黄芪甲苷调控p62-Nrf2通路对抗小鼠淋巴细胞白血病耐药株的机制研究

Mechanism research of astragaloside Ⅳ on drug-resistant strain of mouse lymphocytic leukemia based on p62-Nrf2 pathway

关键词:  p62-Nrf2通路  黄芪甲苷  化疗耐药

Key words:  p62-Nrf2 signaling pathway  astragaloside Ⅳ  chemoresistance

摘要:

[目的] 基于p62-核因子红细胞样2相关因子2（Nrf2）通路探讨黄芪甲苷（AST Ⅳ）对抗小鼠淋巴细胞白血病耐药株的机制。[方法] 复制白血病细胞耐药模型，将细胞分为6组，即空白组（blank）、模型组（model）、黄芪甲苷组（AST Ⅳ）、盐酸维拉帕米（VER）、Nrf2通路抑制剂（ML385）组及抑制剂与黄芪甲苷组（ML385+AST Ⅳ），药物干预48 h。细胞计数试剂盒-8（CCK-8）检测耐药细胞对顺铂的敏感性，流式细胞仪检测细胞内活性氧（ROS）变化，明确AST Ⅳ对耐药细胞的影响；流式细胞仪检测细胞周期及凋亡率变化，荧光实时定量聚合酶链式反应（Real time PCR）、蛋白免疫印迹（Western Blot）检测p62-Nrf2通路p62、Nrf2、血红素氧化酶-1（HO-1）、B淋巴细胞瘤-2基因（bcl-2）、bcl-2相关蛋白X（bax）及半胱氨酸蛋白酶-3（Caspase-3）等表达变化明析AST Ⅳ对耐药细胞的作用机制。[结果] AST Ⅳ通过下调p62-Nrf2通路节点基因p62、HO-1、bcl-2/bax，上调Caspase-3的表达量，降低耐药细胞ROS水平，增加其对顺铂敏感性，使其增殖受抑，细胞周期呈现DNA合成期（S）、合成后期（G2）/有丝分裂期（M）阻滞，凋亡率升高。[结论] AST Ⅳ具有逆转白血病耐药的功效，可能与p62-Nrf2通路调控相关，但细胞最终命运受多条通路共同调控仍需探讨。

Abstract:

[Objective] To investigate the effect of astragaloside Ⅳ/AST Ⅳ on drug-resistant strain of mouse lymphocytic leukemia based on p62-Nrf2 pathway.[Methods] The drug-resistant leukemia model was replicated in vitro, and drug-resistant cells were divided into 6 groups, namely blank group, model group, AST Ⅳ group, VER group, ML385 group, and ML385+AST Ⅳ group. They were subsequently treated with drugs for 48 hours. CCK-8 assay and ROS quantification were performed to observe the effects of AST Ⅳ on drug-resistance. To analyze the reversal mechanism of AST Ⅳ on drug resistance, Flow cytometry was performed to detect the changes of cell cycle and apoptosis rate, and real time RT-PCR and Western blot were performed to detect the expression of p62, Nrf2, HO-1, bcl-2, bax and Caspase-3 in p62-Nrf2 pathway.[Results] AST Ⅳ could down regulate the expression of p62, HO-1, bcl-2/bax, but up regulate the expression of Caspase-3, resulting in that drug-resistant cells were more sensitive to cisplatin because of the decreased ROS level. The proliferation of drug-resistant cells was inhibited. The cell cycle showed S-phase, G2, M phase arrest and the apoptosis rate increased.[Conclusion] AST Ⅳ can have the effect of reversing drug-resistance of leukemia cells, which may be related to the regulation of p62-Nrf2 pathway. However, the co-regulation of multiple pathways in cell fate still needs to be discussed.