摘要
目的探讨银杏二萜内酯葡胺注射液(DGMI)抑制缺糖缺氧(OGD)诱导人源性神经母细胞瘤细胞(SH-SY5Y)的氧化应激损伤及其机制。方法 SH-SY5Y细胞随机分为正常细胞对照组,OGD模型组,OGD 4 h+DGMI 25 mg·L^(-1)组,OGD 4 h+银杏叶提取物注射液(EGBLI)25 mg·L^(-1)组,OGD 4 h+银杏内酯注射液(LGBI)25 mg·L^(-1)组。药物作用6 h后,CCK-8法检测细胞存活,水溶性四唑盐1(WST-1)显色法法检测SOD活性,荧光探针法(DCFH-DA)检测ROS生成,Western蛋白印迹法检测细胞内血红素加氧酶(HO-1)、醌氧化还原酶(Nqo1)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、细胞内核因子E2相关因子2(Nrf2)、磷酸化Nrf2(p-Nrf2)的表达;OGD 4 h+DGMI 25 mg·L^(-1)组加入PI3K抑制剂LY294002(终浓度为0,12.5,25和50μmol·L^(-1)),检测1 h后Akt,p-Akt,Nrf2和p-Nrf2的蛋白表达。结果与正常细胞对照组相比,OGD模型组SH-SY5Y细胞存活和SOD活性降低(P<0.01),ROS含量升高(P<0.01),降低p-Akt,Nrf2,p-Nrf2,HO-1和Nqo1的表达(P<0.01);与OGD模型组相比,OGD 4 h+药物组作用6 h,细胞存活率和SOD活性显著提高(P<0.01),ROS含量降低(P<0.05,P<0.01),p-Akt,Nrf2,p-Nrf2,HO-1和Nqo1蛋白表达水平提高(P<0.01),且DGMI 25 mg·L^(-1)效果优于EGBLI 25 mg·L^(-1)和LGBI 25 mg·L^(-1)(P<0.05,P<0.01);相较DGMI组,DGMI和LY294002作用1 h后p-Akt和p-Nrf2的表达被显著抑制(P<0.01),抑制效果呈浓度依赖性。结论 DGMI 25 mg·L^(-1)对OGD诱导的SH-SY5Y细胞具有抗氧化保护作用,其机制可能与激活Akt/Nrf2通路有关。
OBJECTIVE To investigate the protective effects and mechanism of diterpene ginkgolides meglumine injection（DGMI） against oxidative stress induced by oxygen-glucose deprivation（OGD） in SH-SY5 Y cel s. METHODS SH-SY5 Y cel s were divided into five groups： normal control, model control（OGD group） and drug（25 mg · L（-1）） administration groups including DGMI group, extract of ginkgo biloba leaves injection group（EGBLI） and lactones ginkgo biloba injection group（LGBI）. The cells suffered from oxygen-glucose deprivation（OGD） for 4 h, followed by reoxygenation with drugs for 6 h.Then, cell viabilities were detect using CCK-8 assays, reactive oxygen species（ROS） levels using fluorescence probe DCFH-DA and superoxide dismutase（SOD） activities using WST-1 test. Western blotting was used to detected protein levels of hemeoxygenase-1（HO-1）, NAD（P）H, quinone oxidoreductase l（Nqo1）, protein kinase B（Akt）, phosphorylated Akt（p-Akt）, nuclear factor-E2-related factor2（Nrf2） and phosphorylated Nrf2（p-Nrf2）. The cells were induced by OGD for 4 h, followed by reoxygenation and DGMI for 1 h, combined with different concentrations of PI3 K inhibitor（LY294002）（at the final concentration of 12.5, 25 and 50 μmol·L（-1）） before the protein levels of AKT, p-AKT, Nrf2 and p-Nrf2 were detected by Western blotting. RESULTS SH-SY5 Y cells induced by OGD for 4 h resulted in an increase in ROS（P〈0.01）, but a decrease in cell viabilities（P〈0.01）, SOD activities（P〈0.01）, and antioxidant protein levels（ Akt, p-Akt, Nrf2, p-Nrf2, HO-1 and Nqo1）（P〈0.01）. Compared with OGD group, treatment with reoxygenation and drugs（DGMI,EGBLI and LGBI respectively） for 6 h resulted in a decrease in ROS（P〈0.01）, but an increase in cell viabilities, SOD activities and antioxidant protein levels of p-Nrf2,HO-1, Nqo1 and p-Akt（P〈0.05,P〈0.01）. DGMI group showed the best efficiently. Moreover, after OGD for 4 h, compared with DGMI group, combin
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2017年第1期65-72,共8页
Chinese Journal of Pharmacology and Toxicology
基金
国家科技重大专项(2013ZX09402203)~~
