王馨悦, 王素华, 王丽, 赵宇航, 高艳荣. 亚砷酸钠致人胚肺成纤维细胞损伤中自噬和凋亡的关系[J]. 环境与职业医学, 2019, 36(9): 853-857, 863. DOI: 10.13213/j.cnki.jeom.2019.19239
引用本文: 王馨悦, 王素华, 王丽, 赵宇航, 高艳荣. 亚砷酸钠致人胚肺成纤维细胞损伤中自噬和凋亡的关系[J]. 环境与职业医学, 2019, 36(9): 853-857, 863. DOI: 10.13213/j.cnki.jeom.2019.19239
WANG Xin-yue, WANG Su-hua, WANG Li, ZHAO Yu-hang, GAO Yan-rong. Relatonship between autophagy and apoptosis of human embryonic lung fbroblasts induced by sodium arsenite[J]. Journal of Environmental and Occupational Medicine, 2019, 36(9): 853-857, 863. DOI: 10.13213/j.cnki.jeom.2019.19239
Citation: WANG Xin-yue, WANG Su-hua, WANG Li, ZHAO Yu-hang, GAO Yan-rong. Relatonship between autophagy and apoptosis of human embryonic lung fbroblasts induced by sodium arsenite[J]. Journal of Environmental and Occupational Medicine, 2019, 36(9): 853-857, 863. DOI: 10.13213/j.cnki.jeom.2019.19239

亚砷酸钠致人胚肺成纤维细胞损伤中自噬和凋亡的关系

Relatonship between autophagy and apoptosis of human embryonic lung fbroblasts induced by sodium arsenite

  • 摘要: 背景 砷可导致细胞损伤,引起细胞自噬和凋亡,而细胞自噬与凋亡之间目前没有明确界限,二者关系目前尚不明确。

    目的 探讨亚砷酸钠致人胚肺成纤维细胞(HELF)损伤中细胞自噬与凋亡的关系。

    方法 HELF细胞株培养3 d后传代,随机分为对照组、亚砷酸钠处理组(亚砷酸钠浓度分别为5、10、20 μmol/L)、自噬抑制组(20 μmol/L NaAsO2+5 mmol/L 3-甲基腺嘌呤)、凋亡抑制组(20 μmol/L NaAsO2+20 μmol/L Caspase抑制剂z-VAD-FMK),染毒48 h后,MTT实验检测细胞存活率,倒置显微镜观察细胞形态学改变。提取细胞上清液、蛋白,ELISA法测定上清液中白介素(IL)-1β、IL-6、干扰素受体(IFN)-α含量。Western blot检测自噬相关蛋白p62和LC3的表达水平。Caspase试剂盒检测凋亡相关蛋白caspase3/7。

    结果 各组细胞存活率在染毒12、24、48 h后均低于对照组(P < 0.05)。随着染毒浓度增加细胞形态学改变,由紧密的梭形逐渐变为松散的圆形。10、20 μmol/L砷染毒组HELF细胞上清液中IL-1β表达量升高(P < 0.05);各砷染毒组细胞培养上清液中促炎因子IL-6、IFN-α表达量高于对照组(P < 0.05);与20 μmol/L亚砷酸钠染毒组相比,自噬抑制组呈现促炎因子表达量升高的趋势,凋亡抑制组呈现降低的趋势。与对照组相比,砷染毒组细胞中LC3-Ⅰ、LC3-Ⅱ蛋白表达量呈升高的趋势,p62蛋白的表达量呈降低的趋势;与20 μmol/L亚砷酸钠染毒组相比,自噬抑制组细胞中LC3-Ⅰ、LC3-Ⅱ蛋白表达量降低(P < 0.05),p62蛋白表达量呈升高的趋势;凋亡抑制组细胞中LC3-Ⅰ、LC3-Ⅱ蛋白表达水平呈升高趋势,p62蛋白的表达水平呈降低趋势。随着染毒浓度增加,与对照组相比,10、20 μmol/L砷暴露组凋亡相关蛋白caspase3/7表达量增多(P < 0.05);与20 μmol/L亚砷酸钠染毒组相比,自噬抑制组caspase3/7表达量升高(P < 0.05),凋亡抑制组表达量降低(P < 0.05)。

    结论 亚砷酸钠染毒后可引起HELF发生炎症反应、细胞自噬和凋亡,且细胞自噬与凋亡之间可能存在一定的拮抗关系。

     

    Abstract: Background Arsenic can cause cell damage, autophagy, and apoptosis, but there is no clear boundary between autophagy and apoptosis, and their relatonship is unclear.

    Objective This study is designed to investigate the relationship between autophagy and apoptosis in human embryonic lung fbroblasts (HELF) treated with sodium arsenite.

    Methods HELF cells were cultured for three days and then randomly divided into a control group, three sodium arsenite treatment groups (5, 10, and 20 μmol/L), an autophagy inhibiton group (20 μmol/L NaAsO2+5 mmol/L 3-methyladenine), and an apoptosis inhibition group (20 μmol/L NaAsO2+20 μmol/L Caspase inhibitor z-VAD-FMK). After 48 hours of the designed exposure protocol, the cell survival rate was detected by MTT assay, and the morphological changes were observed under inverted microscope. Proteins were extracted from supernatants to determine the contents of interleukin (IL)-1β, IL-6, interferon (IFN)-α in supernatants by ELISA. The expression levels of autophagy related proteins p62 and LC3 were detected by Western blot and apoptosis related protein caspase3/7 by Caspase kit.

    Results The cell survival rates of the sodium arsenite treatment groups, the autophagy inhibition group, and the apoptosis inhibition group were signifcantly lower than that of the control group at 12, 24, and 48 h afer treatment (P < 0.05). With the increase of exposure concentraton, the morphology changed from tghtly arranged spindle cells to loosely arranged round cells. The expressions levels of IL-1β in the supernatant of HELF cells in the 10 and 20 μmol/L arsenic exposure groups were increased (P < 0.05). The expression levels of proinflammatory cytokins IL-6 and IFN-α in the supernatant of arsenic treated groups were signifcantly higher than those of the control group (P < 0.05). Compared with the 20 μmol/L sodium arsenite treatment group, the expression levels of pro-inflammatory cytokines in the autophagy inhibiton group were higher, and the expression levels in the apoptosis inhibiton group were lower. Compared with the control group, the expression levels of LC3-Ⅰ and LC3-Ⅱ proteins showed an upward trend, while the expression levels of p62 protein showed a downward trend in the arsenic exposure groups. Compared with the 20 μmol/L sodium arsenite treatment group, the expression levels of LC3-Ⅰ and LC3-Ⅱ proteins were decreased (P < 0.05), and the expression level of p62 protein showed an increasing trend in the autophagy inhibiton group; the expression levels of LC3-Ⅰ and LC3-Ⅱ proteins shouled an upward trend, while the expression level of p62 protein showed a downward trend in the apoptosis inhibiton group. With the increase of arsenite exposure concentraton, compared with the control group, the expression of apoptosis-related protein caspase3/7 in the 10 and 20 μmol/L arsenic exposure groups increased (P < 0.05). Compared with the 20μmol/L sodium arsenite treatment group, the expression of caspase3/7 in the autophagy inhibiton group was increased (P < 0.05), and the expression of caspase3/7 in the apoptosis inhibiton group was decreased (P < 0.05).

    Conclusion Sodium arsenite can induce inflammatory reacton, autophagy, and apoptosis of HELF cells, and there may be an antagonistc relatonship between autophagy and apoptosis.

     

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