银杏叶提取物对慢性间歇性低氧大鼠胰岛β细胞氧化应激的影响及机制探讨

Effects of ginkgo biloba extracts on oxidative stress of islet β-cells in chronic intermittent hypoxic rats

目的探讨银杏叶提取物(EGB)对慢性间歇性低氧(CIH)大鼠胰岛β细胞氧化应激反应的影响及其机制。方法取10只SD大鼠置于低压氧舱内模拟间歇性低氧条件制备CIH模型,另取10只大鼠在常氧条件下饲养。于造模12周结束后,提取CIH模型大鼠及常氧饲养大鼠的原代胰岛β细胞,将常氧饲养大鼠的原代胰岛β细胞作为常氧对照组,将CIH模型大鼠的原代胰岛β细胞分为CIH模型组、抗核因子2相关因子(Nrf2)抗体组、Nrf2激活剂组、EGB低剂量组、EGB中剂量组、EGB高剂量组。常氧对照组、CIH模型组用普通DMEM培养基培养,抗Nrf2抗体组加入终浓度为20μg/mL的Nrf2抗体,Nrf2激活剂组加入终浓度为20μmol/L的Nrf2激活剂莱菔硫烷,EGB低、中、高剂量组分别加入EGB 5、10、20 mg/mL。各组均干预24 h后,取各组细胞,检测细胞中氧自由基(ROS)含量、细胞上清液中丙二醛(MDA)及谷胱甘肽过氧化物酶(GSH-Px)水平。采用Western blotting法检测Nrf2-抗氧化反应元件(ARE)信号通路相关蛋白Nrf2及下游抗氧化酶靶蛋白血红素加氧酶-1(HO-1)、醌氧化还原酶(NQO1)、γ-谷氨酰半胱氨酸合成酶(γ-GCS)的表达。结果与常氧对照组对比,CIH模型组MDA及ROS均升高,GSH-Px降低(P均<0.01)。与CIH模型组比较,抗Nrf2抗体组MDA及ROS均升高,GSH-Px降低(P<0.05或0.01);Nrf2激活剂组、EGB中剂量组、EGB高剂量组MDA及ROS均降低,GSH-Px均升高(P<0.05或0.01)。EGB高剂量组MDA及ROS均低于EGB低剂量组,GSH-Px高于EGB低剂量组(P<0.01或0.05)。与常氧对照组对比,CIH模型组Nrf2升高,HO-1、NQO1、γ-GCS均降低(P均<0.01)。与CIH模型组比较,抗Nrf2抗体组HO-1、NQO1均降低(P均<0.01);Nrf2激活剂组、EGB中剂量组、EGB高剂量组Nrf2、HO-1、NQO1、γ-GCS均升高(P<0.05或P<0.01)。EGB高剂量组Nrf2、HO-1、NQO1、γ-GCS均高于EGB中剂量组(P<0.05或0.01)。结论EGB可减轻CIH导致的胰岛β细胞氧化应激反应水平,其机制可能与激活Nrf2-ARE信号通路有关。

Objective To investigate the effects of ginkgo biloba extracts（ EGB） on oxidative stress of islet β-cells in rats with chronic intermittent hypoxia（ CIH） and its mechanism. Methods Ten SD rats were placed in hypoxia chamber to simulate intermittent hypoxia to prepare CIH models,and the other 10 rats were fed under normoxic conditions. At the end of 12 weeks,the primary islet β-cells from CIH model rats and normoxic rats were extracted. The primary islet cells of the normoxic rats were used as the normoxic control group; the primary islet cells of CIH model rats were divided into the CIH model group,anti-Nrf2 antibody group,Nrf2 activator group,low-dose EGB group,medium-dose EGB group,and high-dose EGB group. Rats in the oxygen control group and CIH model group were cultured in DMEM medium; Nrf2 antibody was added to the Nrf2 antibody group; Nrf2 activator sulforaphane with final concentration of 20 μg/mL was added to the Nrf2 activator group; 5,10,and 20 mg/mL EGB were added to the low-dose EGB group,medium-dose EGB group,and high-dose EGB group,respectively. The cells were taken from rats in each group after 24-hour intervention. The content of reactive oxygen species（ ROS）,the content of malondialdehyde（ MDA） and glutathione peroxidase（ GSH-Px） in the supernatant was measured. The expression levels of Nrf2-ARE signal pathway related protein Nrf2 and downstream antioxidant enzyme-target protein HO-1,NQO1 and γ-GCS were detected by Western blotting. Results Compared with the normoxic control group,MDA and ROS increased and GSH-Px decreased in the CIH model group（ all P〈0. 01）. Compared with the CIH model group,MDA and ROS increased and GSH-Px decreased in anti-Nrf2 antibody group（ P〈0. 01 or P〈0. 05）. In the Nrf2 activator group,medium-dose EGB group,and high-dose EGB group,MDA and ROS were reduced,and GSH-Px increased（ P〈0. 01 or P〈0. 05）. MDA and ROS were lower and GSH-Px was higher in the highdose EGB group than in the low-dose EGB group（ P〈0. 01 or P〈0. 05）. Compared with the normoxic control group,the level of Nrf2 increased,and HO-1,NQO1 and γ-GCS decreased in the CIH model group（ all P〈0. 01）. Compared with the CIH model group,HO-1 and NQO1 decreased in the anti-Nrf2 antibody group（ both P〈0. 01）. In the Nrf2 activator group,medium-dose EGB group,and high-dose EGB group,Nrf2,HO-1,NQO1,and γ-GCS increased（ P〈0. 01 or P〈0. 05）. The levels of Nrf2,HO-1,NQO1,and γ-GCS in the high-dose EGB group were higher than those of the mediumdose EGB group（ P〈0. 01 or P〈0. 05）. Conclusion EGB can reduce the level of oxidative stress in islet cells caused by CIH,and its mechanism may be related to the activation of Nrf2-ARE signaling pathway.