Aim To investigate the roles of intracellu-lar reactive oxygen species ( ROS ) and Nrf2 pathway in shikonin-induced A549 cell apoptosis. Methods The cytotoxicity was analyzed by MTT assay. The ap-optosis of A549 cells was analyzed by both cellular morphological and biochemical methods. The relative changes of the redox marks ( ROS/GSH) were studied by fluorescence assay in the shikonin-treated A549 cells in accompany with the changes of the intracellular redox homeostasis by GSH/GSSG ratio. ROS inhibitor was also employed in the treatment to find the role of ROS in shikonin-induced A549 cell apoptosis. Real-time PCR analysis and ELISA assay were performed as well to determine the role of Nrf2 pathway in the shiko-nin-induced A549 cell apoptosis. Results The IC50 of shikonin on A549 cells was 3. 2 mg·L-1 . The cellu-lar redox homeostasis shifted toward oxidation signifi-cantly in shikonin treatment in a time-dependent man-ner. The expression of the Nrf2 pathway related genes was up-regulated by shikonin ( 3 . 2 mg · L-1 , 8 h ) . The expression of the anti-apoptotic genes was down-regulated , and proapoptotic genes were up-regulated by shikonin (3. 2 mg·L-1, 24h). Futhermore, the inhi-bition of intracellular ROS alleviated the cytotoxicity of shikonin in A549 cells. Conclusion The critical role of shikonin-induced redox imblance in A549 cell, coped with the secondary produced ROS and Nrf2 path-way antioxidants, result in A549 cell apoptosis.%目的：探讨活性氧和Nrf2途径在紫草素诱导A549细胞凋亡过程中的作用。方法 MTT法评价紫草素对 A549细胞的细胞毒活性。采用细胞形态学和生物化学方法检测细胞凋亡。通过荧光探针对细胞内活性氧、还原型谷胱甘肽的变化进行标示,并用 GSH/GSSG比率对氧化还原态的变化进行评价。通过活性氧的清除剂分析活性氧在凋亡过程中的决定因素。通过实时荧光定量PCR和ELISA分析来确定Nrf2途径在紫草素诱导A549细胞凋亡中的作用。结果紫草素对A549细胞的IC50为3．2 mg·L-1。0~24 h时间内,随紫草素处理时间增加A549细胞内氧化还原平衡明显向氧化方向切换。3．2 mg·L-1紫草素作用A549细胞8 h, Nrf2途径相关基因表达量上调,24 h时抗凋亡基因表达量下降,促凋亡基因表达上升。抑制细胞内活性氧的产生会减轻紫草素对A549细胞增殖的抑制作用。结论紫草素诱导A549细胞产生的活性氧超过了Nrf2途径抗氧化应激的临界阈值,进而引起A549细胞凋亡。
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