王玉荣;杨康;崔伟业;朴香兰;
WANG Yu-rong;YANG Kang;CUI Wei-ye;PIAO Xiang-lan;School of Pharmacy,Minzu University of China;

• 1:中央民族大学药学院

摘要(Abstract)：

该文主要研究绞股蓝黄酮对H_2O_2氧化损伤的人肺腺癌A549细胞的修复作用并探讨其可能的作用机制。分别使用浓度为200⁷00μmol·L700μmol·L^(-1)的H_2O_2诱导A549细胞不同时间,从而建立氧化损伤模型,再用50 mg·L(-1)的H_2O_2诱导A549细胞不同时间,从而建立氧化损伤模型,再用50 mg·L^(-1)绞股蓝黄酮保护10 h。利用MTT法检测6个绞股蓝黄酮化合物对H_2O_2损伤A549细胞活力修复的影响;DCFH-DA荧光探针检测细胞中ROS的含量;分别采用TBA法、NBT法和TNB法检测细胞内MDA,SOD和GSH的含量,Western blot法检测细胞内Nrf2,NOQ1,HO-1蛋白的表达。结果表明,500μmol·L(-1)绞股蓝黄酮保护10 h。利用MTT法检测6个绞股蓝黄酮化合物对H_2O_2损伤A549细胞活力修复的影响;DCFH-DA荧光探针检测细胞中ROS的含量;分别采用TBA法、NBT法和TNB法检测细胞内MDA,SOD和GSH的含量,Western blot法检测细胞内Nrf2,NOQ1,HO-1蛋白的表达。结果表明,500μmol·L^(-1)H_2O_2作用10 h,细胞存活率降低至60.4%,在50 mg·L(-1)H_2O_2作用10 h,细胞存活率降低至60.4%,在50 mg·L^(-1)芦丁(1),4'-O-甲基-山柰酚-3-O-芸香糖(2),异鼠李素-3-O-β-D-芸香糖(5),槲皮素-3-O-β-D-葡萄糖(6)作用10 h后,细胞存活率显著提高。与正常组比较,H_2O_2损伤使SOD,GSH含量以及HO-1蛋白表达量降低,使ROS,MDA含量增加,引起细胞氧化损伤。与模型组比较,绞股蓝黄酮组可降低细胞内ROS,MDA的含量,提高SOD,GSH的含量,激活Nrf2,NOQ1,HO-1蛋白的表达,从而改善H_2O_2诱导的A549细胞氧化应激损伤。
This study focuses on the therapeutical effect of flavonoids from Gynostemma pentaphyllum on human lung carcinoma A549 cells induced by H_2O_2 oxidative stress and its possible mechanisms. The oxidative damage model was established using different concentrations H_2O_2 to induce A549 cell for different hours,and then treated with the flavonoids for 10 hours. The effects of flavonoids from G. pentaphyllum on cell viability of A549 cell damaged by H_2O_2 were detected by MTT assay. The contents of ROS were detected by DCFH-DA fluorescent probe method via flow cytometer. The contents of MDA,SOD and GSH were detected by TBA,NBT and DTNB-linked colorimetry assay,respectively. Expressions levels of Nrf2,NQO1 and HO-1 in A549 cells were evaluated by Western blot.The results showed that the cell activity was decreasing with the rise of H_2O_2 concentration within the range of 200-700 μmol·L(-1)芦丁(1),4'-O-甲基-山柰酚-3-O-芸香糖(2),异鼠李素-3-O-β-D-芸香糖(5),槲皮素-3-O-β-D-葡萄糖(6)作用10 h后,细胞存活率显著提高。与正常组比较,H_2O_2损伤使SOD,GSH含量以及HO-1蛋白表达量降低,使ROS,MDA含量增加,引起细胞氧化损伤。与模型组比较,绞股蓝黄酮组可降低细胞内ROS,MDA的含量,提高SOD,GSH的含量,激活Nrf2,NOQ1,HO-1蛋白的表达,从而改善H_2O_2诱导的A549细胞氧化应激损伤。
This study focuses on the therapeutical effect of flavonoids from Gynostemma pentaphyllum on human lung carcinoma A549 cells induced by H_2O_2 oxidative stress and its possible mechanisms. The oxidative damage model was established using different concentrations H_2O_2 to induce A549 cell for different hours,and then treated with the flavonoids for 10 hours. The effects of flavonoids from G. pentaphyllum on cell viability of A549 cell damaged by H_2O_2 were detected by MTT assay. The contents of ROS were detected by DCFH-DA fluorescent probe method via flow cytometer. The contents of MDA,SOD and GSH were detected by TBA,NBT and DTNB-linked colorimetry assay,respectively. Expressions levels of Nrf2,NQO1 and HO-1 in A549 cells were evaluated by Western blot.The results showed that the cell activity was decreasing with the rise of H_2O_2 concentration within the range of 200-700 μmol·L^(-1). The cell viability was 60. 4% after treated with 500 μmol·L(-1). The cell viability was 60. 4% after treated with 500 μmol·L^(-1) H_2O_2 for 10 h,so it was chosen to be as an oxidant stress model. Compared with normal group,the contents of SOD,GSH and HO-1 expressions were lower after damaged with H_2O_2. On the contrary,the contents of ROS and MDA expressions were increased. Compared with model group,the contents of SOD,GSH and the expressions of Nrf2,NQO1 and HO-1 were increased after treated with flavonoids from G. pentaphyllum. The above results demonstrate that flavonoids from G. pentaphyllum may attenuate the effect of H_2O_2-induced oxidative stress on A549 cell by resisting oxidation. The finding may provide a biological evidence for the application of the G. pentaphyllum to fight the oxidative stress related diseases.

关键词(KeyWords)： 绞股蓝;黄酮;过氧化氢;A549细胞;抗氧化
Gynostemma pentaphyllum;flavonoid;H_2O_2;A549 cell;antioxidant

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81673692)

作者(Authors): 王玉荣;杨康;崔伟业;朴香兰;
WANG Yu-rong;YANG Kang;CUI Wei-ye;PIAO Xiang-lan;School of Pharmacy,Minzu University of China;

DOI: 10.19540/j.cnki.cjcmm.2018.0034

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