The Antagonistic Effect And Mechanism Of GLP-1 Against The Damage Of Vascular Endothelial Cells Induced By AGEs

BackgroundAdvanced glycation endoproducts (AGEs)play an important role in the occurrence and development of diabetic vascular complications.At a pathological concentration, AGEs induce the generation ofReactive oxygen species (ROS) via activating the oxidative stress, which lead to the dysfunction of endothelial cells. The new hypoglycemic drug, glucagon like peptide-1 (GLP-1), can not only improve the micro environment to protect endothelial cells indirectly by regulating blood glucose, but also improve the endothelialfunction directly through the receptor or non-receptor dependent pathwayto inhibit the occurrence and development of diabetic atherosclerosis.The previous studies have shown that GLP-1 could protect endothelial cells from hypercoagulable state and inflammation by lowering the level of plasminogen activator inhibitor 1 (PAI-1),intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1). Furthermore, GLP-1 was found to decreasethe level of ROS in endothelial cells in a dose-dependent manner, which induced by AGEs; Protein kinase A-Endothelial nitric oxide synthase(PKA-eNOS) signal channel plays an important role in GLP-1 inhibiting endothelial damage and improve endothelial proliferation. In addition, GLP-1 could also lower the oxidative damage of renal vascularendothelial cells by reducing the synthesis of Triphopyridine nucleotide (NADPH) through the Cyclic Adenosine monophosphate/Protein kinase A system(cAMP/PKA) signal pathway;ObjectiveThis present studybuilt the model of human umbilical vein endothelial cells(HUVECs) induced by AGEs to explore whether GLP-1 could protect vascular endothelial cells against oxidativestress and whether GLP-1 could antagonize oxidation, apoptosis and mitochondria damage through NADPH- or PKA-eNOS signal pathway.Methods1. The separation, culture,identification of HUVECs.Umbilical cords were all originated from normal full-term newborns at theObstetric Department in The First Affiliated Hospital of Guangdong Pharmaceutical University. Isolated cells were obtained after disinfection, washing, and 0.1% type I collagenase’s digestion, collection.After cells developed to a degree at which more than 80% got fusion, the cells had digestive transfer. The cells in the third generation with a good growth state should be chosen for the identification.The expression of typical endothelial markers-von Willebrand factor (vWF) was detected by immunofluorescence, which was a hallmark of endothelial cells.2.Establish the model of oxidative damage of HUVECs induced by AGEs to investigate the damage of AGEs on HUVECs.Cells were divided into six groups:one blank control group and five groups of AGEs (100,200,400,600,800 ug/mL). The above six groups acted on the endothelial cells of human umbilical vein for 24 h (this part will determine the concentration of AGEs, which was the same as AGEs and Bovine serum albumin (BSA) used in the following experiments without any additional special instructions). AGEs effected on HUVEC respectively for 12,24,36,48 h and set up another blank control group. With each hole being added 10μM DCFH-DA, the fluorescence intensity was detected by flow cytometry.After confirming the action concentration and time of BSA and AGEs, cells were divided into 3 groups, including blank control group, BSA group and AGEs group. We configured the JC-1 staining solution, which then incubated with HUVECs, and finally madefluorescence detection by flow cytometry.After disposing the cells via the Annexin V/PI apoptosis kit, we detectedcell apoptosis through flow cytometry.3.The effect of GLP-1 on the oxidative damage of endothelial cells induced by AGEs and the function of Triphopyridine nucleotideoxidase 4 (NOX4) signal pathExperimental groups:blank control group, BSA group, AGEs group, GLP-1 group, AGEs+GLP-1 group. Detect the expression of NOX4 by Western Blotting method.After pretreated with 100 nM GLP-1 for 30min,the cellswere added with AGEs (The prophase research showed that 100 nM GLP-1 could significantly inhibit cell apoptosis induced by AGEs)(The following would operate in these steps). Experimental groups:blank control group, AGEs group, GLP-1 group, AGEs+GLP-1 group.Detect the expression of NOX4mRNA of HUVECs with Reverse transcription PCR (RT-PCR)Experimental groups:blank control group, AGEs group, AGEs+negativecontrol siRNA group, AGEs+NOX4siRNA group. Detect the apoptosis rate of HUVECs, the levels of ROS and mitochondria membrane potential before and after inhibiting the expression of NOX4. Carry out the transfection of NOX4Small interfering RNA (siRNA) by using lipo-2000. As for the negative interference group, we used scrambling siRNA as the mismatch control group.The transfection results should be identified by RT-PCR.4.The role of PKA-, eNOS- in the protection mechanisms of GLP-1 against the damage of endothelial cells induced by AGEs.Blank control group, BSA group, AGEs group, GLP-1 group, and AGEs+GLP-1 groupwere used to detect the expression of PKA protein.Blank control group, AGEs group, GLP-1 group, AGEs+GLP-1 group, AGEs+PKA inhibitor group, and AGEs+GLP-1+PKA inhibitor group were used to detect the expression of eNOS protein, the level of ROS production and apoptosis rate.Blank control group, BSA group, AGEs group, GLP-1 group, AGEs+GLP-1 group, AGEs+GLP-1+eNOS inhibitor group were used to detect the level of Ntrogen monoxide (NO) generation and apoptosis rate.The cells were pretreated by inhibitor for 1 h, then added (or not) 100 nM GLP-1 to intervene it for 30 min, finally added AGEs.Statistical processingThe data were statistically analyzed by using SPSS 17.0, showed by mean± standard deviation (x±s). Besides, one-way analysis of variance (One-Way ANOVA) was used as statistical analysis technique. As for pairwise comparison, we used LSD method. In addition, Spearman correlation analysis method was adopted in the correlation analysis. If P<0.050, the difference got statistical significance.Results1. If proper time of digestion, HUVECs can be obtained with higher purity.2.When the concentration of AGEs got to400 ug/mLor the action time reached 24 h, the fluorescence value of ROS in cells reached to the maximum level.3.GLP-1 inhibits the damage of Vascular endothelial cells(VECs) induced by AGEs.Compared with the blank group, BSA did not affect the ROS generation (P=0.996).The ROS generation of AGEs in treatment group increased significantly when compared with the blank group (P=0.007), while the fluorescence intensity of ROS in co-incubated group of GLP-1 and AGEs was obviously decreased compared with the AGEs treatment group (P=0.010)AGEs could induce the apoptosis of endothelial cells and the decrease of mitochondria membrane potential. However, after joining the GLP-1, the apoptosis rate was decreased and mitochondrial membrane potential was increasedsignificantly (P=0.000) (P=0.014).4.The function of NOX4 on the protective action of GLP-1 against AGEs.That AGEs promoted the expression of NOX4 gene and protein synthesis in endothelial cells had a statistical difference compared with the blank control group (P=0.000,P=0.001).After added GLP-1 into the AGEs group, it significantly inhibited the expression of NOX4 gene and protein synthesis in endothelial cells, which had a statistically significant difference compared with the AGEs group (P=0.000,P=0.003).Under the induction of AGEs, DCF fluorescence in endothelial cells decreased significantly after the inhibition of NOX4mRNA expression, which was statistically significant difference compared with the AGEs treated group and AGEs+ negative interference group (P=0.011,P=0.010).The cell apoptosis and the decrease of the mitochondrial membrane potential induced by AGEs were significantly antagonized after the interference of NOX4mRNA gene expression (P=0.000,P=0.017), while the treatment of negative NOX4siRNA did not affect both the above (P=0.169,P=0.741)5.The role of PKA in VECs induced by AGEs with GLP-1.BSA did not affect the expression of PKA protein in endothelial cell comparing with the blank control group (P=0.186), while AGEs induced the significant decrease of the expression of PKA protein in endothelial cells (P=0.003).After joined the GLP-1 to AGEs, the expression of PKA protein was significantly increased when compared with AGEs group (P=0.003)Comparing AGEs group with the one adding GLP-1, DCF fluorescence was significantly reduced (P=0.013). After adding H-89-2HCl into AGEs+GLP-1 group, the ROS generation was significantly higher than the one added before (P=0.045). Therefore, inhibiting the expression of PKA could partly antagonize the inhibition of GLP-1 on the ROS generation of endothelial cells induced by AGEs.The apoptosis rate of AGEs+GLP-1 was significantly lowered than AGEs group (P=0.001). The inhibition of GLP-1 on the cell apoptosis induced by AGEs was significantly antagonized by H-89·2HCl (P=0.010).The mitochondrial membrane potential of AGEs+GLP-1 group was significantly higher than AGEs group (P=0.011). After adding the PKA inhibitor (H-89·2HC1), the mitochondrial membrane potential decreased significantly when compared with AGEs+GLP-1 group (P=0.021)6.The role of eNOS on VECs induced by AGEs with GLP-1.Compared with the blank group, AGEs significantlydown-regulated the protein expression of eNOS in endothelial cells (P=0.004). After adding the GLP-1 to AGEs group, the inhibited expression level of eNOS protein raised up(P=0.035). There was no obvious change in the inhibition of AGEs on the expression of eNOS protein whenintervened by H-89-2HC1 (P=0.908). However, the expression of eNOS protein was inhibited after adding H89 2HCl to AGEs+GLP-1 group, (P=0.045).Before the intervention of L-NAME, the NO level of AGEs+GLP-1 group was higher than that of AGEs group (P=0.017) and the rate of cell apoptosis was lower (P=0.000). After the intervention of L NAME, the NO generation in AGEs+GLP-1 group was significantly inhibited (P=0.017)and the apoptosis rate was increased(P=0.011)Conclusion1.A culture model in vitro is successfully established to isolate human umbilical vein endothelial cells (HUVECs).2. GLP-1 can protect HUVECsfrom oxidative damage and mitochondria damage induced by AGEs through inhibiting NOX4 signaling channel or activating PKA signaling channel and thus protect endothelial cells.3.GLP-1 plays a protective role by PKA-eNOS-NO signal channel in the damage of HUVECs induced by AGEs.