目的：观察槲皮素（quercetin ，QUE）诱导的血红素氧合酶-1（heme oxygenase-1，HO-1）对离体星形胶质细胞（astrocytes ，AS）缺氧缺糖（oxygen/glucose deprivation ，OGD）损伤的保护作用。方法（1）新生24 h内的Sprague-Dawley （SD）大鼠，取其新鲜大脑皮质组织，体外培养并纯化获得纯度达90％以上的AS ，采用神经胶质纤维酸性蛋白（glia fibrillary acid protein ，GFAP）免疫荧光技术鉴定AS。（2）应用2 mmol/L连二亚硫酸钠（Na2 S2 O4）联合无糖培养基建立细胞OGD模型，将AS分为Control组、OGD组、OGD＋QUE组，QUE终浓度分别为1μmol/L、3μmol/L、10μmol/L 和30μmol/L ，培养6 h后CCK-8法检测细胞相对存活率，使用相应试剂盒检测各组LDH漏出量、MDA和SOD水平。（3）Western blotting法检测10μmol/L、25μmol/L、50μmol/L 和100μmol/L的QUE共孵育正常培养的AS 8 h后对HO-1蛋白表达水平的影响，并观察10μmol/L QUE共孵育正常培养的AS 3 h、6 h和12 h后对其表达水平的影响。结果（1）细胞免疫荧光鉴定显示大多数细胞为GFAP阳性其纯度达95％以上。（2）10μmol/L和30μmol/L的QUE共孵育能显著提高OGD损伤后AS的相对存活率，并能显著降低OGD损伤后AS的LDH漏出量、MDA水平，提高SOD水平，与OGD组相比差异均有统计学意义（P＜0.05）。（3）与Control组相比，10μmol/L QUE孵育正常培养的AS 8 h后较25μmol/L、50μmol/L和100μmol/L的QUE显著提高了HO-1蛋白的表达水平（P＜0.01）；10μmol/L QUE共孵育AS 6 h和12 h后较Control组HO-1蛋白表达水平显著升高（P＜0.01），以6 h组为著。结论10～30μmol/L QUE可保护Na2 S2 O4联合无糖培养基对AS的OGD损伤；QUE对AS OGD损伤的保护作用与其能诱导 AS高表达 HO-1蛋白有关。%Objective To investigate the protective effect of Quercetin (QUE) on astrocytes (AS) injured by oxygen/glu-cose deprivation (OGD) in vitro and explore whether heme oxygenase-1 (HO-1) was involved in this effect. Methods The AS obtained from cerebral cortex of newborn 24 h SD rat were cultured in vitro. After being purified to over 90% ,cells were iden-tified by immunofluorescent staining with GFAP antibody.OGD injury model of AS was established by 2 mmol/L Na2 S2 O4 combined with sugar-free medium ,AS were divided into control group ,OGD group and OGD+ QUE (1 μmol/L ,3 μmol/L , 10 μmol/L and 30 μmol/L) groups ;AS were treated by different concentrations of QUE under OGD condition for 6 h;the via-bility of AS was measured by CCK-8 assay and LDH release magnitude was measured by LDH assay ;relative MDA level was measured by MDA assay and relative SOD level was measured by SOD assay.In this work ,the levels of HO-1 protein were quantified using by western blotting ;we investigated HO-1 protein expression induced by different concentrations of QUE (10μmol/L ,25 μmol/L ,50 μmol/L and 100 μmol/L) after incubation for 8 h and 10 μmol/L QUE at 3 h ,6 h and 12 h course.Results (1) Immunofluorescence staining showed that most of the cells were GFAP-positive and the purity was over 95%.(2) The viability of AS ,the magnitude of LDH release and MDA relative level were significantly suppressed while the SOD relative level improved by QUE at 10 μmol/L and 30μmol/L compared with the OGD group (P< 0.05). (3) Compared to the control group ,the expression levels of HO-1 protein were significantly up-regulated by QUE at 10 μmol/L for 8 h incubation ,which were higher than the 25 μmol/L group ,50 μmol/L group and 100 μmol/L group (P< 0.01);QUE at 10 μmol/L incubated AS for 6 h and 12 h could statistical significantly up-regulated the HO-1 protein level compared with the control group (P<0.01) ,and the levels in 6 h group were much higher than 12 group. Conclusion 10~30μmol/L QUE can protect AS from the injury of OGD induced by Na2 S2 O4 combined with sugar-free medium. The protection of QUE on AS OGD may be associated with its induction of the HO-1 protein.
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