AS), stroke, heart failure, and so on, which were the main causes for disability and even death. Endothelial cell dysfunction has been postulated to be an early signal of diabetes combined with AS, therefore, the protection of vascular endothelial cells plays an important role in diabetes treatment and prevention of vascular complications. Advanced glycation endoproducts also be critical in the occurrence and development of diabetic vascular complications. Studies have shown that a variety of abnormal cytokine secretion and metabolic disorder caused by AGEs is the main reason of inducing abnormal structure and function of endothelial cells. The large accumulation of AGEs accumulate with aging and abundantly increase while hyperglycemia, deposit on blood vessel wall, AGEs and RAGE interaction can activate the oxidative stress, induce the significant generation of ROS, destroy the functions of endothelial cells, lead to mitochondrial dysfunction and thus promoting its apoptosis. Therefore, How to combat the vascular damage caused by AGEs has been a concern of many scholars.As an important member of mitogen-activated protein kinase(MAPK) gene family, p38 MAPK signaling pathway play the role through phosphorylation(p- p38 MAPK). Studies have shown that activation of p38 MAPK signaling pathway is closely related to vascular injury and organ microcirculation changes. For example, AGEs can induce the renal microvascular endothelial damage and blood barrier via p38 MAPK. Inhibition of p38 MAPK activation can reduce oxidative stress of mesangial cells induced by high glucose in mesangial cells, and then reduce the production of ROS. Hyperglycemia can enhance the activity of NADPH oxidase in coronary artery, increase the level of ROS, moreover, both of hyperglycemia and ROS can induce cell injury by p38 MAPK phosphorylation. It has been reached consensus that high-glucose and ROS can promote apoptosis, but the mechanism is unclear. It is reported that a certain concentration of AGEs mediated downregulation of e NOS, weakened production of NO via p38 MAPK signaling pathway, thereby promoting apoptosis.Glucagon like peptide-1(GLP-1), a kind of Peptide hormone secreted by intestinal endocrine cells and nerve cells, is a new promising pharmaceutical in treating diabetes, which can lower blood glucose by promoting the insulin secretion and inhibiting the glucagon release, but also protect the cardiovascular system. In addition to the effect of anti-inflammation, GLP-1 can also perform the antioxidant function via c AMP/PKA,PI3K/Akt,NADPH,p38 MAPK signal pathway to protect endothelial cells effectively. Researches showed that, GLP-1 and GLP-1 simulation Exendin-4 stimulated proliferation of endothelial cells by promoting NO production through PKA-PI3K/Akt activation pathways. p38 MAPK-e NOS also played an important role in the protection of GLP-1 induced by high glucose in endothelial cells injury. But the related rearch is still poorly.Thus, in a variety of metabolic disorders of diabetes,the activation of oxidative stress of AGEs, is the key factor of vascular endothelial damage. When GLP-1 reduces the glucose level, at the same time, it can antagonize the oxidative stress induced by AGEs to protect VECs(vascular endothelial cells). However, its specific protection mechanism is not clear very much. With the human umbilical vein endothelial cells(HUVEC) induced by AGEs as the research model, this study discuss the Interventional mechanism of GLP-1 on the damaged endothelial cells induced by AGEs via the signal pathway of p38 MAPK, which can show a broad prospect for the studies on occurrence and treatment of disease.Objective:We aim to on the base of apoptotic model induced by AGEs, to investigate the effects of GLP-1 on p38 MAPK pathway and e NOS protein expression, observe the effects of GLP-1 or p38 MAPK inhibitor on ROS level and apoptosis of endothelial cells, to explore the mechanism of GLP-1 against cell damage via p38 MAPK signaling pathway.Methods:1. The separation, culture, identification of human umbilical vein endothelial cells(human umbilical vein endothelial cells, HUVECs) According to the experience, isolated and cultured HUVECs after acquisition, cells were identified using v WF antibody staining. Third-fifth generation of cells were taken for subsequent experiments. 2. Preparation and identify of AGEs in vitro BSA was dissolved in PBS with D-glucose, and incubated for 12 weeks in the dark. AGEs was identified using a fluorescence spectrophotometer. Without D-glucose in the same method, BSA was prepared as the negative control group. 3. Detection of p38 MAPK and e NOS phosphorylation by western blotting The protein expression of p-p38 MAPK and t-p38 MAPK were detected in negative control group, AGEs group, GLP-1 group, and AGEs+GLP-1 group. The protein expression of p-e NOS and t-e NOS were detected in negative control group, AGEs group, AGEs+GLP-1 group, AGEs+p38 MAPK group, and AGEs+GLP-1+p38 MAPK group. The cells were pretreated by p38 MAPK inhibitor for 1 h, then added(or without) GLP-1 100 n M to intervene it for 30 min, finally added AGEs for 24 h(This topic earlier research showed that 400 ug/m L AGEs significantly induced endothelial cells apoptosis when effected for 24 h. 100 n M GLP-1 could significantly inhibit cell apoptosis which induced by AGEs). BSA under the same concentration like AGEs was prepared as the negative control group.(The following parts would be in the same order of operation) 4. Detection of ROS generation, apoptosis of cells handled with GLP-1 and p38 MAPK inhibitor by flow cytometry Experimental groups: negative control group, AGEs group, GLP-1 group, AGEs+GLP-1 group, AGEs+p38 MAPK inhibitor group, AGEs+GLP-1+p38 MAPK inhibitor group. Cells were planted in 6-well plates, after each experimental treatment, To detect the level of ROS by DCFH-DA staining, apoptosis by Annexin V/PI staining. 5. Detect the generation of NO by nitric acid deoxidize enzyme method on AGEs and AGEs+GLP-1 group before and after adding e NOS inhibitor. 6. Detection of apoptosis by Annexin V/PI staining before and after adding p38 MAPK and e NOS inhibitor.Statistical processing:The data were statistically analyzed by using SPSS17.0, showed by mean ± standard deviation( ±s). And one-way analysis of variance(One-Way ANOVA) was used for multigroup comparison. Pairwise comparison used LSD method. P<0.050 was of statistical significance.Results:1. The effect of GLP-1 on p38 MAPK phosphorylation of HUVECs induced by AGEs It showed that, the protein expression of p-p38 MAPK were significant different between these groups(F=56.989,P=0.000), as t-p38 MAPK had no change(F=0.568,P=0.652). Compared with the control group, AGEs activated p38 MAPK phosphorylation(P=0.001), but it could be inhibited by GLP-1(P=0.000). 2. The effect of p38 MAPK inhibitor(SB203580) on e NOS phosphorylation of HUVECs The protein expression of p-e NOS were significant different between them(F=6.645,P=0.007), as t-e NOS had no change(F=1.677,P=0.231). Compared with the control group, AGEs inhibited e NOS phosphorylation(P=0.007).Compared with the AGEs group, both of GLP-1 and p38 MAPK inhibitor could restored the activation of p-e NOS(P=0.004)(P=0.011). 3. The effects of GLP-1 and p38 MAPK inhibitor on ROS generation and apoptosis of HUVECs induced by AGEs Compared with the BSA control group, AGEs increased ROS generation and apoptosis rate significantly( P=0.000)(P=0.000),GLP-1 did not effect ROS generation and apoptosis rate separately(P=0.170)(P=0.234). When added GLP-1 or SB203580 to AGEs group, the ROS generation attenuated significantly compared with AGEs treatment group(P=0.000)(P=0.006), while cell apoptosis rate was decreased significantly(P=0.000)(P=0.000). As pretreated with SB203580, the effect of GLP-1 on ROS generation and apoptosis of cells induced by AGEs did not change(P=0.828)(P=0.424). 4. The roles of GLP-1 and e NOS inhibitor on the level of NO when induced by AGEs After ANOVA analysis, compared with the control group, AGEs reduced the NO generation of cells significantly(P=0.000),when GLP-1 did not change it separately(P=0.055). GLP-1 restored NO synthesis, which was reduced by AGEs(P=0.000),but it could be inhibited by adding L-NAME in advance(P=0.011). 5. The effect of e NOS inhibitor on anti-apoptosis of GLP-1 in HUVECs The cells apoptosis rate of AGEs+GLP-1 group was lower than AGEs group(P=0.000), but it could be destroyed when pretreated with L NAME(P=0.002).Conclusion:1. AGEs can activate p38 MAPK signal pathway on endothelial cells, and GLP-1 can antagonize the effect. 2. GLP-1 can reverse the oxidation damage and apoptosis of VECs induced by AGEs via inhibiting p38 MAPK signaling pathway. 3. GLP-1 can reverse the NO synthesis disorder and cell apoptosis induced by AGEs through activating e NOS, and the effects of e NOS are involved in p38 MAPK signaling pathway. |
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