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蓝莓花色苷对H2O2诱导A549细胞氧化损伤的保护作用
杨兆艳;胡红娟;李新; 王玲丽;田艳花1,2,3
1.山西药科职业学院食品工程系;2.山西省食品工业研究所;3.运城学院生命科学系
摘要:
为探究蓝莓花色苷(BA)对H2O2诱导A549细胞氧化损伤的保护作用及其机制,通过MTT法测定A549细胞存活率,采用ELISA法检测细胞内丙二醛(MDA)水平、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,采用DCFH-DA荧光探针检测细胞内活性氧(ROS)水平,采用流式细胞分析仪测定细胞凋亡率,通过RT-PCR技术测定细胞内抗氧化酶和凋亡相关蛋白的mRNA表达量,用免疫印迹法(Western blot)测定凋亡相关蛋白表达量。结果表明:选择H2O2浓度400 μmol/L、处理时间24 h来构建细胞氧化损伤模型。BA质量浓度为30,60,90 mg/mL对A549细胞无毒性作用;BA能显著抑制H2O2造成的A549细胞存活率降低(P<0.05),同时显著降低细胞凋亡率、MDA和胞内ROS水平(P<0.05),显著增加GSH-Px、SOD和CAT活性及其相对mRNA表达量(P<0.05),并显著上调Bcl-2/Bax的比值和下调Cytochrome c、Caspase-9和Caspase-3相对mRNA和蛋白表达量(P<0.05)。BA对H2O2诱导A549细胞氧化损伤具有保护作用,其作用机制可能与抑制细胞凋亡通路相关蛋白的表达,提高胞内抗氧化酶活性,清除胞内过量ROS,降低细胞凋亡率有关。
关键词:  蓝莓花青素  A549细胞  H2O2  氧化损伤  凋亡通路
DOI:
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基金项目:特色农产品发展山西省服务产业创新学科群项目(2018);山西省高等学校科技创新项目(2019L1012)
Protective Effects of Blueberry Anthocyanins on H2O2-Induced Oxidative Damage in A549 Cells
Abstract:
The aim of this study is to evaluate the cytoprotection and its mechanisms of blueberry anthocyanins (BA) on hydrogen peroxide(H2O2)-induced oxidative damage in A549 cells. The survival rate of A549 cells was determined by MTT. MDA levels and the activities of GSH-Px, SOD and CAT were detected by ELISA. The level of intracellular reactive oxygen species (ROS) was monitored by the DCFH-DA. The apoptosis rate was measured by flow cytometry. RT-PCR was employed to determine mRNA relative expression levels of antioxidant enzymes and apoptosis related proteins in A549 cells. Finally, western blot was used to measure the relative expression levels of apoptosis related proteins. The results showed that H2O2 concentration and treatment time were 400 μmol/L and 24 h, respectively, to establish A549 cells oxidative damage model. BA concentration of 30, 60 and 90 mg/mL had no toxic effect on A549 cells. BA could significantly inhibit the decrease of survival rate of A549 cells induced by H2O2 (P<0.05), decrease apoptosis rate, the levels of MDA and intracellular ROS(P<0.05), increase the activities and mRNA relative expression levels of GSH-Px, SOD and CAT (P<0.05), and significantly up regulate the ratio of Bcl-2/Bax and down regulate the mRNA and proteins relative expression levels of cytochrome c, caspase-9 and caspase-3(P<0.05). BA can repair and protect A549 cells from oxidative damage induced by H2O2, and its mechanism may be related to inhibiting the expression of apoptosis pathway related proteins, increasing the activities of intracellular antioxidant enzymes, clearing excessive ROS and reducing the rate of apoptosis.
Key words:  blueberry anthocyanin  A549 cell  H2O2  oxidative damage  apoptosis pathway

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