Oxidative Injury Caused By Glucose Reperfusion In Human Umbilical Vein Endothelial Cells And Its Potencial Mechanisms

BackgroundNowadays, the prevalence of diabetes mellitus is growing rapidly. Diabetes has become a major public health problem especially in China. With the increasing prevalence of diabetes mellitus, the incidence of hypoglycemia in clinical has also increased. Scholar Cryer has pointed out that, the benefits of maintenance of blood glucose in the normal scope in a lifetime may be offset by a severe low blood sugar or cardiovascular events as a result of that. It has long been kown that hypoglycemia can damage central nervous system. Currently, some studies have suggested, hypoglycemia can induce acute cardiovascular disease and can aggravate diabetic macrovascular disease. The mechanism may be related to neurohumoral stress response at hypoglycaemia state and also related to injury of vascular endothelial cells induced by hpoglycemia.But in recent years, some basic studies discovered that glucose reperfusion can induce oxidative stress to damage cells or tissue. More serious injury of cells or tissues is triggered by glucose reperfusion rather than hypoglycaiemia is called glucose reperfusion injury. Scholar Haces ML used insulin-injected hypoglycemic rats found that 3-nitrotyrosine (NT) residues in proteins were observed after glucose reperfusion 9h and 21h in different rat brain regions. And 3-NT-posi-tive cells were only observed after glucose reperfusion, when the blood glucose was normal. Scholar Suh has found that the injury of neuronas by glucose reperfusion was more serious than by hypoglycemia, which is attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. there is a popular belief that incidence of cardioascular disease and endothelial dysfunction has the close relation, and our previous research suggested low glucose can induce oxidative stress and directly caused endothelial cells ECV304 damage. So can glucose reperfusion damage vascular endothelial cells? And what is the mechanism? Now, such research has yet to see on the report.The dysfunction and injury of endothelial cells are important in cardiovascular diseases and diabetic vascular disease. eNOS-mediated nitric oxide(NO) production is an important endothelial function mark. Reduced production of eNOS-mediated NO in patients with diabetes is thought to be of major importance in the development of diabetic complications and is a surrogate marker for endothelial cell dysfunction. A large number studies have confirmed that there are several mechanisms of endothelial dysfunction caused by high glucose including insulin resistance, dyslipidemia, oxidative stress, inflammation and so on. Among them oxidative stress plays a key role. Increased ROS concentrations reduce the amount of bioactive NO by chemical inactivation to effect endothelial function. High glucose can increase diester triglyceride of endothelial cells, which inhibite the eNOS activity and decrease NO level to damage endothelial function. High glucose also can promoted increasing apoptosis of endothelial cells to change vascular structure. In pre-test, we have confirmed that dysfunction of endothelial cells can be induced by low glucose. So is oxidative stress involved in injury of endothelia cells by glucose reperfusion?Oxidative stress is the disorder between reactive oxygen species(ROS) generation and anti-oxidant systems, and the disorder which causes damage to tissues and cells. Vascular oxidative stress with an increased production of ROS also contributes to mechanisms of vascular dysfunction. Oxidative stress is mainly caused by an imbalance between the activity of endogenous pro-oxidative enzymes such as NADPH oxidase, xanthine oxidase, uncoupled nitric oxide synthase or the mitochondrial respiratory chain and anti-oxidative enzymes such as superoxide dismutase, glutathione peroxidase or catalase in favor of the former. In recent years, considerable evidences pointed out that, NADPH oxidase complex and uncoupled eNOS are present in vascular endothelial cells, and are major sources of ROS. NOX is a complex enzyme composed of several subunits, including the membrane, subunits of cytochrome b558 (p22Phox and gp91Phox protein), several cytoplasmic subunits (p47Phox, p40Phox, p67Phox) and small G protein Rac. It mediates the occurrence of oxidative stress by transfer two electron of NADPH continuously to oxygen molecules, the process can be expressed as (2O2+NADPH-2O22-+NADP++ 2H+). Endothelial nitric oxide synthase(eNOS) are usually referred to as homodimers in their active form, which can generate endothelium-derived NO. But a series of pathological factors can make dimeirs dissociate into monomers. Monomers can not complete electronic chain transfer and generate O2-. Increased ROS concentrations reduce the amount of bioactive NO by chemical inactivation to form toxic peroxynitrite. Peroxynitrite in turn can "uncouple"endothelial NO synthase to become a dysfunctional superoxide generating enzyme that contributes to vascular oxidative stress.Studies have confirmed that high glucose and low glucose can induce oxidative stress to injure endothelial cells, which is associated with these two pathways. So can glucose reperfusion also damage endothelial cells? Is the mechamism associated with NADPH oxidase and uncoupled eNOS? There are few studies concerning these aspects, and further studies are needed to clarify these questions. In this study, we use human umbilical vein endothelial cell line (HUVEC-12) to investigate the effects of glucose reperfusion on human umbilical vein endothelial cell and its mechanism, thus provide new ideas for studies on preventing and treatment of hypoglycemia.Chapter 1 Oxidative injury caused by glucose reperfusion in human umbilical vein endothelial cellsObjectiveTo investigate the dysfunction effect and oxidative stress caused by glucose reperfusion in human umbilical vein endothelial cell line (HUVEC-12).Methods1. The experiment object was human umbilical vein endothelial cell line (HUVEC-12). HUVEC-12 cells were cultured in RPMI1640 containing 10% fetal bovine serum (FBS),100ug/ml penicillin and 100ug/ml streptomycin at 37℃,the cells were passaged every other day.2. There were seven groups according to different concentrations of glucose:(1). The normal control group (Ctr group,5.5mM, cells were cultured in medium containing 5.5 mM glucose);(2). The glucose deprived group (GD group, OmM, cells were cultured in medium containing 0 mM glucose);(3). The glucose reperfusion groupⅠ(GRⅠgroup,0-5.5mM, cells were cultured in medium containing OmM glucose for two hours and then in medium containing 5.5 mM glucose); (4). The glucose reperfusion groupⅡ(GRⅡgroup,0-11.1mM, cells were cultured in medium containing OmM glucose for two hours and then in medium containing 11.1 mM glucose);(5). The glucose reperfusion groupⅢ(GRⅢgroup,0-25mM, cells were cultured in medium containing OmM glucose for two hours and then in medium containing 25mM glucose);(6). The constant high glucose groupⅠ(HGⅠgroup,11.1 mM, cells were cultured in medium containing 11.1 mM glucose);(7). The constant high glucose groupⅡ(HGⅡgroup,25mM, cells were cultured in medium containing 25 mM glucose);HUVEC-12 cells of these seven groups were cultured in basal medium for 24 hours before intervention to keep synchronized growth of cells.3. The total nitric oxide (NO) level of HUVEC-12 cells in the supernatant of cell culture at 15min,30min,45min,1h was measured at 15min,30min,45min,1hby nitate reductase method.4. The endothelia nitric oxide synthase (eNOS) activity in the cells was measured at 15min by chemical chromatometry.5. ROS level of HUVEC-12 cells at 15min,30min,45min,1h, detecting the mean fluorescence intensity of samples by making use of SpectraMax M5/M5e system:using of dihydroethidium fluorescent probe, excitation wavelength and emission wavelength was set at 535 nm and 610 nm respectively. So compare the production of ROS in HUVEC-12 cells.6,All values are expressed as means±SD. Statistical analysis the repeated measure ANOVA and one way ANOVA was performed using SPSS 13.0 software. Multiple comparisons were carried out using LSD method. Statistical significance was defined as P<0.05.Results 1. The NO level of HUVEC-12 cells:There were significant differences among the seven groups at different time (F=1936.989, P=0.000; F=43.040, P=0.000).(1 Concentration effect:①At the same time, compared with 5.5mM group, the NO levels of OmM group,11.1 Mm group and 25mM group were decreased significantly(P=0.000).②At the same time, comparations among NO level of GD group,HG groups and of different GR groups:0-11.1 mM group<0mM group; 0-25mM group<0mM group. At 15min and 30min,0-11.1mM group< 11.1mM group; 0-25mM group< 25mM group.③NO levels of different GR groups: 0-25mM group< 0-11.1mM group< 0-5.5mM group.(2) Time effect:NO level of 0-5.5mM group was gradually normal with the time, and NO levels of other groups were decreased to the same level at 1h.2. The eNOS activities of the seven groups:There were significant differences among the seven groups (F=38.636, P=0.000). (1)Compared with 5.5mM group (8.740±0.507), the eNOS activities of OmM group (:4.971±0.545), 11.1mM group (4.971±0.545) and 25mM group (4.830±0.168) were decreased significantly (P=0.000). (2)Comparations among eNOS activity of GD group, HG groups and of different GR groups:0-25mM group<0mM group, (P=0.000).0-11.1 mM group< 11.1mM group; 0-25mM group< 25mM group, (P=0.011,P=0.000). (3) eNOS activities of different GR groups:0-25mM group (2.040±0.437)< 0-11.1mM group (3.983±0.717)< 0-5.5mM group (7.677±0.891), (P=0.000,P=0.002).3. The ROS level of HUVEC-12 cells:There were significant differences among the seven groups at different time (F=70.086, P=0.000; F=7.309, P=0.009).(1)Concentration effect:①At the same time, compared with 5.5mM group, the ROS levels of OmM group and 25mM group were increased significantly(P=0.000).②Comparations between ROS level of GD group, HG groups and of GR groups: 0-25mM group>0mM group; 0-25mM group> 25mM group.③ROS levels of different GR groups:0-25mM group> 0-5.5mM group; 0-25mM group> 0-11.1mM group.(2) Time effect:ROS level of OmM group,25mM group and 0-25mM group were gradually increasing with the time, but the differences were not significantly.4. The correlation analysis showed that there was significantly correlation between the NO level and ROS level (r=0.-0.733 P=0.000,N=21), the same as eNOS activity and ROS level (r=-0.801,P=0.000,N=21). And there was significantly correlation between the NO level and eNOS activity(r=0.951,P=0.000,N=21).There was significantly correlation between NO level and glucose concentration (r=-0.802,P=0.000,N=36), the same as ROS level and glucose concentration (r=0.902,P=0.000,N=36).Conclusion1. Glucose reperfusion can cause dysfunction and oxidative injury in HUVEC-12 cells.2. The highier glucose concentrations, the more severe injury was3. There was nagtive correlation between NO and ROS, which showed that oxidative stress may be one of the mechanisms of endothelial cell injury.Chapter 2 Mechanisms of oxidative stress induced by glucose reperfusion in human umbilical vein endothelial cellsObjective1. To observe the total antioxidant capacity changes of HUVEC-12 cells under condition of glucose reperfusion 2. To explore role of NADPH oxidase and uncouped nitric oxide synthase in possible mechanisms of oxidative stress induced by glucose reperfusionMethods1. The experiment object was human umbilical vein endothelial cell line (HUVEC-12), they were cultured like the first chapter.2. The experimental group of total antioxidant capacity test like the first chapter, other experiments were divided into four groups:(1). The normal control group (5.5mM group, cells were cultured in medium containing 5.5 mM glucose);(2). The glucose reperfusion group (0-25mM group, cells were cultured in medium containing OmM glucose for two hours and then in medium containing 25mM glucose)(3). APO treated with glucose reperfusion group (APO group:500umol/l of apocynin pre-incubated for 1hr and then adding OmM of medium for two hours and then adding 25mM of medium);(4).L-NAME treated with glucose reperfusion group (L-NAME group:100umol/l of L-NAME pre-incubated for 1hr and then adding OmM of medium for two hours and then adding 25mM of medium);The HUVEC-12 cells of these seven groups were cultured in basal medium for 24 hours before intervention to keep synchronized growth of cells. Each group was officially intervened for 15min.3. The total antioxidant capacity of HUVEC-12 cells was measured by chemical chromatometry.4. ROS level was detected by Dihydroethidium fluorescent probe labeling method.5. Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. The data was expressed as the mean±S.D. Statistical significance of differences among groups was evaluated by One-way ANOVA and multiple comparisons were carried out using the Least-significant Difference (LSD) method. P<0.05 was considered as significant.Results1. The total antioxidant capacity (T-AOC) of the seven groups:There were significant differences among seven groups (F=24.126, P=0.000). (1)Compared with 5.5mM group (1.5920.247), the T-AOC of OmM group (0.571±0.090), 11.1mM group (0.860±0.272) and 25mM group (0.625±0.127) were decreased significantly (P=0.001). (2)Comparations among T-AOC of GD group, HG groups and of different GR groups:OmM group> 0-25mM group, OmM group< 0-11.1mM group, (P=0.042,P=0.025).25mM group> 0-25mM group, (P=0.022). (3)T-AOC of different GR group:0-5.5mM group (1.477±0.214)> 0-11.1mM group (0.960±0.195)> 0-25mM group (0.224±0.094), (P=0.001. P=0.000).2. The ROS level of each group was:0.663±0.124,1.398±0.132,0.789±0.054, 0.917±0.058. There were significant differences among the four groups (F=31.99, P=0.000). Compared with 5.5mM group, the ROS level of 0-25mM group was inreased significantly, and after APO and L-NAME treatment, ROS level was dereased respectively 44% and 34%(P=0.000).3. The total antioxidant capacity (T-AOC) of each group was:0.852±0.089, 0.205±0.128,0.714±0.132,0.456±0.111. There were significant differences among the four groups (F=24.027, P=0.000). Compared with 5.5mM group, the T-AOC of 0-25mM group was decreased significantly, and after APO and L-NAME treatment, T-AOC was inreased respectively 338% and 219%(P=0.000, P=0.01).Conclusions1. Glucose reperfusion can decrease the total antioxidant capacity of HUVEC-12 cells. 2. Mechanisms of oxidative stress induced by glucose reperfusion is associated with NADPH oxidase and uncoupled eNOS.