苍术素通过激活RIPK1/RIPK3/MLKL信号通路诱导非小细胞肺癌A549细胞程序性坏死并抑制裸鼠移植瘤生长

Atractylodin induces programmed necrosis of non-small cell lung cancer A549 cells and inhibits xenograft growth in nude mice by activating the RIPK1/RIPK3/MLKL signaling pathway

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0~160μmol/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-VADFMK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、40μmol/L ATR处理)、ATR+Nec-1组(40μmol/L ATR+50μmol/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:10~160μmol/L的ATR可显著抑制A549细胞增殖,选择10、20、40μmol/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。

Objective: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 0~160μmol/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 40μmol/L ATR,respectively)and the ATR+Nec-1 group(treated with 40μmol/L atractylodin and 50μmol/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:10-160μmol/L ATR could significantly inhibit the proliferation of A549 cells,and the concentrations of 10,20 and 40μmol/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.