紫草素通过促进ROS的产生诱导人非小细胞性肺癌A549细胞凋亡的机制
投稿时间:2018-01-20  修订日期:2018-03-02  点此下载全文
引用本文:马建文,冯伟,张立平,李伟.紫草素通过促进ROS的产生诱导人非小细胞性肺癌A549细胞凋亡的机制[J].医学研究杂志,2018,47(12):103-109
DOI: 10.11969/j.issn.1673-548X.2018.12.025
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作者单位E-mail
马建文 830001 乌鲁木齐, 新疆医科大学附属中医医院药学部  
冯伟 830001 乌鲁木齐市妇幼保健医院药剂科  
张立平 830001 乌鲁木齐, 新疆医科大学附属中医医院肿瘤二科  
李伟 830001 乌鲁木齐, 新疆医科大学附属中医医院药学部 liwei13483942@sina.com 
中文摘要:目的 研究紫草素对人非小细胞性肺癌A549细胞的抗肿瘤作用,并探讨其可能的机制。方法 不同浓度紫草素处理A549细胞和人正常肺MRC-5细胞,四氮唑盐还原(MTT)法检测细胞的活性;流式细胞术结合PI-FITC双染色测定细胞凋亡;2'7'-二氯双乙酸盐(DCFH-DA)荧光探针测定活性氧(reactive oxygen species,ROS);Western blot法测定caspase-8、caspase-9、caspase-3、Bcl-2和Bcl-xL蛋白表达。结果 紫草素对A549细胞的增殖有明显的抑制作用,且呈现出时间和剂量依赖性(P<0.05,P<0.01);当剂量≤10μmol/L时,其对MRC-5细胞的增殖没有明显的影响(P>0.05)。不同浓度紫草素处理24h后,A549细胞的凋亡率(早期凋亡+晚期凋亡)显著增加(P<0.05或P<0.01),caspase-8、caspase-9和caspase-3蛋白被诱导活化,caspase抑制剂(Z-VAD-FMK)预处理则明显降低A549细胞的凋亡率(P<0.01),并抑制以上3种蛋白的活化。另外,紫草素处理可引起A549细胞ROS的产生,且呈现出一定的剂量依赖性。ROS抑制剂N-乙酰-L-半胱氨酸(NAC)和L-谷胱甘肽(GSH)预处理可使A549细胞的凋亡率下降(P<0.01)。同时,紫草素处理可明显下调抗凋亡因子Bcl-2和Bcl-xL的表达(P<0.01),NAC和GSH预处理亦能上调Bcl-2和Bcl-xL的表达(P<0.01)。结论 紫草素可明显抑制肺癌A549细胞的增殖,其作用机制与诱导A549细胞产生大量的ROS,抑制抗凋亡因子Bcl-2和Bcl-xL表达,进而激活caspase依赖的凋亡途径有关。
中文关键词:紫草素  A549细胞  ROS  细胞凋亡  Caspase
 
Mechanisms of Shikonin Inducing Apoptosis in Human Non-small Cell Lung Cancer A549 Cells by Promoting ROS Production
Abstract:Objective To study the anti-tumor effects of shikonin on human non-small cell lung cancer A549 cells and its mechanisms. Methods A549 cells and human normal lung MRC-5 cells were treated with different concentrations of shikonin. The cell viability was measured by MTT assay. Apoptosis was detected by flow cytometry and PI-FITC double staining. Fluorescence probe of 2'7'-dichloro diacetate (DCFH-DA) was used to detect the reactive oxygen species (ROS). The expression of caspase-8, caspase-9, caspase-3, Bcl-2 and Bcl-xL protein was measured by Western blot. Results The proliferation of A549 cells was significantly inhibited by Shikonin in a time-and dose-dependent manner (P<0.05 or P<0.01). Shikonin had no significant effects on the proliferation of MRC-5 cells at a dose of ≤ 10μmol/L (P>0.05). The apoptotic rates of A549 cells (early apoptosis and late apoptosis) were significantly increased after treated with different concentrations of shikonin for 24 h (P<0.05 or P<0.01). The protein expressions of caspase-8, caspase-9 and caspase-3 proteins were induced to be activated. The pretreatment with caspase inhibitor (Z-VAD-FMK) significantly decreased the apoptosis rate of A549 cells and inhibited the activation of these three proteins (P<0.01). In addition, the treatment of shikonin could cause the production of ROS in A549 cell, and showed a certain dose-dependent. The apoptosis rate of A549 cells was decreased with ROS inhibitor N-acetyl-L-cysteine (NAC) and L-glutathione (GSH) pretreatment (P<0.01). At the same time, shikonin significantly down-regulated the expression of anti-apoptotic factors Bcl-2 and Bcl-xL (P<0.01). NAC and GSH pretreatment also upregulated the expression of Bcl-2 and Bcl-xL. Conclusion Shikonin can significantly inhibit the proliferation of A549 cells, and its mechanisms is related to inducing a large amount of ROS, inhibiting the expression of anti-apoptotic factors Bcl-2 and Bcl-xL, and then activating caspase-dependent apoptotic pathway.
keywords:Shikonin  A549 cells  ROS  Apoptosis  Caspase
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