| Diabetes mellitus is a chronic metabolic disease in recent year,which harms human health and life seriously. According to statistics, vascular disease is the main cause of death for patient with diabetes mellitus. How to decrease the occurrence of vascular disease is a matter of great urgency. Controlling blood glucose is the first measure in diabetes treatment, but hypoglycemia is not avoided which reduces benefits, even increases the risk of death. Some results indicated that, Intensive blood-glucose control not only increased the incidence of cardioascular disease, but also increased all-cause mortality. This series of evidence-based medicine worth every clinicians ponder:hypoglycemia cause by intensive treatment may be an important factor leading to an increased risk of cardioascular disease.Previous studies showed, low glucose can direct damage cells of nerves, islet, tumor and endothelium, the damage mechanism and oxidative stress related. Our previous research suggested low glucose was able to induce oxidative stress and directly caused human umbilical vein endothelial cells ECV304 damage. Therefore, decreasing the occurrence hypoglycemia and actively correcting hypoglycemia is worth causing clinical doctors are highly valued. Glucose reperfusion is a positive, received way to deal with hypoglycemia by the clinician. But foreign studies showed, tissues or cells dysfunction and structural damage are more serious by reperfusing glucose after hypoglycemia, which is called glucose reperfusion injury. As mentioned previously, hypoglycemia can significantly increase diabetes cardiovascular events. 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 glucose reperfusion whether can induce and (or) accentuate further endothelial function damage, leading to increased risk of cardioascular disease, has yet to see any reports.Numerous studies confirm endothelial dysfunction and oxidative stress related. Reactive oxygen species (ROS) product by oxidative stress mainly includes superoxide radical(O2-), hydrogen peroxide(H2O2), hydroxyl radicals(OH-), nitric oxide(NO). Excessive ROS can directly cause cell membrane lipid peroxidation, protein degeneration, DNA fracture, inducing endothelial cell injury and dysfunction. ROS overmuch can also lead to a low density lipoprotein by oxidative modification into oxidative-low density lipoprotein (ox-LDL), and ox-LDL on endothelial cells has cytotoxic effects.Mitochondria is one of an important source of Reactive oxygen species. Mitochondria membrane potential (MMP) for maintaining mitochondria normal function is necessary, and is one of an important target to study mitochondrial function and its oxidative status. Foreign studies show that oxidatie stress level and MMP were positively correlated, Any make protons returned to the matrix changes can reduce MMP, resulting in decreasing the formation of mitochondrial ROS. Physiological condition, electron go through mitochondria respiratory chain, protons (H+) returne to the matrix,driving ADP and PI generate ATP. Most electrons are normally enter respiratory chain and eventually with oxygen and proton to form water. But in some sites of the respiratory chain, small electrons are directly with oxygen to form superoxide. Mitochondria produce a small number of reactive oxygen species used to maintain the cell physiological function in normal physiological condition. But in pathologic conditions, electrons are not able to go through mitochondria respiratory chain smoothly, which causes electronic leakage and produces the massive ROS generation. Excessive ROS cause cell damage.The excessive ROS can be cleared away by antioxidant system in normal condition. Antioxidant ability declines obviously in pathologic condition, which aggravates oxidative damage. Superoxide dismutase (SOD) as an important matter of antioxidant, can unite superoxide, making superoxide into an inactive product. More and more researches show that in pathologic condition SOD plays the antioxidant effect, causing its activity decline. In eukaryote, SOD includes MnSOD located in mitochondrial and Cu-ZnSOD within the enchylema.Therefore, MnSOD become the first line to protect mitochondrial from oxidative damage, and studying changes of MnSOD activity is of great significance.To sum up, such conditions of high glucose, anoxia reperfusion, low glucose and so on can induced oxidative stress, which mediated endothelial cell injury. Whether glucose reperfusion cause endothelial cells oxidative damage and how the relationship between damage and mitochondria, about which the reports have not be seen at present. Therefore, our study observing the levels of oxidative stress and the changes of mitochondrial function by glucose reperfusion in HUVEC-12 as endothelial cell research model, explore endothelial cell injury by glucose reperfusion and the possible mechanism, to protect endothelial cells. Chapterl Effect of glucose reperfusion on oxidative stress in human umbilical vein endothelial cell[Objective]To observe the changes of nitric oxide and ROS in HUVEC-12 cells,explores the relationship between oxidatie stress and cells injury induced by glucose reperfusion.[Methods]1,The experiment object is HUVEC-12 cell, which is cultured in RPMI1640 complete medium with 10% fetal bovin serum, placed in 5% CO2-95% air at 37℃2,There are seven group according to different conditions:①The normal control group(5.5mM group, cells were cultured in RPIM-1640 complete medium containing 5.5mM glucose)②The glucose deprivation group(0mM group, cells were cultured in RPIM-1640 complete medium containing OmM glucose)③The glucose reperfusion group-Ⅰ(0-5.5mM group, cells were reperfused by 5.5mM glucose in 15min after 2h cultured in OmM glucose)㏕he glucose reperfusion group-Ⅱ(0-11.1 mM group, cells were reperfused by 11.1 mM glucose in 15min after 2h cultured in OmM glucose)⑤The glucose reperfusion group-III(0-25mM group, cells were reperfused by 25mM glucose in 15min after 2h cultured in OmM glucose)⑥The persistent high glucose group-Ⅰ(11.1mM group, cells were cultured in RPIM-1640 complete medium containing 11.1mM glucose)⑦The persistent high glucose group-Ⅱ(25mM group, cells were cultured in RPIM-1640 complete medium containing 25mM glucose) The HUVEC-12 cells of there seven group were cultured in basal medium for 24 hour before intervention to keep synchronized growth of cells. Each group officially intervention for 135 minimum.3,NO was detected by nitrate reductase method4,ROS was detected the mean fluorescence intensity of samples by making use of SpectraMax M5/M5e:Dihydroethidium (DHE) was fluorescence detector, excitation wavelength and emission wavelength was set at 535 nm and 610 nm respectively. So compare the production of ROS in HUVEC-12 cells.5,All values are expressed as means±S.D. Statistical analysis,the one way ANOVA was performed using SPSS 13.0 software,and multiple comparisons were carried out using the Least-significant difference test(LSD). Statistical significance was defined as p<0.05.[Result]1,NO levels:The NO levels of the 5.5mM group, OmM group,0-5.5mM group, 0-11.1mM group,0-25mM group, 11.1mM group and 25mM group were 103.63±5.82,45.06±2.73,105.56±5.10,19.57±1.60,17.47±1.74,29.45±0.74 and 26.35±6.28 respectively. Under the different condition, NO levels between groups was statistically significant (P=0.000). In comparison with the 5.5mM group, the NO levels in 0-11.1mM group and 0-25mM group were decreased significantly (P<0.000, respectively),with the 0-11.1mM group lower than the OmM group and 11.1mM group (P<0.01, respectively),.with the 0-25mM group lower than the OmM group and 25mM group (P<0.05, respectively)2,ROS levels:The ROS levels of the 5.5mM group, OmM group,0-5.5mM group, 0-11.1mM group,0-25mM group,11.1mM group and 25mM group were 0.56±0.06,0.88±0.07,0.66±0.08,0.67±0.16,1.35±0.12,0.47±0.06 and 0.82±0.26 respectively.Under the different condition,ROS levels between groups was statistically significant (P=0.000). Comparing to 0-5.5mM group and 0-11.1mM group, the ROS levels of 0-25mM group was increased significantly (P=0.000, respectively), with the 0-25mM group lower than the OmM group and 25mM group (P<0.01, respectively).[Conclusions]1,glucose reperfusion can cause the NO level decreasing. It is suggested glucose reperfusion may induce HEVEC-12 cells injury.2,Glucose reperfusion can induce significant oxidative stress of HEVEC-12 cells. In comparison with the persistent low glucose group and the persistent high glucose group, glucose reperfusion group may trigger more severe oxidative stress, which is negatively correlated to the ROS level. Therefore, oxidative stress induced by glucose reperfusion may lead to the endothelial dysfunction.Chapter 2 Relationship between Oxidative stress induced by Glucose Reperfusion and Mitochondrial Function in human umbilical vein endothelial cell[Objective]To observe the effect of MMP on oxidative stress in glucose reperfusion, describe the relationship between MMP and ROS in different time with reperfuing by different concentration glucose in HUVEC-12 cells. After regulating the level of MMP under the condition of glucose reperfusion, to investigate into the changes of ROS and MnSOD activity.[Methods]1,Cell culture was just the same as those of the chapter 1.2,There are two part according to different conditions:The HUVEC-12 cells of there seven group were cultured in basal medium for 24 hour before intervention to keep synchronized growth of cells.First part:①The normal control group(5.5mM group, cells were cultured in RPIM-1640 complete medium containing 5.5mM glucose)②The glucose reperfusion group-Ⅰ(0-5.5mM group, cells were reperfused by 5.5mM glucose in 15min after 2h cultured in OmM glucose)③The glucose reperfusion group-Ⅱ(0-11.1mM group, cells were reperfused by 11.1 mM glucose in 15min after 2h cultured in OmM glucose)④The glucose reperfusion group-Ⅲ(0-25mM group, cells were reperfused by 25mM glucose in 15min after 2h cultured in OmM glucose)Assay ROS and MMP of HUVEC-12 cells reperfused by different glucose at 5min,10min,15min and detecting MnSOD activity at glucose reperfusing for 15min.Second part:①The normal control group(5.5mM group, cells were cultured in RPIM-1640 complete medium containing 5.5mM glucose)②The glucose reperfusion group-A(0-25mM group, cells were reperfused by 25mM glucose in 15min after 2h cultured in OmM glucose) ③The glucose reperfusion group-B(0-25Mm+CCCP group, cells were reperfused by 25mM glucose in 15min after 2h cultured in OmM glucose with 0.05uM CCCP pre-treatment)Each group officially intervention for 135 minimum and detecting ROS, MMP and MnSOD activity at glucose reperfusing for 15min.3,Detecting ROS was just the same as those of the chapter 1.4,MMP was detected the mean fluorescence intensity of samples by making use of SpectraMax M5/M5e:Rhodamine 123 was fluorescence detector, excitation wavelength and emission wavelength was set at 508 nm and 529 nm respectively. So compare the level of MMP in HUVEC-12 cells.5,MnSOD activity was detected by xanthine oxidase method6,All values are expressed as means±SD. Statistical analysis, the repeated measure AN OVA and one way ANOVA was performed using SPSS 13.0 software. Statistical significance was defined as p<0.05.[Result]1,Comparing to 5.5mM group,0-5.5mM group and 0-11.1mM group, the levels of ROS and MMP in 0-25mM group were increased significantly (P< 0.000, respectively). Along with reperfusing time, the levels of ROS and MMP are gradually increased, which are to the highest level inl5min. With 0.05uM CCCP pre-treatment, comparing to 0-25mM group, the levels of ROS and MMP in 0-25Mm+CCCP group were significantly decreased 17%(P<0.05) and 45% (P=0.001) respectively.2,Changes of MnSOD activity:Comparing to 5.5mM group, MnSOD activity in 0-5.5mM group,0-11.1mM group and 0-25mM group was decreased 11%,12%,25% respectively (P<0.05, respectively).And MnSOD activity in 0-25mM group was the worst in the three glucose reperfusion group (P<0.01, respectively). With 0.05uM CCCP pre-treatment, comparing to 0-25mM group, the MnSOD activity in 0-25Mm+CCCP group were significantly increased 23% (P<0.01)[Conclusions]1,Glucose reperfusion can cause mitochondrial membrane potential increasing2,The increasing ROS and MMP in HUVEC-12 cells induced by glucose reperfusion were positive correlation. Oxidative stress caused by glucose reperfusion may be affected by the mitochondrial membrane potential in HUVEC-12 cells.3,Glucose reperfusion can lead to MnSOD activity decreasing.4,Controlling the level of MMP, the levels of ROS and MnSOD activity in GR group were significantly decreased. Therefore, mitochondrial dysfunction may be one of the mechanism of oxidative stress induced by glucose reperfusion. |
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