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目的:应用高浓度葡萄糖孵育大鼠骨髓内皮祖细胞(EPCs),测定培养上清的丙二醛(MDA)、一氧化氮(NO)水平,同时观察EPCs GPx-1、eNOS的表达,然后应用抗氧化剂ALA进行干预,来探讨葡萄糖引发EPCs损伤的机制及治疗措施。方法:清洁级健康雄性Wistar大鼠(体质量180～200g)15只,分离培养EPCs,用2.5 g/L胰酶/0.2 g/LEDTA消化培养4 d的EPCs,分组贴壁24 h后分别给予葡萄糖5 mmol/L作为正常对照组(NC)、30 mmol/L葡萄糖作为高糖组(HS)以及葡萄糖(30 mmol/L)+α硫辛酸(40μg/L)作为ALA组,孵育EPCs 48 h,实时荧光定量PCR技术及Western blot法测定EPCs GPx-1及eNOS的mRNA及蛋白表达,测定培养液上清的一氧化氮(NO)及MDA水平。结果:(1)不同干预措施对EPCs分泌MDA的影响:高浓度葡萄糖干预48 h后培养上清MDA水平高于正常对照组(P<0.05);应用ALA干预后与高糖组比较MDA水平下降(P<0.05)。(2)不同干预措施对EPCs GPx-1表达的影响:高糖干预48 h后EPCs GPx-1表达显著低于对照组,而ALA干预后EPCs GPx-1表达较高糖组显著增加(P<0.05)。(3)不同干预措施对EPCs eNOS表达及分泌NO的影响:高糖干预48 h后EPCs eNOS表达显著低于对照组,培养上清NO水平低正常对照组(P<0.05),而ALA干预后EPCs eNOS表达较高糖组显著增加(P<0.05),培养上清NO水平升高(P<0.05)。结论:高浓度葡萄糖可以诱发EPCs产生氧化应激,减弱EPCs的抗氧化能力及eNOS表达,使EPCs分泌NO减少,α硫辛酸能够改善高糖导致的EPCs抗氧化能力减弱及NO的分泌。
OBJECTIVE: To investigate the effect of high concentration glucose on EPCs of rat bone marrow, the levels of malondialdehyde (MDA) and nitric oxide (NO) in cultured supernatants were determined, and the expression of GPx-1 and eNOS in EPCs was observed. Antioxidant ALA intervention to explore the mechanism of glucose-induced EPCs injury and treatment. Methods: Fifteen healthy male Wistar rats (body weight 180-200g) were cleaned, EPCs were isolated and cultured, and EPCs were cultured for 4 days with 2.5 g / L trypsin / 0.2 g / LEDTA. Glucose, 5 mmol / L glucose as control group, 30 mmol / L glucose as high glucose group and 30 mmol / L glucose and 40 μg / L α-lipoic acid as ALA group. EPCs were incubated for 48 h The mRNA and protein expressions of GPx-1 and eNOS in EPCs were determined by real-time fluorescence quantitative PCR and Western blot. The contents of nitric oxide (NO) and MDA in supernatants were determined. Results: (1) The effect of different interventions on the secretion of MDA in EPCs: The level of MDA in the culture supernatant after 48 h intervention with high glucose was higher than that in the normal control (P <0.05) (P <0.05). (2) The effect of different interventions on the expression of GPx-1 in EPCs: The GPx-1 expression in EPCs after 48 h intervention was significantly lower than that in control group, while the expression of GPx-1 in EPCs increased significantly after ALA intervention (P < 0.05). (3) The effect of different interventions on the expression of eNOS and the secretion of nitric oxide in EPCs: The expression of eNOS in EPCs after 48 h treatment was significantly lower than that in control group, and the NO level in culture supernatant was lower than that in normal control group (P <0.05) EPCs eNOS expression increased significantly higher glucose group (P <0.05), the level of NO in culture supernatant increased (P <0.05). CONCLUSION: High concentrations of glucose can induce oxidative stress in EPCs, attenuate the anti-oxidative capacity and eNOS expression of EPCs, decrease the NO secretion of EPCs, and α-lipoic acid can improve the anti-oxidation ability and NO secretion of EPCs induced by high glucose.