蠲瘤散结方对人肺腺癌A549细胞增殖和糖酵解的调控作用及机制研究  

Effect of Juanliu Sanjie Formula(蠲瘤散结方)on Proliferation and Glycolysis in Human A549 Lung Adenocarcinoma Cells

作　　者：刘艳霞 吴建春[1] 骆莹滨 李雁[1] LIU Yanxia;WU Jianchun;LUO Yingbin;LI Yan(Affiliated Hospital of Shanghai University of Traditional Chinese Medicine,Shanghai 200071,China)

机构地区：[1]上海中医药大学附属市中医医院肿瘤科,上海200071

出　　处：《辽宁中医杂志》2024年第8期18-23,I0001,I0002,共8页Liaoning Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(81973795,82174183);上海申康医院发展中心临床三年行动计划项目(SHDC2020CR4052)。

摘　　要：目的研究蠲瘤散结方对肺腺癌A549细胞增殖和糖酵解得影响,并探讨其潜在的分子机制。方法CCK-8法(cell counting kit-8,CCK-8)检测不同浓度蠲瘤散结方(2、4、6、8、10、12 mg·mL^(-1))处理A549细胞24 h和48 h后的细胞活力。将A549细胞分为对照组和蠲瘤散结方低、高(3、6 mg·mL^(-1))浓度组,加药干预24 h,细胞克隆形成实验检测A549细胞增殖情况;流式细胞术检测A549细胞周期;试剂盒检测细胞上清液中葡萄糖消耗量、乳酸生成水平;测量仪检测细胞上清液酸碱度(potential of hydrogen,pH值);实时荧光定量逆转录PCR(real-time RT-PCR,RT-qPCR)和蛋白质免疫印迹(western blot,WB)检测蛋白激酶B(protein kinase B,AKT)、磷酸化的蛋白激酶B(phosphorylation protein kinase B,p-AKT)、缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)、葡萄糖转运体1(facilitative glucose transporter,Glut1)、己糖激酶2(hexokinase2,HK2)、丙酮酸激酶(pyruvate kinase isozyme type M2,PKM2)、乳酸脱氢酶A(lactate dehydrogenase A,LDHA)基因和蛋白的表达。使用AKT激活剂后,试剂盒检测细胞上清液液中葡萄糖消耗量、乳酸生成水平;pH测量仪检测细胞上清液pH值;CCK-8检测A549细胞增殖,RT-qPCR和Western blot检测AKT(p-AKT)、Hif-1α、Glut1、HK2、PKM2、LDHA基因和蛋白的表达。结果蠲瘤散结方抑制A549细胞增殖(P<0.05),与对照组比较,蠲瘤散结方低、高(3、6 mg·mL^(-1))浓度组细胞克隆形成率降低(P<0.05,P<0.01),G0/G1期细胞比率下降(P<0.05,P<0.01),细胞上清液中葡萄糖消耗量以及乳酸生成量下降(P<0.05,P<0.01),AKT(p-AKT)、Hif-1α、Glut1、HK2、PKM2、LDHA基因和蛋白的表达下降(P<0.05,P<0.01)。使用AKT激活剂后,与蠲瘤散结方高浓度组比较,蠲瘤散结方联合AKT激活剂用药组细胞上清液葡萄糖消耗量和乳酸生成量上升(P<0.05,P<0.01),pH值下降(P<0.05),细胞增殖活力上升(P<0.05),AKT(p-AKT)、Hif-1α、Glut1、HK2、PKM2、LDHA基因和蛋白的表Objective The aim of this study is to investigate the effects and molecular mechanisms of Juanliu Sanjie Formula(蠲瘤散结方)on the proliferation and glycolysis of lung adenocarcinoma A549 cells.Methods The cell counting kit-8(CCK-8)method was used to detect the cell viability of A549 cells treated with different concentrations of Juanliu Sanjie Formula(2,4,6,8,10,12 mg·mL^(-1))for 24 h and 48 h.A549 cells were divided into control group and Juanliu Sanjie Formula low and highconcentration groups(3,6 mg·mL^(-1)).Drug intervention was added for 24 hours and the proliferation of A549 cells was detected through cell clone formation experiments.A549 cell cycle was detected by flow cytometry.The reagent kit detected glucose consumption and lactate production levels in the cell supernatant.The potential of hydrogen(pH)value of cell supernatant was detected by using a pH meter.CCK-8 detected A549 cell proliferation.Real-time RT-PCR(RT-qPCR)and Western blot(WB)were used to detect the expressions of protein kinase B(AKT),phosphorylation protein kinase B(p-AKT),hypoxia inducible factor-1α(HIF-1α),facilitative glucose transporter(Glut1),hexokinase 2(HK2),pyruvate kinase isozyme type M2(PKM2),lactate dehydrogenase A(LDHA)genes and proteins.Results Juanliu Sanjie Formula inhibited the proliferation of A549 cells(P<0.05,P<0.01).Compared with the control group,the Juanliu Sanjie Formula low and high concentrations groups(3,6 mg·mL-1)lowered the cell clone formation rate(P<0.05,P<0.01),the G0/G1 phase cell ratio(P<0.05,P<0.01),the glucose consumption and lactic acid production in the cell supernatant(P<0.05,P<0.01).The expressions of AKT(p-AKT),Hif-1α,Glut1,HK2,PKM2,LDHA genes and proteins decreased(P<0.05,P<0.01).After using AKT activator,compared with the Juanliu Sanjie Formula high concentration group,the Juanliu Sanjie Formula combined with AKT activator increased glucose consumption and lactic acid production in cell supernatant(P<0.05,P<0.01),decreased pH value(P<0.05)and increased cell proliferation activity(P<0

关 键 词：蠲瘤散结方 肺腺癌 增殖 糖酵解 AKT 

分 类 号：R256.13[医药卫生—中医内科学]