摘要
目的探讨人参皂苷Rg3通过抑制哺乳动物雷帕霉素靶蛋白(mTOR)通路介导的磷酸戊糖途径(PPP),对肺癌细胞放射敏感性的影响。方法以0、10、20、40、60、80 mg/L的人参皂苷Rg3处理肺癌细胞A549;MTT法检测细胞增殖情况;将细胞分为对照组(正常培养,不照射)、放射组(X射线照射处理)、人参皂苷Rg3组(60 mg/L人参皂苷Rg3,不照射)、联合组(X射线照射+60 mg/L人参皂苷Rg3)、激活剂组(X射线照射+60 mg/L人参皂苷Rg3+100 nmol/L mTOR通路激活剂MHY1485);均为加入相对应药物培养48 h后放射组、联合组和激活剂组采用8 Gy X射线照射。平板克隆实验检测各组细胞克隆形成率;酶联免疫吸附试验(ELISA)测定各组细胞葡萄糖-6-磷酸脱氢酶(G6PD)、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)水平;DCFH-DA荧光探针法检测细胞内活性氧(ROS)水平;流式细胞仪检测细胞凋亡;γ-H2AX免疫荧光染色分析DNA损伤修复情况;Western blot检测细胞中mTOR、p-mTOR、增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(caspase-3)、γ-H2AX蛋白的表达。结果人参皂苷Rg3以剂量依赖性的方式抑制A549细胞的增殖(P<0.05);与对照组比较,放射组、人参皂苷Rg3组细胞克隆形成率、G6PD、ROS、NADPH水平下降,p-mTOR/mTOR、PCNA蛋白表达水平降低,细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达水平升高(P<0.05);与放射组、人参皂苷Rg3组比较,联合组细胞克隆形成率、G6PD、ROS、NADPH水平下降,p-mTOR/mTOR、PCNA蛋白表达水平降低,细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达升高(P<0.05);与联合组比较,激活剂组细胞克隆形成率、G6PD、ROS、NADPH水平升高,p-mTOR/mTOR、PCNA蛋白表达水平升高,细胞凋亡率、γ-H2AX焦点数、Bax、caspase-3、γ-H2AX蛋白表达水平降低(P<0.05)。结论人参皂苷Rg3发挥抑制肺癌作用可能是通过抑制mTOR介导的PPP实现的。
Objective To investigate the influence of ginsenoside Rg3 on the radiosensitivity of lung cancer cells by inhibiting the pentose phosphate pathway(PPP)mediated by mammalian target of rapamycin(mTOR)pathway.Methods A549 lung cancer cells were treated with 0,10,20,40,60,80 mg/L ginsenoside Rg3.The proliferation of A549 cells was detected by MTT method.Cells were divided into the control group(normal culture,no irradiation),the radiation group(X-ray irradiation),the ginsenoside Rg3 group(60 mg/L ginsenoside Rg3,no irradiation),the combination group(X-ray irradiation+60 mg/L ginsenoside Rg3)and the activator group(X-ray irradiation+60 mg/L ginsenoside Rg3+100 nmol/L mTOR pathway activator MHY1485).All of groups were irradiated by 8 GyX ray after 48 h of culture with corresponding drugs.Plate cloning experiment was applied to detect the formation rate of cell clones in each group.Enzyme-linked immunosorbent assay(ELISA)was applied to determine levels of glucose-6-phosphate dehydrogenase(G6PD)and reduced nicotinamide adenine dinucleotide phosphate(NADPH)in cell supernatant of each group.DCFH-DA fluorescent probe method was applied to detect the level of intracellular reactive oxygen species(ROS).Cell apoptosis was detected by flow cytometry.γ-H2AX immunofluorescence staining was applied to analyze DNA damage repair.Western blot assay was applied to detect expression levels of mTOR,p-mTOR,proliferating cell nuclear antigen(PCNA),Bcl-2-associated X protein(Bax),caspase-3 andγ-H2AX protein in cells.Results Ginsenoside Rg3 inhibited the proliferation of A549 cells in a dose-dependent manner(P<0.05).Compared with the control group,the cell clone formation rate,G6PD,ROS,NADPH levels,p-mTOR/mTOR and PCNA protein expressions were significantly decreased in the radiation group and the ginsenoside Rg3 group,and the cell apoptosis rate,γ-H2AX foci number,Bax,caspase-3,γ-H2AX protein expressions were significantly increased(P<0.05).Compared with the radiation group and the ginsenoside Rg3 group,the cell clone formation rate,G6PD,ROS,NADPH levels,p-mTOR/mTOR,and PCNA protein expressions were significantly decreased in the combination group,the cell apoptosis rate,γ-H2AX foci number,Bax,caspase-3,γ-H2AX protein expressions were significantly increased(P<0.05).Compared with the combination group,the cell clone formation rate,G6PD,ROS,NADPH levels,p-mTOR/mTOR and PCNA protein expressions were significantly increased in the activator group,and the cell apoptosis rate,γ-H2AX foci number,Bax,caspase-3,γ-H2AX protein expressions were significantly decreased(P<0.05).Conclusion The inhibitory effect of ginsenoside Rg3 on lung cancer cells may be realized through inhibition of PPP mediated by mTOR.
作者
黄琳
李彬
胡作为
HUANG Lin;LI Bin;HU Zuowei(Department of Oncology,Wuhan NO.1 Hospital,Wuhan 430000,China)
出处
《天津医药》
CAS
北大核心
2023年第8期791-796,共6页
Tianjin Medical Journal
基金
武汉市医学科研项目(WZ20Q04)。
