人参皂苷F2对过氧化氢诱导细胞损伤的保护作用

Protective effects of ginsenoside F2 on hydrogen peroxide induced cell injury

目的研究人参皂苷F2对人胚肾293(HEK-293)细胞氧化应激损伤的保护作用。方法以0.4 mmol/L过氧化氢(H_2O_2)诱导HEK-293细胞氧化应激,1.25、5和20μmol/L人参皂苷F2预处理,MTS法检测细胞活力;DCFH-DA荧光探针考察细胞活性氧(reactive oxygen species, ROS)含量;试剂盒检测丙二醛(malondialchehyche, MDA)水平和抗氧化酶超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)活性;蛋白质印迹法(Western blot)和qRT-PCR检测核因子E2相关因子2 (nuclear factor erythroid 2-related factor 2, Nrf2)/抑制蛋白Kelch样环氧氯丙烷相关蛋白-1 (kelch-like ECH-associated protein 1, Keap1)信号的蛋白和mRNA表达量。结果 1.25、5和20μmol/L人参皂苷F2处理正常HEK-293细胞后对细胞无毒性或促增殖作用;人参皂苷F2预处理氧化应激细胞后,细胞活力显著高于损伤组,各组间差异均有统计学意义(P<0.05);氧化损伤组的DCF荧光较对照组相比显著增强(P<0.05),人参皂苷F2预处理后细胞ROS相对量呈浓度依赖性降低,各组间差异均有统计学意义(P<0.05);人参皂苷F2预处理后细胞MDA水平呈浓度依赖性降低,各组间差异均有统计学意义(P<0.05),SOD和GSH-Px活性显著高于损伤组(P<0.05),5和20μmol/L人参皂苷F2能显著提高CAT活性(P<0.05);人参皂苷F2处理后,Nrf2的mRNA和蛋白表达量均显著升高(P<0.05),Keap1的mRNA和蛋白表达量显著低于损伤组(P<0.05)。结论人参皂苷F2具有细胞保护作用,可降低HEK-293细胞ROS和MDA水平,提高抗氧化酶活性,其作用机制可能是通过调节Nrf2/Keap1信号通路以抵抗过氧化氢导致的细胞氧化应激损伤。

OBJECTIVE To investigate the inhibitive effects of ginsenoside F2 on oxidative stress in human embryonic kidney cells(HEK-293).METHODSHydrogen peroxide induced oxidative stress of HEK-293 cell was used as the research object.HEK-293 cells were pretreated with different concentrations of ginsenoside F2(1.25,5,20μmol/L).Cell viability was measured by MTS assay.Malondialchehyche(MDA)level and activities of antioxidant enzymes(superoxide dismutase SOD,glutathione peroxidase GSH-Px,catalase CAT)were measured by corresponding assay kits.DCFH-DA fluorescent probe assay was used to measure the level of intracellular reactive oxygen species(ROS).Quantitative real-time PCR and Western blot were used to detect the expression of nuclear factor erythroid 2-related factor 2(Nrf2)and kelch-like ECH associated protein 1(Keap1).RESULTS After treated with 1.25,5,20μmol/L ginsenoside F2,no cytotoxic or proliferative effects were shown on normal HEK-293 cells.After pretreatment with ginsenoside F2,the cell viability was significantly higher than that of the injury group(P<0.05)and increased in a concentration-dependent manner.The fluorescence intensity of oxidative DCF in injured group was significantly increased compared with control group(P<0.05).The fluorescence intensity of cells which pretreated with different concentrations of ginsenoside F2 was gradually weakened(P<0.05).The ROS content of control group was chosen as the standard,and the relative amount of ROS pretreated by ginsenoside F2 decreased in a concentration-dependent manner.After pretreatment of ginsenoside F2,the MDA levels decreased in a concentration-dependent manner and the activities of SOD and GSH-Px were significantly higher than those of the injured group(P<0.05).The activity of CAT was significantly increased with pretreatment of higher concentrations of ginsenoside F2(P<0.05).Furthermore,ginsenoside F2 significantly enhanced the protein and mRNA expressions of Nrf2 and reduced the expressions of Keap1 in a dose-dependent manner(P<0.05).CONCLUSION Ginsenoside F2 protect HEK-293 cells against H2O2-induced oxidative stress through reducing intracellular ROS and MDA,as well as activating Nrf2/Keap1 signaling pathway and antioxidant enzymes.