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目的:研究金丝桃苷对过氧化氢(H_2O_2)诱导的A549细胞氧化损伤的保护作用,并探讨其相关的作用机制。方法:体外培养A549细胞,采用H_2O_2(400μmol·L~(-1))作用24 h,建立A549细胞氧化损伤模型,分为空白组,模型组(400μmol·L~(-1)H_2O_2),阳性药(100μmol·L~(-1)N-乙酰半胱氨酸)组和不同浓度金丝桃苷(1,10,100μmol·L~(-1))组。采用噻唑蓝(MTT)法检测细胞活力,通过酶联免疫吸附测定(ELISA)法检测活性氧(ROS),丙二醛(MDA)含量,乳酸脱氢酶(LDH),过氧化氢酶(CAT),谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)活性。通过罗丹明123染色法检测细胞线粒体膜电位改变。采用蛋白质免疫印迹(Western blot)检测沉默信息调节因子3(Sirt3),异柠檬酸脱氢酶2(IDH2)和锰超氧化物岐化酶(Mn SOD)蛋白表达。结果:与模型组比较,金丝桃苷显著提高细胞存活率(P<0.01),降低LDH的外漏,ROS和MDA含量(P<0.05,P<0.01),明显增加CAT,SOD,GSH-Px活性(P<0.05,P<0.01)。金丝桃苷能够显著提高线粒体膜电位,上调Sirt3,IDH2和Mn SOD蛋白表达(P<0.05,P<0.01)。结论:金丝桃苷能够保护H_2O_2诱导的A549细胞氧化损伤,其作用可能与提高清除ROS能力,调控Sirt3线粒体途径相关。
Objective: To study the protective effect of hyperoside on the oxidative damage of A549 cells induced by hydrogen peroxide (H_2O_2) and to explore its related mechanism. Methods: The A549 cells were cultured in vitro and the oxidative injury model of A549 cells was established by H 2 O 2 (400 μmol·L -1) for 24 h. The cells were divided into blank group and model group (400 μmol·L -1 H 2 O 2) (100μmol·L -1 N-acetylcysteine) group and hyperoside (1,10,100μmol·L -1) group. Cell viability was measured by MTT assay. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), lactate dehydrogenase (LDH) and catalase (CAT) were detected by enzyme linked immunosorbent assay (ELISA) ), Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity. Mitochondrial membrane potential was detected by rhodamine 123 staining. Protein expression of Sirt3, IDH2 and Mn SOD were detected by Western blot. Results: Compared with the model group, hyperoside significantly increased the cell viability (P <0.01), reduced the leakage of LDH, the content of ROS and MDA (P <0.05, P <0.01) Px activity (P <0.05, P <0.01). Hyperin increased the mitochondrial membrane potential and up-regulated the expression of Sirt3, IDH2 and Mn SOD (P <0.05, P <0.01). Conclusions: Hyperin can protect H 2 O 2 -induced oxidative injury in A549 cells, which may be related to its ability of scavenging ROS and regulating the mitochondrial pathway of Sirt3.