| BackgroundThe prevalence rate of diabetes mellitus (DM) is rising year and year, and has become the third chronic non-communicable diseases like cardiovascular disease and tumor which were the global threat of human health. With the improvement of people’s living and the changes of life style, the morbidity of type2diabetes (T2DM) presents a tendency of rapid increasing in our country. According to the latest result from epidemiological survey in2013, the estimated prevalence of DM was11.6%which meant there were about1.139hundred million patients in adults of18years old and above diagnosed with the newest international clinical criteria. The more amazed result was that the prevalence of impaired glucose tolerance (IGT) was up to50.1%in Chinese adult and the onset of DM became younger in age. Acute and chronic complications caused by DM, specially the diabetic macro-and micro-vascular complications, become the main cause of deaths and disabilities and severely impact the quality of life and lifetime.Insulin resistance (IR) and β cells hypofunction are two major reasons for T2DM. Current treatment includes promoting insulin secretion, improving insulin resistance, inhibiting the absorption of glucose and inhibiting hepatic glucose output. These treatments still cannot stop progressive failure of β cells function with the course of disease proceeding, even they can improve the blood glucose. In recent years, more studies supposed that micro-circulation disorder of β cells was closely related to the injury and hypofunction of (3cells. Islet is a organ of high vascularity. Islet capillaries are not only the crucial link between islet cells and the body, but also the microenvironment for cells to live by. Islet has unique microvascular system which contains intensive capillary network with the density of5times as peripheric exocrine organization. The abundant blood supply could not only satisfy its need for oxygen and nutrition, but also could regulate the metabolic balance in the body when it rapidly percept the changes of metabolic environment and distribute various hormones to relevant target tissue via blood circulation. Therefore the microcirculatory system is a important regulator for islet endocrine function. The intact structure and function of islet microcirculation depend on the islet microvascular endothelial cells (IMECs) which have caused researcher’s concern for these years. IMECs, the most elements of islet microcirculation-tissue barriers, have a close relationship with islet endocrine function and directly participate in the development of islet hypofunction. With the effect of islet capillaries in the pathogenesis of T2DM being revealed, Tal MG proposed that β cells hypofunction was probably the representation of diseased islet capillaries in2009. He said that the dysfunction and injury of IMECs was the important factor resulting in the disease of islet capillaries, which could cause anoxia and ischemic injury in islet microcirculation and eventually cause progressive dysfunction of β cells. Therefore, to deeply study the physiopathologic mechanism in dysfunction and injury of IMECs and the effective treatment has exerting great impact on protecting the function of β cells and looking for new targets to prevent T2DM.The dysfunction and injury of vascular endothelial cells (VEC) is the precondition and basis of the development in diabetic microvascular complications. Glucotoxicity, lipotoxicity, the change of hemarheology and haemodynamics and reduced antioxidant capacity of tissue are closely linked to dysfunction of vascular endothelial cells. The vasodilator factor NO reduce synthesis and the vasoconstrictor factor endothelin-1increase production, which can directly stimulate the hyperplasia of intimal arterial smooth muscle and the expression of fibronectin and collagen IV. In the state of hyperglycemia, the synthesis and release of intercellular adhersion molecule1(ICAM-1) and vascular cell adhesion molecule1(VCAM-1) increased by VECs, which could enhance the adherence of leucocyte and platelet resulting in activation of inflammatory cells and intimal inflammation. The change could induce medial smooth muscle cells migrating under intima and lipid depositing in cells, which accelerated formation of arterial atherosclerosis. As a kind of VECs, IMECs should be damaged by different pathogenic factors In the state of hyperglycemia, which could result in dysfunction of endothelial cells and islet microcirculation, specially the dysfunction of islet cells.Advanced oxidative protein products (AOPPs), considered as a novel marker of oxidant-mediated protein damage, are dityrosine-containing and cross-linking protein products mainly formed during oxidative stress by reaction of plasma albumin with chlorinated oxidants. AOPPs were firstly found in the plasma of patients undergoing dialysis and were subsequently found in subjects with diabetes. The structure and biological function of AOPPs was similar to advanced glycation end products (AGEs) which could combine with RAGE (receptor of AGEs) to activate NADPH oxidase, intra-cellular signaling of redox reaction such as ERK1/2, p38MAPK and translocation of nuclear factor-KB resulting in inflammation and oxidative stress in VECs. Chronic accumulation of AOPPs aggravated accumulation of oxidized low density lipoprotein (LDL) in arterial wall and worsen inflammation and oxidative stress in artery to accelerate atherosclerosis in a hyperlipidemic model. The level of AOPPs closely correlated with the degree of carotid atherosclerosis in patients with CKD, which meant that accumulation of AOPPs could play a role in atherosclerosis of patients with CKD. Gradinaru D and his team discussed the relation between level of AOPPs and the factors leading to atherosclerosis such as endothelium NO synthetase, oxidative stress and metabolic profiling in patients with diabetes or prediabetes. They found that AOPPs significantly increased compared with normal control group and inhibited activity of endothelium NO synthetase to reduce synthesis of NO, which suggested that the level of AOPPs closely correlated with the degree of diabetic atherosclerosis in old patients with DM. Bansal S and team conducted an analysis of gene polymorphism of RAGE and its relation with oxidative stress products, paraoxonase and diabetic macro-vascular complications in265Indian patients with T2DM. They found that there existed a positive correlation between the genotype of429T/C and level of oxidative stress products, development of diabetic macro-vascular complications. Thus, apart from being regarded as oxidative stress makers, AOPPs has also been shown to be a pro-inflammatory factor in accelerating the progress of atherosclerosis and damaging vascular system in our body. However, few studies aimed at the effect of AOPPs in islet microcirculation, especially in the function of IMECs.GLP-1is a polypeptide secreted by the intestinal L cell. GLP-1is best known as a safe and effective insulinotropic hormone and has been proposed as prospective approach to clinical treatment of T2DM. More and more studies indicated that GLP-1not only stimulated survival and proliferation, increased insulin secretion, inhibited appetite, retarded peristalsis of gastrointestinal smooth muscle and inhibited release of glucagon, but also plays an important role in diabetic cardiovascular complications. The receptor of GLP-1(GLP-1R) expressed in vascular smooth muscle, cardiac muscle, endocardium and coronary endothelium. The agonist of GLP-1R could protect cardiac muscle from ischemic-reperfusion and improved the function of heart. On one hand, exendin-4significantly reduced the area of myocardial infarction in vitro, on the other hand, exendin-4and GLP-1could enhance left ventricular systolic function. Timmers and his team also found that exendin-4sustaining treatment could significantly reduce the area of myocardial infarction and promote the recovery of myocardial contractive and systolic function in a pig model after ligation of coronary artery. GLP-1could inhibit monocyte from adhering to aortic vascular endothelial cells, alleviate vascular inflammatory injury and stop the progress of AS. Supplementing GLP-1in rats with IR and hypertension could improve the function of vascular endothelium, blood pressure and cardiac function. In the clinical study, GLP-1could improve the vascular endothelial function of patients with T2DM and stable CAD. In recent years, researchers suggested that GLP-1significantly blocked the oxidative stress of endothelial cell induced by AGEs-RAGE through the down-regulation of RAGE and ICAM-1and reduced intimal hyperplasia and smooth muscle hyperplasia after the injury of endothelial cells induced by AGEs in vitro, which meant that GLP-1had potential effect of anti-inflammatory and anti-atherosclerosis. Exendin-4promoted the proliferation of endothelial cells through PI3K/Akt-eNOS signal pathway. In the study of rats model with endothelial cell dysfunction and AS, liraglutide, GLP-1receptor agonist, could improve dysfunction of VECs and alleviate the degree of AS. In2012, chinese researchers found that GLP-1could alleviate the apoptosis of human umbilical vein endothelial cells (HUVECs) induced by AGEs through activation of PI3K/Akt signal pathway by the means of regulating the expression of Bcl-2, Bax and cyto-c in the endothelial cells. In2013, Erdogadu.O and his team conducted a study about the effect of GLP-1in human coronary artery endothelial cells (HCAECs) induced by lipotoxicity, and found that GLP-1could improve the proliferation, metastasis and vasculogenesis of aortic endothelial cells and reduced the apoptosis through PKA/PI3K/Akt/eNOS, p38MAPK and JNK kinase-dependent pathway.Thus, apart from the hypoglycemic effect, GLP-1also play a important role in protecting endothelial cells and improving the function of them, which through PKA/PI3K/Akt pathway. However, did GLP-1have the same protective effect in the injury of IMECs induced by AOPPs under the state of DM? Did it can improve the function of IMECs and islet microcirculation so that delay the progressive dysfunction of Pcells? All of these are important issues for our discussion.It is very important to investigate the effects of GLP-1on islet microcirculation and its possible mechanisms for discovering a new therapeutic target to protect the function of islet cells in T2DM patients. In this study, we established the damaged model by treating IMECs with AOPP-modified RSA (AOPP-RSA), in order to explore the effects and related mechanisms of GLP-1in the apoptosis, proliferation, migration and revascularization of IMECs induced by AOPPs, and try to provide a new viewpoint for GLP-1treatment which could delay the progressive dysfunction of β-cells.ObjectivesIn this project we aim to on the base of damaged model induced by AOPPs, to observe the effects of GLP-1on the apoptosis, proliferation, migration and revascularization of IMECs induced by AOPPs, and to investigate the effect of GLP-1on apoptosis-relative proteins expression and PI3K/Akt/eNOS pathway, also to discuss the protective role of GLP-1and its molecular mechanisms in the dysfunction of IMECs induced by AOPPs.ContentThe whole project includes three parts:Part I The effect of AOPPs on the apoptosis of islet microvascular endothelial cells (IMECs) and its possible mechanismObjectivesTo explore the effect and mechanism of AOPPs on the apoptosis of IMECsMethods1. Preparation of AOPP-RSA20mg/ml rat serum albumin (RSA) was mixed with the same volume of hypochlorous (40mmol/1), and then was placed for30min. The molar ratio of RSA and hypochlorous in AOPP was1:140. Excessive dissociative hypochlorous was removed by dialysis with non-endotoxin PBS for24h. The solution was sterilized by passing it through a0.22micron bacteria-retentive filter.2. Isolation and digestion of rat islet cellsWe used a modified method of collagenase digestion and Ficoll density gradient separation for isolation and digestion of islet from the rat. The cells were stained with DTZ and typan blue, and the concentration of cells was adjusted to500IEQ/ml. These cells were cultured at34℃in a humidified atmosphere of5%CO2.3. Purification and identification of IMECsThe growth of spindle cells in the islet was observed after culturing3-5days in vitro and the proliferation of cells was observed in7-9days. IMECs which were carried by the2%gelatin were cultured after immunomagnetic beads. These cells were identified for von Willebrand factor (vWF) antigen and Dil-acetylated low density lipoprotein (Dil-Ac-LDL) by immunofluorescence.4. Groups:The effect of different concentrations of AOPPs on the apoptosis of IMECs: AOPPs-RSA treated the cells at concentrations of100,200,300μg/mL for48h. The effect of AOPPs on the apoptosis of IMECs for different time:AOPPs-RSA alone (final concentration200μg/mL) incubated with cells for0,12,24,48,72h respectively.5. The apoptosis of IMECs with Hoechst33258dye staining and AnnexinV-FITC/PICells were treated with4%paraformaldehyde for5min in4℃after indicated treatment. The morphology of cells apoptosis were observed by fluorescence microscope after Hoechst33258dye staining. Cells were added AnnexinV and PI after the digestion with trypsin, and then placed for15min protecting from light. The early apoptosis rate was detected with flow cytometry and analyzed by Cell Quest software.6. The ROS level in IMECs detected by DHE fluorescent probeThe cells were divided into4groups:normal control group, RSA control group,200μg/ml AOPPs group and200μg/ml AOPPs+apocynin group, and all the groups were accepted identified treatment for48h. Cells were collected and cultured in the solution of DHE at37℃for30min. After the removal of too much solution, the distributions of DCF in cells were observed by laser scanning confocal microscope. All the groups were selected3horizons to be scanned and analyzed the relative intensity of fluorescence.7. The expression of BcI-2and Bax in IMECs with Western blotThe total protein of all groups were extracted and detected by BCA method. After electrophoresis in12%SDS-PAGE, transfer, blocking and washing, all the groups were incubated with anti-Bcl-2monoclonal antibody(1:1000) and anti-Bax monoclonal antibody(1:2000) at4℃over night. Cells were conducted hybridization with secondary antibodies labeled with horseradish peroxidase for1h and coloration, and the results were analyzed by ImageJ software.8. The activities of caspase-9and-3were detected by ELISACells of all the groups were accepted identified treatment for48h and then were collected. Cells were then added to the reaction substrate correspondingly. Incubated in37℃water bath for2h, finally using ELISA meter measured OD values at the detection wavelength of405nm.Statistical AnalysisAll analyses were carried out with SPSS13.0software. Data were expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. We used Spearman correlation analysis for correlation analysis. Statistical significance was defined as two-sided P<0.05.Results1. The effect of different concentration of AOPPs on the apoptosis of IMECs for48hAfter treatment with100,200,300μg/ml AOPPs for48h, the apoptosis rate were14.57±4.7,24.20±4.30,35.53±6.47, respectively, and significantly higher than those of normal control group (5.87±1.51) and RSA control group (6.90±1.42)(P<0.05). Spearman correlation analysis showed that a significant positive correlation existed between the apoptosis rate of IMECs and the AOPPs concentrations (100,200,300ug/ml)(r=0.949, P=0.000). 2. The effect of AOPPs on the apoptosis of IMECs for different timeThe apoptosis rate of200μg/ml AOPPs incubated for different time (12,24,48,72h) were11.61±1.94,16.69±3.17,23.48±4.69,32.80±7.13, respectively, and significantly higher than control group (0h)(6.10±2.07)(F=17.854, P=0.000). ntrol group (5.87±1.51) and RSA control group (6.90±1.42)(P<0.05). Spearman correlation analysis showed that a significant positive correlation existed between the apoptosis rate of IMECs and time (r=0.950, P=0.000).3. The effect of AOPPs on ROS level of IMECsThe fluorescence value of ROS in200μg/ml AOPPs group was58.43±2.71, and significantly higher than that of RSA control group (27.49±4.75)(P=0.000), which suggested that AOPPs stimulated the prodiction of ROS in IMECs. Treated IMECs with apocynin (inhibitor of NADPH oxidase) for48h, the fluorescence value of ROS was significantly down to30.45±4.35compared with the200μg/ml AOPPs group (P=0.000).4. The effect of AOPPs on the expression of Bcl-2, Bax of IMECsAfter identified treatment for48h, the expressions of Bcl-2were significantly different among the groups (F=4.675, P=0.036). The expression of Bcl-2in200μg/ml AOPPs group was0.60±0.09, which significantly lower than that of RSA control group (P=0.021). Treated IMECs with apocynin (inhibitor of NADPH oxidase) for48h, the expression of Bcl-2was significantly up to0.87±0.14compared with the200μg/ml AOPPs group (P=0.033). The expression of Bax in200μg/ml AOPPs group was1.35±0.19, which significantly higher than that of RSA control group (P=0.008). Treated IMECs with apocynin, the expression of Bax was significantly down to0.97±0.15compared with the200μg/ml AOPPs group (P=0.021).5. The effect of AOPPs on the activities of caspase-9,-3in IMECsAfter identified treatment for48h, the activities of caspase-9,-3were significantly different among the groups (F=144.224and311.617, P=0.000and P=0.000). The activities of caspase-9,-3were1.24±0.08and1.01±0.04, which significantly higher than those of normal control group (P<0.05) and RSA control group (P<0.05). Pretreatment IMECs with apocynin, the activities of caspase-9,-3were down to 0.45±0.06and0.40±0.04compared with the200μg/ml AOPPs group (P<0.05and P<0.05).ConclusionsApoptosis was induced by AOPPs in a dose, time-dependent manner. Exposure to AOPPs for48h caused a significant increase in ROS generation, expression of Bax and activities of caspase-9and-3, but a decrease in expression of Bcl-2. However, co-incubation with apocynin (inhibitor of NADPH oxidase) attenuated these AOPPs-induced effects in IMECs. Therefore, we speculated that AOPPs aggravated the oxidative stress injury through activity of NADPH oxidase and increasing the level of ROS, and then induced the apoptosis of IMECs by the regulation of Bcl-2/Bax, caspase-9and-3.Part Ⅱ The effect and mechanism of AOPPs on the proliferation, migration activity and revascularization of IMECsObjectivesTo explore the effect of AOPPs on the proliferation, migration activity and revascularization of IMECs, and then to investigate the mechanism of AOPPs on the injury and dysfunction of IMECsMethod1. Group:The effect of different concentrations of AOPPs on the proliferation, migration activity and revascularization of IMECs:the cells were divided into6groups:normal control group, RSA control group,100μg/ml AOPPs group,200μg/ml AOPPs group,300μg/ml AOPPs group and200μg/ml AOPPs+apocynin group. We chose24h as intervention time to observe the migration activity of IMECs excluding the effect of apoptosis and48h to observe the the proliferation and revascularization of IMECs.The effect of AOPPs on the proliferation of IMECs for different time:200μg/ml AOPPs alone incubated with cells forO,12,24,48,72h respectively.2. The proliferation activity of IMECs with CCK-8method The cells were planted in96-well plates. After each experimental treatment, we added10μl cell counting kit-8solution into each hole, and incubated at37℃in a humidified atmosphere of5%CO2for1h. The OD value was detected by ELISA with wavelengths of450nm to reflect the proliferation activity.3. The effect of AOPPs on the migration activity of IMECs with Transwell assayThe cells were cultured in serum-free medium for12-24h and the density of cells was adjusted to1×105. The upper-well was added in200μl cell suspension of IMECs, RSA, different concentration of AOPPs (100μg/ml,200μg/ml,300μg/ml) and apocynin, and the lower-well was added in600μl DMEM with10%fetal bovine serum. The cells were cultured at37℃for24h. After the fixation and staining, we counted the cells and gently erased the non-migrated cells in the upper-well by swab.4. The effect of AOPPs on the revascularization of IMECs with matrigelThe high concentration of matrigel was placed in96-well plates (70μl/hole) and formed into gel in incubator for30min after melting over night. The IMECs were planted in each hole with serum-free medium and added in RSA, different concentration of AOPPs (100μg/ml,200μg/ml,300μg/ml) and apocynin. The cells were incubated at37℃in a humidified atmosphere of5%CO2for48h and detected by invert microscope with3views for each hole.5. The effect of AOPPs on the activity of NADPH oxidase in IMECsAfter each experimental treatment for48h, we prepared the cell homogenate and detected the protein content in them.100μg proteins of each group were planted in96-well plates, and added in5μm lucigenin and100μm substrate of NADPH. The fluorescence quantum produced by NADPH-dependent O2-was detected with VICTOR1420multi-label analyzer, which indirectly reflected the production of O2-and activity of NAPDH oxidase. The data were expressed as counts per second (CPS).6. The effect of AOPPs on the expression of p-Akt and p-eNOS in IMECs with Western blot The cells were treated with200μg/ml RSA and different concentration of AOPPs (100,200,300μg/ml) and apocynin for48h. The detailed experimental procedure and methods referred to part1.7. The effect of AOPPs on the level of NO in IMECsAccording to standard operating procedure of NO detection kit produced from Beyotime, we detected the level of NO in the cells. The standard substance was diluted with DMEM and10%FBS, and placed50μl of each hole in96-well plates with the supernatant. The absorbance was detected by the wavelength of540nm after adding Griess Reagent I and Griess Reagent Ⅱ at room temperature.Statistical AnalysisAll analyses were carried out with SPSS13.0software. Data were expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided P<0.05.Results1. The effect of different concentration of AOPPs on the proliferation of IMECsAfter treatment with100,200,300μg/ml AOPPs for48h, the OD value were1.02±0.06,0.75±0.07,0.55±0.08, respectively, and significantly lower than that of RSA control group (1.29±0.08)(P=0.000). Pretreated IMECs with apocynin, the OD value was up to1.22±0.12compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the OD value of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000).2. The effect of AOPPs on the proliferation of IMECs for different timeThe OD values of200μg/ml AOPPs incubated for different time (12,24,48,72h) were1.11±0.08,0.96±0.07,0.75±0.07,0.53±0.11, respectively, and significantly lower than that of RSA control group (1.29±0.08)(P=0.000). A significant negative correlation existed between the proliferation of IMECs and time (r=-0.972, P=0.000).3. The effect of AOPPs on the migration activity of IMECs After treatment with100,200,300μg/ml AOPPs for48h, the cell counts of penetrating to lower-well were266.00±11.14,184.00±9.17,135.00±10.44, respectively, and significantly lower than those of nomal control group and RSA control group (P=0.000and P=0.000). Pretreated IMECs with apocynin, the cell count of penetrating to lower-well was up to305.67±12.22compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the migration activity of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.972, P=0.000).4. The effect of AOPPs on the capacity of forming3d structure of IMECs in matrigelAfter treatment with100,200,300μg/ml AOPPs for48h, the counts of forming3d structure of IMECs in matrigel were7.33±0.58,5.67±0.57,3.33±0.57, respectively, and significantly lower than those of nomal control group (P<0.05) and RSA control group (P<0.05). Pretreated IMECs with apocynin, the count of forming3d structure in matrigel was up to9.33±1.53compared with the200μg/ml AOPPs group (P<0.05). A significant negative correlation existed between the migration activity of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.978, P=0.000).5. The effect of AOPPs on the activity of NADPH oxidase of IMECsThe production of NADPH-dependent O32-increased with the increase of AOPPs concentration, and the300μg/ml AOPPs group reached the peak. The productions of NADPH-dependent O2-were significantly different among the groups (F=59.389, P=0.000). A significant negative correlation existed between the activity of NADPH oxidase of IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000).6. The effect of AOPPs on the expression of p-Akt and p-eNOS of IMECsThe expressions of p-Akt and p-eNOS of IMECs were significantly different among the groups (F=32.631, P=0.000) and (F=21.791, P=0.000). With the AOPPs concentration increasing, the expression of p-Akt and p-eNOS significantly decreased, compared with those of RSA control group (P<0.05) and (P<0.05). Treated IMECs with apocynin, the expressions of p-Akt and p-eNOS significantly increased compared with other groups. A significant negative correlation existed between the expression of p-Akt, p-eNOS and the AOPPs concentrations (100,200,300μg/ml)(r=-0.949, P=0.000) and (r=-0.926, P=0.000).7. The effect of AOPPs on the level of NO in IMECsThe levels of NO of IMECs were significantly different among the groups (F=27.649, P=0.000). After treatment with100,200,300μg/ml AOPPs for48h, the levels of NO were15.13±1.48,12.14±1.44,8.87±1.47, respectively, and significantly lower than those of nomal control group (19.56±0.81)(P<0.05) and RSA control group (18.64±1.36)(P<0.05). Pretreated IMECs with apocynin, the level of NO was up to17.89±1.25compared with the200μg/ml AOPPs group. A significant negative correlation existed between the levels of NO in IMECs and the AOPPs concentrations (100,200,300μg/ml)(r=-0.896, P=0.000).ConclusionsAOPPs could significantly lower the proliferation, migration activity and revascularization of IMECs, enhanced the activity of NADPH oxidase, and decreased the expressions of p-Akt, p-eNOS and the level of NO. Therefore, we speculated that AOPPS decreased the level of NO not only through the down-regulation of p-Akt and p-eNOS in PI3K/Akt pathway but also via increasing the activity of NADPH oxidase to enhance eNOS uncoupling. More importantly, the decreased NO level induced the injury and dysfunction of IMECs.Part III The protection of GLP-1and its mechanism against the damage and dysfunction of IMECs cells induced by AOPPsObjective:Our study investigated the protective effect of GLP-1on the apoptosis, proliferation, migration, the capacity of revascularization and the influence on PI3K/Akt and its downstream molecule signal pathway in IMECs cells induced by AOPPsMethod: 1. Group:The whole experiment divided into negative control group (200μg/mLRSA), AOPPs200μg/mL group, AOPPs200μg/mL±100nmol/LGLP-1group, AOPPs200μg/mL+100nmol/L GLP-1±10μmol/L LY294002group (added APOO-RSA and GLP-1after preprocessing1hour by LY294002, the specific inhibitor of PI3K). Serum-free medium was used to attenuaate all kinds of stock solution, and then collected cells after effect for a certain time. We chose24hours as the intervention time in case to exclude the influence of apoptosis when we observed the capacity of migration, while we chose48hours as the intervention time when we observed the apoptosis, proliferation, and the capacity of revascularization.2. Apoptosis detected by Annexin V-FITC/PI staining techniqueThe method was the same as we mentioned in the first part.3. Cell proliferation activity detected by using CCK-8The method was the same as we mentioned in the second part.4. The effect of GLP-1on the migration in vitro of IMECs cells induced by AOPPs was measured by Transwell chamberThe method was the same as we mentioned in the second part.5. The capacity of revascularization of IMECs cells detected by using MatrigelThe method was the same as we mentioned in the second part.6. The ROS level in IMECs cells was measured by DHE fluorescence probeThe method was the same as we mentioned in the first part.7. The expressions of RAGE, Bcl-2, Bax, p-Akt, p-eNOS were detected by Western blottingThe method was the same as we mentioned in the first and second part.8. The NO level in islet microvascular endothelial cellThe method was the same as we mentioned in the first and second part.Statistical analysesStatistical analyse was performed using SPSS version13.0software. Quantitative data normality was assessed with the One-Way ANOVA test and is expressed as mean±SD. Comparisons between quantitative data were conducted using LSD tests. A value of p<0.05was considered statistically significant.Results1. The influence of GLP-1on the apoptosis in AOPPs induced IMECs cellsThe apoptosis rate of IMECs cells have significant difference between treatment groups (F=43.433, P=43.433). Apoptosis rate was25.05±3.46%about48hours after200μg/mL AOPPs effect on IMECs cells, and was significantly higher than the RSA control group8.57±0.86%(P<0.05). The apoptosis rate was significantly lower (P<0.05) after giving the treatment of GLP-1(100nmol/L). The apoptosis rate increased significantly (P<0.05) in the group which was first pre-processed by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1.2. The influence of GLP-1on the proliferation vitality in IMECs cells induced by AOPPsThe OD value of AOPPs200mu g/mL inducing pancreatic microvascular endothelial cells after48h was0.65±0.08, and was significant differences (P=0.000) compared with RSA control group. The OD value of the combined treatment group giving GLP-1was1.13±0.14, significantly higher than that of AOPPs individual intervention group (P=0.001). The OD value of LY294002pre-processing group was0.72±0.10, decreased significantly (P=0.001) compared with AOPPs±glp-1group, and suggested that LY294002pre-processing could partially blocks the effect of GLP-1to restore cell vitality.3. The influence of GLP-1to the ability of migration in IMECs cells induced by AOPPs.After stimulation by giving AOPPs200μg/mL for24hours, the quantity of cells which migrated from Transwell upper chamber to the lower chamber was196.33±15.50, and significantly reduced compared with RSA control group (306.00±13.11)(P<0.05). The quantity of cells which migrated after giving GLP-1(100nmol/L) was296.67±17.62, and was significantly increased (P<0.05). And the cell migration rate of the group first pre-processing by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1was significantly reduced (216.00±21.07)(P<0.05).4. The influence of GLP-1on the three-dimensional structure which formated in the Matrigel in IMECs cells induced by AOPPsThe quantity of the three-dimensional structure was4.67±0.58in the group of AOPPs200μg/mL intervented48hours, and was significantly reduced compared with RSA negative control (9.33±0.58)the number of cells form a three-dimensional structure (4.67±0.58)(P=0.000). The quantity of the three-dimensional structure was significantly increased (8.00±1.00)(P<0.05) by giving GLP-1(100nmol/L). And the quantity of the three-dimensional structure of the group first pre-processing by PI3K inhibitors LY294002and then intervened by AOPPs and GLP-1did not see a significant increased (6.00±1.00)(P<0.05).5. The influence of GLP-1on the activity of NADPH oxidase in IMECs cells induced by AOPPsThe NADPH dependent02-production in islet microvascular endothelial cells was (367.67±21.94) by giving AOPPs200μg/mL, and significantly increased compared with RSA control group (P=0.000). The NADPH dependent O2production of the combined treatment group giving GLP-1was significantly reduce (158.00+13.00). And the NADPH dependent O2-production in the LY294002pre-processing group was348.33-18.2, s... |
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