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目的探讨晚期蛋白氧化产物(AOPPs)对大鼠胰岛微血管内皮细胞(IMECs)体外增殖、迁移及再血管化功能的影响及可能机制。方法分为空白对照(NC)组、鼠血清白蛋白(RSA)组、不同浓度AOPP组(100、200、300μg/ml)及AOPPs 200μg/ml+NADPH氧化酶抑制剂(Apocynin)组。CCK8试剂盒检测IMECs增殖活性;Transwell小室、Matrigal胶及显微镜下细胞计数法检测AOPPs对IMECs体外迁移及再血管化的影响;化学发光法检测还原型辅酶-Ⅱ(NADPH)氧化酶活性;免疫印迹检测磷酸化丝-苏氨酸激酶(p-Akt)、磷酸化内皮型一氧化氮合酶(p-eNOS)蛋白的表达及ELISA检测一氧化氮(NO)水平。结果 AOPPs可呈剂量、时间依赖性降低IMECs体外增殖能力、迁移能力及再血管化能力,增强细胞内NADPH氧化酶的活性,降低细胞内p-Akt、p-eNOS的表达水平以及细胞内NO的水平(P<0.05)。结论 AOPPs可导致胰岛微血管内皮细胞增殖、迁移及再血管化功能受损,可能与下调p-Akt及p-eNOS的表达水平以及加强NADPH氧化酶介导的eNOS脱偶联有关。
Objective To investigate the effects of advanced protein oxidation products (AOPPs) on the proliferation, migration and revascularization of pancreatic islet microvascular endothelial cells (IMECs) in vitro and its possible mechanism. Methods The rats were divided into blank control group (NC), rat serum albumin (RSA) group, AOPP group (100,200,300μg / ml) and AOPPs 200μg / ml + Apocynin group. CCK8 kit was used to detect the proliferation activity of IMECs; the effect of AOPPs on the migration and revascularization of IMECs was detected by Transwell chamber, Matrigal gel and cell counting under microscope; the chemiluminescence method was used to detect the activity of NADPH oxidase; The expressions of p-Akt and p-eNOS were detected by enzyme linked immunosorbent assay (ELISA). The level of nitric oxide (NO) was detected by ELISA. Results AOPPs could reduce the proliferation, migration and revascularization ability of IMECs in vitro in a dose-and time-dependent manner, increase the activity of NADPH oxidase and decrease the expression of p-Akt, p-eNOS and intracellular NO Level (P <0.05). CONCLUSION: AOPPs can lead to impaired proliferation, migration and vascularization of islet microvascular endothelial cells, which may be related to down-regulation of p-Akt and p-eNOS expression and enhancement of NADPH oxidase-mediated eNOS decoupling.