人参皂苷F2对过氧化氢诱导细胞损伤的保护作用
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摘要
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, 20 μmol/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, 20 μmol/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 H_2O_2-induced oxidative stress through reducing intracellular ROS and MDA, as well as activating Nrf2/Keap1 signaling pathway and antioxidant enzymes.
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, 20 μmol/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, 20 μmol/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 H_2O_2-induced oxidative stress through reducing intracellular ROS and MDA, as well as activating Nrf2/Keap1 signaling pathway and antioxidant enzymes.
引文
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[2] NI N,LIU Q,REN H,et al.Ginsenoside Rb1 protects rat neural progenitor cells against oxidative injury[J].Molecules,2014,19(3):3012-3024.
[3] SONG Z M,LIU Y,HAO B S,et al.Ginsenoside Rb1 prevents H2O2-induced HUVEC senescence by stimulating sirtuin-1 pathway[J].PLoS One,2014,9(11):9.
[4] LIU D,ZHANG T,CHEN Z,et al.The beneficial effect of ginsenosides extracted by pulsed electric field against hydrogen peroxide-induced oxidative stress in HEK-293 cells[J].J Ginseng Res,2017,41(2):169-179.
[5] LO Y T,TSAI Y H,WU S J,et al.Ginsenoside Rb1 inhibits cell activation and liver fibrosis in rat hepatic stellate cells[J].J Med Food,2011,14(10):1135-1143.
[6] KANG H J,HUANG Y H,LIM H W,et al.Stereospecificity of ginsenoside Rg2 epimers in the protective response against UV-B radiation-induced oxidative stress in human epidermal keratinocytes[J].J Photochem Photobiol B,2016 (165):232-239.
[7] ZHU Y H C,ZHENG P.Ginsenoside Rb1 alleviates aluminum chloride-induced rat osteoblasts dysfunction[J].Toxicol Appl Pharmacol,2016 (368):183-188.
[8] LI W Y M,LIU Y.Ginsenoside Rg5 ameliorates cisplatin-induced nephrotoxicity in mice through inhibition of inflammation,oxidative stress,and apoptosis[J].Nutrients,2016,8(9):566.
[9] WANG X,CHEN L,WANG T,et al.Ginsenoside Rg3 antagonizes adriamycin-induced cardiotoxicity by improving endothelial dysfunction from oxidative stress via upregulating the Nrf2-ARE pathway through the activation of akt[J].Phytomedicine,2015,22(10):875-884.
[10] LI F,ZHANG Y,ZENG D,et al.The combination of three components derived from Sheng MaiSan protects myocardial ischemic diseases and inhibits oxidative stress via modulating MAPKs and JAK2-STAT3 signaling pathways based on bioinformatics approach[J].Front Pharmacol,2017,8:21.
[11] JUNG J S,LEE S Y,KIM D H,et al.Protopanaxatriol ginsenoside Rh1 upregulates phase II antioxidant enzyme gene expression in rat primary astrocytes:involvement of MAP kinases and Nrf2/ARE signaling[J].Biomol Ther (Seoul),2016,24(1):33-39.