| Backgrounds and aimsRecently the morbidity of diabetes has increased rapidly. Vascular complications are the common in diabetes patients, with the characteristics of high prevalence, disability and mortality. Thus it is pivotal to investigate the mechanism of vascular complications of diabetes. Nowadays it is well known that endothelium dysfunction is an early manifestation in the development of diabetic vascular complications. High concentration of glucose may impair the endothelial function via inflammation, oxidative stress, advanced glycosylation endproducts, and subsequent dyslipidemia, and so on.Hydrogen sulfide (H2S), a colorless gas, has been considered as the third gasotransmitter except for nitric oxide (NO) and carbon monoxide (CO). H2S has been shown to possess important roles in the physiology and pathophysiology of several biological systems, especially in cardiovascular system. Endogenous H2S or H2S donor may decrease blood pressure, ameliorate myocardial ischemia and hypoxia or ischemia-reperfusion injury, reduce atherosclerosis lesions and improve the symptoms of heart failure, and so on. For example, administration of exogenous H2S (NaHS) has been shown to limit infarct size and preserve ventricular functioning in a myocardial ischemia-reperfusion mouse model. In apoE(-/-) mice, NaHS may increase plasma H(2)S level, decrease the size of atherosclerotic plaque and plasma and aortic ICAM-1levels. In mammalian, endogenous H2S is generated by three known enzymes, namely cystathionine-β-synthase (CBS), cystathionine-y-lyase (CSE) and3-mercaptopyruvate sulfur transferase (3MST). The former two enzymes are pyridoxal-5’-phosphate dependent, which utilize L-cysteine and homocysteine as substrates to generate H2S. The latter enzyme utilizes L-cysteine and a-ketoglutarate through the metabolism with cysteine aminotransferase (CAT) to produce H2S. CBS and3MST are the main H2S-forming enzyme in the central nervous system, whereas CSE is the principal H2S-forming enzyme in the vasculature and heart.Recently more and more attention has been put on the effects of H2S on endothelium. It has been shown that NaHS suppressed ICAM-1expression in tumor necrosis factor (TNF)-alpha-treated HUVECs via the NF-κB/IκB pathway. H2S has also been reported to suppress superoxide formation, NADPH oxidase-1(NOX-1) expression in endothelial cells, and protect ventricular function from oxidative stress to restore normal remodeling.Therefore, H2S may be involved in the vascular structure disorder and dysfunction. However, up to date, little is known about the role of H2S in the development of diabetic vascular complications. In the present study, we intended to investigate the protective effect of H2S on the injury of human umbilical vein endothelium cells (HUVECs) induced by high glucose levels. Furthermore, we determined the mechanism of H2S on HUVECs from the point of H2S production, apoptosis, oxidative stress and secretion of ET-1.Materials and methods1. HUVECs were isolated from umbilical vein cords of normal pregnancies as previously described. Briefly, umbilical veins were rinsed with sterile saline and digested with0.25%trypsin. Harvested cells were cultured in M199medium supplemented with10%fetal bovine serum,100U/mL penicillin-streptomycin and20ng/mL vascular endothelial growth factor in an atmosphere of5%CO2at37℃. The medium was refreshed at intervals of3days at cell confluence. To maintain uniform condition, the cells in passage2to3were used for experiments.2. After growing to70%confluence, the primary HUVECs were observed under inverted microscope, photoed and identified with Ⅷ factor antigen polyclonal antibody.3. When growing to approximately80%confluence, HUVECs were then cultured in medium containing either normal glucose (5.5mmol/L) which served as a normal control or high glucose (25mmol/L) for48h. To determine the effects of NaHS, an H2S donor, on endothelial cells, HUVECs were pre-treated with NaHS (50μmol/L) for30min before adding high glucose. After48h, the cells were harvested for following experiments.4. Cell viability was determined by MTT. At the same time, cell apoptosis was examined according to the morphological changes in cell nuclei using Hoechst33258staining. Cells were washed with cold PBS and fixed with paraformaldehyde for30min. Hoechst33258(10μg/mL) was added and incubated for20min before being detected by fluorescence microscopy. Apoptotic cells appeared as chromatin condensation and multiple chromatin fragments.5. The content of H2S in cells was tested by UV spectrophotometer. The H2S concentration of each sample was expressed as nanomoles of H2S per milligram soluble protein.6. The mRNA and protein expression of CSE in HUVECs were determined by Real-time PCR and Western blot analysis, respectively.7. The apoptosis-associated proteins expression in HUVECs such as Bax、Bcl-2and Cleaved caspase-3were detected by Western blot analysis.8. The secretion of ET-1of HUVECs was discovered by ELISA method. Results were normalized to cellular protein content in all experiments.9. Intracellular ROS generation was measured by the oxidation-sensitive fluorescent probe (DCF-DA). The MDA level was measured to assess lipid peroxidation and the activity of SOD was measured as per kit instructions to estimate the role of anti-oxidation in the cells by commercial reagent kitsResults1. Morphological features of HUVECs are proper and in line with others. This is validated by Ⅷ factor antibody. 2. Cell viability in the high glucose treated HUVECs (68.5±3.8%) was significantly decreased in comparison with normal cells (P<0.05), which could be ameliorated by NaHS (97.1±7.4%)(P<0.05).HUVECs treated with high glucose showed obvious apoptotic features including chromatin condensation and multiple chromatin fragments. In contrast, nuclei of cells in the normal control group did not show apoptotic morphology. The percentage of apoptotic cells increased significantly in high glucose-treated cells compared with that of the normal glucose treated cells (P<0.05). NaHS administration reduced the apoptotic cells in high glucose cultured HUVECs significantly (P<0.05).This indicated that high glucose induced HUVECs apoptosis and reduced cell viability. H2S exerted the protective effect on HUVECs as proved by decreased apoptosis and increased cell viability.3. Production of H2S was significantly less in25mmol/L glucose treated HUVECs than those in the5.5mmol/L glucose treated cells (10.26±0.51μmol/min vs12.85±0.53μmol/min, P<0.05). The reduced H2S production caused by high glucose was retrieved by50μmol/L NaHS (10.26±0.51μmol/min vs11.90±0.32μmol/min, P<0.05).4. Although the mRNA expression of CSE in high glucose treated HUVECs was somewhat higher than that in normal glucose treated cells, the difference did not achieve statistical significance.The CSE protein expression was shown by Western blot analysis to be down-regulated about53.7%(P<0.05) in25mmol/L glucose treated HUVECs, while the addition of50μmol/L NaHS up-regulated the expression by19%(P<0.05).5. High glucose significantly increased Bax protein expression and decreased Bcl-2protein expression (both P<0.05). The ratio of Bax/Bcl-2was increased about2-fold following exposure to high glucose (P<0.05). Accordingly, the level of cleaved caspase-3, the activated form of caspase-3, was higher in the high glucose group compared with that of control cells (P<0.05).Compared with the high glucose group, the decrease in Bax protein expression and the increase in Bcl-2protein expression were observed in the group pre-treated with NaHS (both P<0.05). Similarly, NaHS attenuated the elevated Bax/Bcl-2ratio induced by high glucose, and inhibited high glucose-induced caspase-3activation (both P<0.05).These results suggested that high glucose-induced endothelial cell apoptosis might be corrected by H2S.6. High glucose resulted a significant elevation of ET-1secretion in HUVECs when compared with normal glucose (P<0.05). The glucose induced increase in ET-1was significantly attenuated by NaHS (P<0.05).7. The production of ROS was measured using fluorescence microscopy, based on ROS-dependent oxidation of DCF-DA to DCF with green fluorescence. Compared with the high glucose group, NaHS-treated group showed a significant decrease in DCF levels (P<0.01). The results indicated that pre-treatment with NaHS could decrease the intracellular ROS generation.Compared with control group, SOD activity was decreased in the high glucose group from23.59±1.08U/mg protein to19.53±2.17U/mg protein (P<0.05). Whereas, pre-treatment with NaHS increased SOD activity from19.53±2.17U/mg protein to23.66±1.16U/mg protein (P<0.05).The amount of MDA was measured to assess the extent of lipid peroxidation. Compared with control group, the high glucose group showed a significant increase in MDA level from1.38±0.33μM/mg protein to2.78±0.59μM/mg protein (P<0.01), which was reversed by pre-treatment with NaHS (from2.78±0.59μM/mg protein to1.32±0.16μM/mg protein, P<0.01).This indicated that H2S may alleviate the oxidative stress and thus protect HUVECs injury from high glucose.Conelus ions1. High glucose induces apoptosis of HUVECs via inhibiting of CSE expression and H2S production, which can be alleviated by H2S donor.2. The cellular mechanisms of high glucose on HUVECs damage include promotion of apoptosis, increase of oxidative stress and elevation of ET-1secretion.3. Exogenous H2S protect HUVECs from high glucose induced injury through inhibition of Bax and Cleaved caspase-3expression, increase Bcl-2expression, elevation of SOD activity, reduce of ROS and MDA, and suppression of ET-1. |
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