ABSTRACT
Objective To investigate the effects and mechanism of advanced glycosylation end products (AGEs)on endothelial cell senescence and endothelial barrier dysfunction.Methods Human umbilical vein endothelial cells (HUVECs)were isolated and cultured.The cells were randomized into three groups:the control group(normal medium),the bovine serum albumin-treated group(BSA control group)and AEGs group(treated with AEGs-BSA).Senescence of HUVECs were detected by senescence-associated β-galactosidase (SA-beta-Gal)staining.The mRNA and protein expressions of senescence-related genes of p53,p21 and p16 in each group were determined by reverse transcription and real-time PCR(RT-qPCR)and Western blot.Reactive oxygen species(ROS)level was determined by dichlorodihdrofluorescence diacetate (DCFH-DA).The transendothelial electric resistance(TER)were measured by endothelial electric resistance meter.The protein levels of myosin light chain kinase (MLCK),phosphorylated myosin light chain (p-MLC),myosin light chain (MLC)were detected by Western blot.Results Compared with the control group and the BSA control group,the AGEs group showed the significantly increased positive rate of senescence-associated SA-beta-Gal staining (67.30 ± 0.75 ％ vs.7.81 ±0.35 ％ and 7.64 ± 0.91％,respectively,P ＜ 0.01)and the expressions of aging-related genes of p53,p21 and p16 were significantly increased (P ＜ 0.05)There was no significant difference in transendothelial electric resistance(TER)between the control group and theBSAgroup(48.0±6.3 Ω· cm2 vs.42.0±7.8 Ω· cm2,P＞0.05),while TER was lower in the AEGs group than in control group and the BSA group[(27.0±4.2)Ω · cm2 vs.(48.0±6.3)Ω · cm2 and (42.0 ± 7.8) Ω · cm2,P ＜0.01].ROS production had no significant difference between the control group and the BSA group[(38.36 ± 8.55) ％ vs.(41.67 ± 6.93) ％,p ＞ 0.05],while was increased in the AEGs group versus control group and the BSA group[(69.31±8.47)％ vs.(38.36±8.55) ％ and (41.67 ± 6.93) ％,P ＜0.05).The protein expression levels of MLK and p-MLC/MLC were higher in the AGEs group than in the control group and the BSA group(P＜0.05).Conclusions AGEs may lead to endothelial cell senescence and endothelial barrier dysfunction by promoting ROS production and oxidative stress,and by regulating MLCK signaling pathway.
ABSTRACT
<p><b>Background</b>Shensong Yangxin Capsule (SSYX), traditional Chinese medicine, has been used to treat arrhythmias, angina, cardiac remodeling, cardiac fibrosis, and so on, but its effect on cardiac energy metabolism is still not clear. The objective of this study was to investigate the effects of SSYX on myocardium energy metabolism in angiotensin (Ang) II-induced cardiac hypertrophy.</p><p><b>Methods</b>We used 2 μl (10 mol/L) AngII to treat neonatal rat cardiomyocytes (NRCMs) for 48 h. Myocardial α-actinin staining showed that the myocardial cell volume increased. Expression of the cardiac hypertrophic marker-brain natriuretic peptide (BNP) messenger RNA (mRNA) also increased by real-time polymerase chain reaction (PCR). Therefore, it can be assumed that the model of hypertrophic cardiomyocytes was successfully constructed. Then, NRCMs were treated with 1 μl of different concentrations of SSYX (0.25, 0.5, and 1.0 μg/ml) for another 24 h. To explore the time-depend effect of SSYX on energy metabolism, 0.5 μg/ml SSYX was added into cells for 0, 6, 12, 24, and 48 h. Mitochondria was assessed by MitoTracker staining and confocal microscopy. mRNA and protein expression of mitochondrial biogenesis-related genes - Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), energy balance key factor - adenosine monophosphate-activated protein kinase (AMPK), fatty acids oxidation factor - carnitine palmitoyltransferase-1 (CPT-1), and glucose oxidation factor - glucose transporter- 4 (GLUT-4) were measured by PCR and Western blotting analysis.</p><p><b>Results</b>With the increase in the concentration of SSYX (from 0.25 to 1.0 μg/ml), an increased mitochondrial density in AngII-induced cardiomyocytes was found compared to that of those treated with AngII only (0.25 μg/ml, 18.3300 ± 0.8895 vs. 24.4900 ± 0.9041, t = 10.240, P < 0.0001; 0.5 μg/ml, 18.3300 ± 0.8895 vs. 25.9800 ± 0.8187, t = 12.710, P < 0.0001; and 1.0 μg/ml, 18.3300 ± 0.8895 vs. 24.2900 ± 1.3120, t = 9.902, P < 0.0001; n = 5 per dosage group). SSYX also increased the mRNA and protein expression of PGC-1α (0.25 μg/ml, 0.8892 ± 0.0848 vs. 1.0970 ± 0.0994, t = 4.319, P = 0.0013; 0.5 μg/ml, 0.8892 ± 0.0848 vs. 1.2330 ± 0.0564, t = 7.150, P < 0.0001; and 1.0 μg/ml, 0.8892 ± 0.0848 vs. 1.1640 ± 0.0755, t = 5.720, P < 0.0001; n = 5 per dosage group), AMPK (0.25 μg/ml, 0.8872 ± 0.0779 vs. 1.1500 ± 0.0507, t = 7.239, P < 0.0001; 0.5 μg/ml, 0.8872 ± 0.0779 vs. 1.2280 ± 0.0623, t = 9.379, P < 0.0001; and 1.0 μg/ml, 0.8872 ± 0.0779 vs. 1.3020 ± 0.0450, t = 11.400, P < 0.0001; n = 5 per dosage group), CPT-1 (1.0 μg/ml, 0.7348 ± 0.0594 vs. 0.9880 ± 0.0851, t = 4.994, P = 0.0007, n = 5), and GLUT-4 (0.5 μg/ml, 1.5640 ± 0.0599 vs. 1.7720 ± 0.0660, t = 3.783, P = 0.0117; 1.0 μg/ml, 1.5640 ± 0.0599 vs. 2.0490 ± 0.1280, t = 8.808, P < 0.0001; n = 5 per dosage group). The effect became more obvious with the increasing concentration of SSYX. When 0.5 μg/ml SSYX was added into cells for 0, 6, 12, 24, and 48 h, the expression of AMPK (6 h, 14.6100 ± 0.6205 vs. 16.5200 ± 0.7450, t = 3.456, P = 0.0250; 12 h, 14.6100 ± 0.6205 vs. 18.3200 ± 0.9965, t = 6.720, P < 0.0001; 24 h, 14.6100 ± 0.6205 vs. 21.8800 ± 0.8208, t = 13.160, P < 0.0001; and 48 h, 14.6100 ± 0.6205 vs. 23.7400 ± 1.0970, t = 16.530, P < 0.0001; n = 5 per dosage group), PGC-1α (12 h, 11.4700 ± 0.7252 vs. 16.9000 ± 1.0150, t = 7.910, P < 0.0001; 24 h, 11.4700 ± 0.7252 vs. 20.8800 ± 1.2340, t = 13.710, P < 0.0001; and 48 h, 11.4700 ± 0.7252 vs. 22.0300 ± 1.4180, t = 15.390; n = 5 per dosage group), CPT-1 (24 h, 15.1600 ± 1.0960 vs. 18.5800 ± 0.9049, t = 6.048, P < 0.0001, n = 5), and GLUT-4 (6 h, 10.2100 ± 0.9485 vs. 12.9700 ± 0.8221, t = 4.763, P = 0.0012; 12 h, 10.2100 ± 0.9485 vs. 16.9100 ± 0.8481, t = 11.590, P < 0.0001; 24 h, 10.2100 ± 0.9485 vs. 19.0900 ± 0.9797, t = 15.360, P < 0.0001; and 48 h, 10.2100 ± 0.9485 vs. 14.1900 ± 0.9611, t = 6.877, P < 0.0001; n = 5 per dosage group) mRNA and protein increased gradually with the prolongation of drug action time.</p><p><b>Conclusions</b>SSYX could increase myocardial energy metabolism in AngII-induced cardiac hypertrophy. Therefore, SSYX might be considered to be an alternative therapeutic remedy for myocardial hypertrophy.</p>