目的：探讨苍术素（ATR）通过调节受体相互作用蛋白激酶（RIPK）1/RIPK3/混合谱系激酶结构域样（MLKL）信号通路对非小细胞肺癌（NSCLC）A549 细胞程序性死亡及裸鼠移植瘤生长的影响。方法：使用0~160 μmol/L 的ATR 处理A549 细胞， MTT 法检测细胞存活率以确定后续实验给药浓度。使用ATR 和/或RIPK1 抑制剂Nec-1（necrostatin-1）、caspase 抑制剂Z-VAD-FMK处理A549 细胞，验证ATR是否诱导A549 细胞发生程序性坏死。将A549 细胞分为对照组、ATR-L 组、ATR-M 组、ATR-H 组（分别用0、10、20、40 μmol/L ATR 处理）、ATR+Nec-1 组（40 μmol/L ATR+50 μmol/L Nec-1 处理），处理24 h 后，采用PI 单染及Hoechst33342/PI 双染法检测细胞死亡情况、透射电镜观察细胞死亡形态、DCFH-DA 荧光探针法检测细胞内ROS水平、JC-1染色法检测线粒体膜电位、WB法检测细胞中RIPK1/RIPK3/MLKL 信号通路相关蛋白质的表达水平。构建A549 细胞裸鼠移植瘤模型，用10 mg/kg ATR（溶于玉米油中）对裸鼠灌胃给药5 周，观察ATR 对移植瘤生长的影响，WB 法检测移植瘤组织中RIPK1/RIPK3/MLKL信号通路相关蛋白质的表达水平。结果：10~160 μmol/L的ATR可显著抑制A549细胞增殖，选择10、20、40 μmol/L的ATR进行后续实验。ATR组A549 细胞存活率显著低于对照组（P<0.01）和ATR+Nec-1组（P<0.01），而ATR+z-VAD组细胞存活率显著低于z-VAD组（P<0.01），说明ATR可诱导A549 细胞发生程序性坏死而非凋亡。与对照组比较，ATR处理组A549 细胞发生肿胀，线粒体内脊消失呈空泡化，细胞内容物向外泄漏，细胞核聚集，表现为坏死特征，ATR-L 组、ATR-M组、ATR-H 组A549 细胞死亡率、ROS水平及p-RIPK1、p-RIPK3、p-MLKL表达水平均显著升高，线粒体膜电位显著降低（均P<0.01），且呈药物浓度依赖性；与ATR-H 组比较，ATR+Nec-1 组细胞死亡率、ROS 及p-RIPK1、p-RIPK3、p-MLKL 表达水平降低，线粒体膜电位显著升高（均P<0.01）。裸鼠移植瘤实验结果显示，与对照组比较，ATR 组裸鼠移植瘤体积、移植瘤质量均降低（P<0.05，或P<0.01），而与瘤组织中p-RIPK1、p-RIPK3、p-MLKL 蛋白表达水平均显著升高（均P<0.01）。结论：ATR可能通过激活RIPK1/RIPK3/MLKL信号通路诱导A549细胞发生程序性坏死，抑制A549细胞及其裸鼠移植瘤的生长。

To investigate the influence of atractylodin (ATR) on programmed death of non-small cell lung cancer (NSCLC) A549 cells and the growth of xenografts in nude mice by regulating receptor-interacting protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain like (MLKL) signaling pathway. Methods: A549 cells were treated with 0~160 μmol/L ATR, and the cell viability was detected by MTT method to determine the concentration of subsequent experiments. A549 cells were treated with ATR and/or RIPK1 inhibitor necrostatin-1 (Nec-1) and caspase inhibitor Z-VAD-FMK, to verify whether ATR induced programmed necrosis in A549 cells. A549 cells were divided into the control group, the ATR-L, ATR-M and ATR-H group (treated with 0, 10, 20 and 40 μmol/L ATR, respectively) and the ATR+Nec-1 group (treated with 40 μmol/L atractylodin and 50 μmol/L Nec-1). After 24 h of treatment, PI single staining and Hoechst33342/PI double staining were used to detect cell death; transmission electron microscopy (TEM) was used to observe the morphology of cell death; DCFH-DA fluorescent probe was used to detect intracellular ROS level; JC-1 staining was used to detect mitochondrial membrane potential, and WB method was used to detect the expression level of RIPK1/RIPK3/MLKL signaling pathway-related proteins in cells. A xenograft model of A549 cells was constructed in nude mice, and 10 mg/kg ATR (dissolved in corn oil) was administered to nude mice by gavage for 5 weeks to observe the effect of atractylodin on xenograft growth. The expression level of RIPK1/RIPK3/MLKL signaling pathway-related proteins in xenograft tissues was detected by WB method. Results: 10-160 μmol/L ATR could significantly inhibit the proliferation of A549 cells, and the concentrations of 10, 20 and 40 μmol/L were selected for follow-up experiments. The survival rate of A549 cells in the ATR group was significantly lower than that in the control group (P<0.01) and ATR+Nec-1 group (P<0.01), while the cell survival rate in the ATR+z-VAD group was significantly lower than that in the z-VAD group (P<0.01), indicating that ATR could induce programmed necrosis of A549 cells instead of apoptosis. Compared with the control group, A549 cells in the ATR-treated groups were swollen; the mitochondria were vacuolated; the inner ridge disappeared, the cell contents leaked outward, and the nuclei were aggregated, showing necrotic characteristics. The mortality rate, ROS level, expression levels of p-RIPK1, p-RIPK3 and p-MLKL in the ATR-L group, ATR-M group and ATR-H group A549 cells increased significantly, while the mitochondrial membrane potential decreased significantly (all P<0.01), all of which were concentration-dependent. Compared with the ATR-H group, the mortality rate, ROS level, and expression levels of p-RIPK1, p-RIPK3 and p-MLKL in the ATR+Nec-1 group decreased, while the mitochondrial membrane potential increased significantly (all P<0.01). The results of nude mouse xenograft experiment showed that compared with the control group, the volume and mass of xenografts were decreased (P<0.05 or P<0.01), and the protein expression levels of p-RIPK1, p-RIPK3 and p-MLKL in the tumor tissues in the ATR group increased significantly (all P<0.01). Conclusion: ATR may induce programmed necrosis of A549 cells by activating the RIPK1/RIPK3/MLKL signaling pathway, and inhibit the growth of A549 cells and their nude mouse xenografts.

海南省自然科学基金项目（No. 822RC868）