PI3K/AKT通路在二烯丙基二硫诱导K562细胞凋亡中的作用机制研究

The role of the PI3K/AKT signaling pathway in DADS-induced apoptosis of K562 cells

摘要:

目的 研究PI3K/AKT 信号通路在二烯丙基二硫（DADS）诱导K562 细胞凋亡中的作用。方法 用 10、20、40、80 mg/L DADS 处理K562 细胞48 h，采用吖啶橙/溴化乙啶（AO/EB）染色法倒置显微镜观察细胞形态学变化；AnnexinV-FITC/PI 双染法检测细胞凋亡率；Western blot 技术检测AKT、p-AKT、Caspases-3 蛋白的表达，并设置空白对照组和溶媒对照组。结果 DADS 作用K562 细胞48 h 后，细胞出现体积缩小、形态不规则、胞膜突出等凋亡特征。AO/EB 染色显示：随DADS 浓度增加，胞体缩小、胞核呈固缩状或圆珠状，染色质凝集，核染色呈橘红色的凋亡细胞数增多，以40 mg/L DADS 组最明显。10、20、40、80 mg/L DADS 组的凋亡率均高于对照组（P < 0.05）。各浓度DADS 处理组的AKT 蛋白表达差异无统计学意义（P > 0.05）；随DADS 浓度增加，p-AKT 蛋白逐渐下降、Caspases 3 蛋白逐渐升高，差异有统计学意义（P < 0.05）。结论 DADS 诱导K562 细胞发生凋亡，其机制可能与DADS 抑制PI3K/AKT 信号通路的表达相关。

Abstract:

Objective To study the role of the PI3K/AKT signaling pathway in the diallyl disulfide (DADS)-induced apoptosis of K562 cells. Methods K562 cells in the logarithmic growth phase were treated with 10, 20, 40, or 80 mg/L DADS for 48 hours, then fixed and stained with acridine orange/ethidium bromide (AO/EB), and examined for cellular morphological changes under an inverted microscope. Annexin V-FITC/PI staining was used for determining the apoptotic rates, and Western blot for measuring the expression of AKT, p-AKT, and Caspase-3. Two control groups, blank and solvent, were used as references. Results K562 cells treated with DADS for 48 hours exhibited the characteristic morphological features of apoptosis including cell shrinkage, irregular cell shape, and membrane blebbing. AO/EB staining results demonstrated that the number of apoptotic cells with cell shrinkage, pyknotic or bead-like nuclei, chromatin condensation, and orange staining increased with the increasing DADS concentration, and 40 mg/L DADS had the most significant effect. The apoptotic rates of cells treated with 10, 20, 40, and 80 mg/L DADS were all significantly higher than those in the control groups (P < 0.05). There were no significant differences in AKT protein expression between the K562 cells treated with different concentrations of DADS; the p-AKT protein expression decreased with the increasing DADS concentration, while the Caspases-3 protein expression increased with the increasing DADS concentration (P < 0.05). Conclusions DADS induces the apoptosis of K562 cells, probably through inhibiting the protein expression in the PI3K/AKT signaling pathway.