Oxidative Injury Caused by Glucose Reperfusion in Human Umbilical Vein Endothelial Cells and Its Potencial Mechanisms
|School||Southern Medical University,|
|Course||Endocrine and Metabolic Diseases|
|Keywords||Glucose reperfusion Human umbilical vein endothelial cells Nitric oxide Nitric oxide synthase Oxidative Stress Total antioxidant capacity NADPH oxidase De - coupling of eNOS|
Especially in China, diabetes has become a major public health problem. With the increasing prevalence of diabetes increased clinical incidence of diabetes-related hypoglycemia increases. As scholars Cryer said: \Hypoglycemia damage to the central nervous system, has long been known. At present, studies have shown that continuous low blood sugar can induce acute cardiovascular disease, aggravate diabetic macroangiopathy, the mechanism may be related to the neurohumoral stress response in the state of hypoglycemia and continuing low sugar directly damage vascular endothelial cells. Recent studies found that the supplemented with glucose to a certain level after the sugar (s) can also be induced oxidative stress, and thus damage the cells or tissue. This low sugar and then supplemented with glucose causes the cells or tissue injury called glucose-reperfusion injury (the glucose reperfusion injury). Haces ML, and other scholars with insulin-induced hypoglycemia, giving 25% glucose infusion, the results show that glucose reperfusion 21h, blood sugar has returned to normal, you can still clearly observed in the rat cerebral cortex necrosis cells. Glucose reperfusion 9h and 21h, the hippocampus of the rat brain, the cortex, striatum able to detect 3-NT protein (3-nitrotyrosine peroxynitrite product: 3 - nitrotyrosine) increased expression. Suh et al. Hypoglycemia caused by insulin injections to mice, glucose reperfusion brain tissue ROS levels detected at 30min, and the glucose reperfusion-induced oxidative stress level is higher than the low-sugar itself. The scholars also found that low-sugar glucose after reperfusion than simply low-sugar greater damage to nerve cells, and its possible mechanism due to the increase in the activity of NADPH oxidase. Endothelial cell damage and functional changes in close contact with the incidence of vascular disease. Our department in the past, studies have shown, low sugar can damage the endothelial cells through the induction of oxidative stress. But glucose reperfusion whether it can lead to vascular endothelial cell injury, has not been reported. Vascular endothelial cell damage and functional changes of diabetic vascular complications and cardiovascular events, an important part of the development. Nitric oxide in endothelial cells (Nitric Oxide, NO) decreased production is an important symbol of the endothelial cell dysfunction. Numerous studies have confirmed the mechanism of vascular endothelial injury caused by the high sugar, including insulin resistance, lipid metabolism, oxidative stress, inflammatory response, which caused by the oxidation of the high sugar should arouse a key role. Oxidative stress generated oxygen radicals can easily react with NO inactivated in an instant, thus affecting the vascular endothelial function. Diacylglycerol elevated high blood sugar can make the blood vessel cells, inhibition of NO synthase activity, lead to reduced NO synthesis and inhibition the ring phosphorus guanosine generated by the NO-mediated vasomotor function changes. In addition, high blood sugar can induce apoptosis in endothelial cells, resulting in structural changes of the vascular wall and endothelial dysfunction. Similarly, low sugar endothelial cell injury, low sugar through the induction of oxidative stress injury in endothelial cells and cause endothelial dysfunction. But it is not yet clear whether the oxidative stress is involved in glucose reperfusion injury of vascular endothelial cells. Oxidative stress (Oxidative Stress, OS) is due to the excess of oxygen free radical generation and / or intracellular antioxidant defense system damage, leading to excessive accumulation of oxygen free radicals and their related metabolites, thereby to produce a variety of toxic effects on the cell. pathological state. Oxidative stress produced by vascular endothelial cells is mainly due to endogenous oxidative enzymes such as: NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase, NOX), xanthine oxidase, mitochondrial electron transport chain uncoupling of nitric oxide together enzyme (uncoupled endothelial nitric oxide synthase eNOS) and antioxidant enzymes such as: superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) lost the balance. In recent years, considerable evidence that NADPH oxidase and the uncoupling of eNOS in endothelial cells is an important source of oxidative stress. NOX which is a complex enzyme composed of multiple subunits, including membrane subunit cytochrome the b558 (p22Phox and gp91Phox protein), multiple cytoplasmic the subunits (p47Phox, p40Phox, p67Phox) and the small G protein Rac. It continuously passed through the two electrons of NADPH to oxygen molecules mediated oxidative stress occurs, the process can be expressed as (2O2 of NADPH-2O2-NADP 2H). eNOS endothelial nitric oxide synthase, and under normal circumstances exist in the form of a dimer, the latter able to catalyze the synthesis of NO, and in a variety of pathological conditions, the dimer dissociation into monomers, monomer the electronic chain transfer can not be completed during the reaction, and thus lead to the generation of the peroxide. Latter further consumption of intracellular NO production, the more toxic superoxide nitrite (ONOO), ONOO continue to eNOS uncoupling of injury, thus creating a vicious cycle there are studies confirm the high sugar and low sugar induced vascular endothelial cells from oxidative stress injury mechanisms associated with these two paths, but glucose reperfusion whether cause oxidative damage to endothelial cells, and its mechanism is related to the NOX and uncoupling of eNOS pathways? currently not been reported yet to be further study of the subject using human umbilical vein endothelial cell line HUVEC-12 study, different concentrations of glucose in the observed low-sugar reperfusion oxidative injury of endothelial cells, and to explore the possible mechanisms of oxidative stress, hypoglycemia prevention provided new experimental data of this study include the following two parts: the first chapter of glucose reperfusion role of oxidative damage in human umbilical vein endothelial cells [Objective] observed glucose reperfusion function in human umbilical vein endothelial cells HUVEC-12 and oxidative stress. objects and methods] 1, human umbilical vein endothelial cell line HUVEC-12 cells as experimental subjects. to the RPMI1640 containing 10% fetal bovine serum medium, placed at 37 ° C for 5? 2 in an incubator. 2, the glucose concentration in the experimental medium were randomly divided into seven groups: (1) normal control group (5.5mmM group, medium the GLU to 5.5 mM); (2) continuous of sugarless group (0mM group training yl GLU 0mm); (3 glucose reperfusion group I (0-5.5mm, i.e. first in the culture medium containing GLU 0mM cultured in the medium two hours after the replacement of the GLU 5.5mM) (4) glucose reperfusion group II (0-11.1mm, i.e. first containing GLU the 0mM cultured in the medium two hours after the replacement GLU11.1mM culture medium) (5) glucose reperfusion group III (0-25mmM, i.e. first in the containing GLU cultured for 2 hours in 0mM medium replace GLU25mM in culture medium) (6) continuous high glucose group Ⅰ (11.1mM group, medium GLU 11.1mM) (7) sustained high glucose group II (25 mM group the, medium GLU 25mM) above groups before formal intervention with serum-free medium and cultured for 24 hours, to maintain the cells in each group are synchronized growth. 2, respectively, in the sugar 2h after reperfusion 15min, 30min, 45min, 1h, of four different culture time points using nitrate reductase method for the determination of NO2-, NO3 level, low-sugar 2h after reperfusion 15min chemical colorimetric determination of eNOS activity. 4, respectively, in the low-sugar 2h after perfusion for 15 min, 30min, 45min , 1h, a total of four different culture time point, the use of superoxide anion fluorescence probe fluorescent probe (Dihydroethidium, DHE), Microplate detection system (SpectraMax M5/M5e) at excitation wavelength 535nm, emission wave 610nm measure intracellular The amount of fluorescence changes indirectly reflect ROS production. 5, the experimental data Measurement data were expressed as mean ± standard deviation (x ± s) said SPSS13.0 software for statistical analysis, data continuity repeated measure analysis of variance and univariate Analysis of variance (One-way ANOVA); multiple comparisons using LSD method taking into account the process of grouping and repeated measurement time point two factors P lt; 0.05 was considered statistically significant difference. [results]. cells in each group different time points NO level of measurement: seven groups between the NO level at each time point are exist significantly with the difference (between-group effect: F = 1936.989, P = 0.000; within the group effect: F = 43.040, P = 0.000). specific analyzed as follows: (1) the concentration effect: (1) at the same point in time, and 5.5mM group, of 0 mM groups, 11.1mM group, 25mM group NO levels are significantly decreased, P lt; 0.05 ② reperfusion group and continued without sugar group, the persistently high level of sugar group NO comparison: 0-11.1mM group at the same time point lt; 0 mM group; the 0-25mmM group lt; 0mM Group, P lt; 0.05 in 15min and 30min when 0-11.1mM group the lt; 11.1mM group; 0-25mmM group lt; 25 mM group, P lt; 0.05 ③ The three groups of different concentrations reperfusion of NO level comparison: 0-25mmM group at the same time point lt; the 0-11.1mM group-lt; 0 -5.5mM group, P lt; 0.05 (2) time effect: With the extension of time ,0-5 .5 mM of NO levels gradually returned to normal levels, the rest of the group, the level of NO decreased gradually reperfusion 1h The basic set of values ??decreased to the same level. cells in each group Determination of eNOS activity: seven groups of cells eNOS activity in the presence of significant difference (F = 38.636, P = 0.000) (1) and 5.5mM group: (8.740 ± 0.507), 0mM the group (4.971 ± 0.545), of 11.1mM group (5.468 ± 0.806), 25 mM group (4.830 ± 0.168), eNOS activity was significantly decreased, P = 0.000 (2) reperfusion group and continued Sugarless sustained high sugar group eNOS activity :0-25mM group lt; 0 mM group, P = 0.000. 0-11.1mM group-lt; 11.1mM group; 0 mM group lt; 25 mM group, P = 0.000. (3) the eNOS activity :0-25mM group different concentrations reperfusion group (2.040 ± 0.437) lt; 0-11.1mM group (3.983 ± 0.717) lt; 0-5.5mmM group (7.677 ± 0.891), P = 0.000. various cells different time points ROS level was measured: ROS level between the seven groups at each time point, a significant difference (between-group effect: F = 70.086, P = 0.000; within the group effect: F = 7.309, P = 0.009). specific analysis is as follows: (1) the concentration effect: (1) at the same point in time, compared with 5.5mmM group 0 mM group, 25mM group ROS levels were significantly higher, P lt; 0.05 ② reperfusion group and continued Sugarless ROS levels of sustained high sugar group comparison: the same time point 0-25mM group gt; 0 mM group, P lt; 0.05; 0 mM group gt; 25 mM group, P lt; 0.05 ③ three different concentrations reperfusion group ROS level comparison: 0-25mM group gt the same time point; 0 - 5.5 mM group; 0 mM group gt; 0-11.1mM group, P lt; 0.05 (2) time effect: With time, 0mM ROS level group, 25 mM ,0-25mM group showed a gradually increasing trend, but no statistically significant differences between each time. 4. correlation analysis: of HUVEC-12 cells in the presence of ROS levels and NO levels significantly negatively related, (r = 0.-0.733, P = 0.000, N = 21), NO levels and eNOS activity in the presence of significantly with sex being related (r = 0.951, P = 0.000, N = 21), the presence of of ROS level and eNOS activity significantly negative correlation (r = -0.801, P = 0.000, N = 21) NO level reperfusion concentration there is a significant negative correlation (r = -0.802, P = 0.000, N = 36) the ROS levels Reperfusion concentrations significantly positively correlated (r = 0.902, P = 0.000, N = 36). [Conclusion] after the low-sugar glucose reperfusion HUVEC-12 cell dysfunction and oxidative stress can lead to this injury than simply low-sugar and pure high sugar is more serious. 2 glucose, the higher the concentration of reperfusion, the higher the level of HUVEC-12 cells from oxidative stress, the more serious dysfunction. 3 glucose reperfusion HUVEC-12 cells caused by changes in NO levels, eNOS activity and ROS content was negative correlation, suggesting that the glucose reperfusion cause endothelial cell dysfunction may be associated with oxidative stress the second chapter glucose reperfusion induced human umbilical vein endothelial cells oxidative stress mechanism of [Objective] 1, observed glucose reperfusion in human umbilical antioxidant capacity vein endothelial cells. 2, to understand the role of NADPH oxidase and the uncoupling of eNOS in glucose reperfusion-induced endothelial cell oxidative stress. objects and methods] people human umbilical vein endothelial cells strains HUVEC-12 cells as experimental subjects. 2, the total antioxidant capacity detection experiments grouped with the first part of the rest of the experimental groups are as follows: (1) normal control group (5.5 mM the group medium GLU 5.5 mM); (2) glucose reperfusion group (0 - 25 mM group, namely the OMM a medium containing GLU culture replaced after 2 hours GLU 25mM culture medium); (3) glucose reperfusion group Joint Apocynin treatment group (APO group: 500umol / l apocynin preincubation 1hr before adding 0mM medium for 2h, after replacement GLU is cultured in a medium of 25 mm); (4) glucose reperfusion joint L-NAME treatment group (L-NAME group: 100umol / l of L-NAME pre-culture and incubated for 1hr then adding 0mM medium 2H replaced after GLU 25mM cultured in a medium) with serum-free medium were cultured for 24 hours in each group of the above formal intervention, in order to maintain the cells in each group synchronized growth in each group after reperfusion after low-sugar 2h 15min detection. 3, experimental methods: the total antioxidant capacity of the cells with chemical colorimetric determination of intracellular ROS production was measured using the fluorescent probe method. 4, the experimental data Measurement data were expressed as mean ± standard deviation (x ± s), using the statistical software SPSS13.0 treatment groups were compared using one-way analysis of variance (One-way ANOVA), group multiple comparisons using LSD method. P lt; 0.05 was considered statistically significant difference.  1, Determination of the total antioxidant capacity (T-AOC): the presence of seven groups of cells total antioxidant capacity significant difference (F = 20.217, P = 0.000) (1) with 5.5mmM group (1.592 ± 0.247), 0 mM group (0.571 ± 0.090), of 11.1mM group (0.860 ± 0.272), 25 mM group (0.625 ± 0.127) T-AOC was significantly decreased (P = 0.001). (2) reperfusion group compared with continuous sugar-free group, sustained high glucose group T-AOC: 0 mM group gt; 0 mM group; the 0 mM groups lt; 0-11.1mM group-, P = 0.042, P = 0.025. 25mM group gt; 0-25mmM group, P = 0.022 (3) different concentrations reperfusion group T-AOC :0-5 .5 mM comparison group (1.477 ± 0.214) gt; 0-11.1mM group (0.960 ± 0.195) gt; 0 -25mM group (0.224 ± 0.094), P = 0.005, P = 0.000 2 inhibitor is added ROS determination: 5.5 mM ,0-25mM group, APO group, ROS levels of L-NAME group were: 0.663 ± 0.124,1.398 ± 0.132,0.789 ± 0.054,0.917 ± 0.058 among the four groups exist significant difference (F = 31.99, P = 0.000), with the compared to 5.5mM group, ,0-25mM ROS in average significantly higher after inhibition agents APO and L-NAME treatment, the APO group, and L-NAME group ROS level decreased (44% and 34%), P = 0.000, total antioxidant capacity (T-AOC) of the inhibitor is added after the determination of: T-AOC the of 5.5mM-group the ,0-25mmM group, the APO group, L-NAME group were: 1.278 ± 0.089,0.308 ± 0.128,1.071 ± 0.132,0.684 ± 0.111 among the four groups for the presence of a significant difference (F = 24.027 , P = 0.000), and the compared to 5.5mM group, ,0-25mM T-AOC water on average a significant decline in the the APO group, and L-NAME group T-AOC levels rose after the inhibitor APO and L-NAME treatment (3.38-fold and 2.19-fold), P = 0.000 and P = 0.01 [Conclusion] glucose reperfusion can lead to decreased HUVEC-12 cells total antioxidant capacity. 2 glucose reperfusion induced HUVEC-12 cells from oxidative stress mechanism with NADPH oxidase pathway and uncoupling eNOS ways related.