健脾养正消癥汤通过抑制有氧糖酵解对胃癌HGC-27细胞增殖及干细胞特性的影响  

Jianpi Yangzheng Xiaozheng Decoction Affect Proliferation and Stemness of Gastric Cancer HGC-27 Cells by Inhibiting Aerobic Glycolysis

作　　者：陶鹤云 刘元杰 李洁玭 曾树宏 张莹[1,2] 刘沈林 邹玺[1] TAO Heyun;LIU Yuanjie;LI Jiepin;ZENG Shuhong;ZHANG Ying;LIU Shenlin;ZOU Xi(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China;The First Clinical Medical College of Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学附属医院,南京210029 [2]南京中医药大学第一临床医学院,南京210023

出　　处：《中国实验方剂学杂志》2023年第11期82-88,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：江苏省2019年省级重点研发计划专项资金(社会发展)项目(BE2019771);江苏省中医院第三批高峰人才培养计划项目(省中科[2021]111号)。

摘　　要：目的:观察健脾养正消癥汤通过抑制有氧糖酵解过程而对人胃癌细胞HGC-27增殖过程及干细胞特性的影响,并探讨其具体机制。方法:采用噻唑蓝(MTT)比色法测定健脾养正消癥汤(0.25、0.5、1、2、4、8、16、32 g·L^(-1))处理胃癌细胞HGC-27的存活率及化疗敏感性的变化;细胞克隆形成实验检测健脾养正消癥汤(2、4、8 g·L^(-1))对细胞克隆形成能力的影响;葡萄糖检测试剂盒和乳酸测定试剂盒检测健脾养正消癥汤处理后胃癌细胞有氧糖酵解水平的变化;流式细胞术检测胃癌HGC-27细胞中干细胞亚群的比例;蛋白免疫印迹法(Western blot)检测胃癌细胞中乳酸脱氢酶(LDH)及己糖激酶2(HK2)、葡萄糖转运蛋白1(GLUT1)、丙酮酸激酶同工酶M2(PKM2)等糖酵解相关蛋白的表达及八聚体结合转录因子4(OCT4)、SRYbox转录因子2(SOX2)、Nanog等干细胞特性相关蛋白的表达。结果:MTT比色法结果表明,与空白组比较,健脾养正消癥汤(0.5、1、2、4、8、16、32 g·L^(-1))组能够抑制胃癌HCG-27细胞活力(P<0.05,P<0.01),其半数抑制浓度(IC_(50))为4.83 g·L^(-1),且健脾养正消癥汤能够提高胃癌HGC-27细胞对顺铂化疗的敏感性;克隆形成实验表明,与空白组比较,健脾养正消癥汤(2、4、8 g·L^(-1))均能显著减少胃癌HGC-27细胞克隆形成数(P<0.01),且呈现浓度依赖性;流式细胞术表明,与空白组比较,健脾养正消癥汤(2、4、8 g·L^(-1))作用后细胞的CD44^(+)CD24^(+)ALDH^(+)细胞亚群比例率明显降低(P<0.05);葡萄糖检测和乳酸检测表明,与空白组比较,健脾养正消癥汤(2、4、8 g·L^(-1))能有效抑制胃癌细胞的葡萄糖摄取和乳酸生成(P<0.01);Western blot表明,与空白组比较,健脾养正消癥汤(2、4、8 g·L^(-1))能下调SOX2、Nanog、OCT4等干性相关蛋白及PKM2、LDH、GLUT1、HK2等有氧糖酵解途径相关蛋白的水平(P<0.05,P<0.01),并呈浓度依赖性。结论:健脾养正消癥汤对胃癌细胞增殖具�Objective:To observe the effect of Jianpi Yangzheng Xiaozheng decoction(JYXD)on the proliferation and stemness of the human gastric cancer(GC)cell line HGC-27 by inhibiting aerobic glycolysis,and explore the underlying mechanism.Method:Methyl thiazolyl tetrazolium(MTT)assay was employed to determine the survival rate and chemotherapy sensitivity of HGC-27 cells treated with JYXD(0.25,0.5,1,2,4,8,16,32 g·L^(-1)).Colony formation assay was employed to detect the effect of JYXD(2,4,8 g·L^(-1))on the colony formation of the cells.The aerobic glycolysis level of HGC-27 cells after treatment with JYXD was measured by glucose assay kit and lactic acid assay kit.The proportion of stem cell subsets in HGC-27 cells was detected by flow cytometry.Western blot was employed to determine the expression of glycolysis-associated proteins such as lactate dehydrogenase(LDH),hexokinase 2(HK2),glucose transporter 1(GLUT1),and pyruvate kinase isozyme M2(PKM2),and the expression of stemness-associated proteins such as octamerbinding transcription factor 4(OCT4),SRY-box transcription factor 2(SOX2),and Nanog.Result:JYXD(0.5,1,2,4,8,16,32 g·L^(-1))inhibited the activity of HGC-27 cells(P<0.05,P<0.01),with the inhibitory concentration 50(IC_(50))of 4.83 g·L^(-1),and it improved the sensitivity of HGC-27 cells to cisplatin chemotherapy.Compared with the control group,JYXD(2,4,8 g·L^(-1))reduced the colony formation number of HGC-27 cells(P<0.01)in a concentration-dependent manner.Flow cytometry showed that compared with that in the control group,the proportion of CD44+CD24+ALDH+population in the cells treated with JYXD(2,4,8 g·L^(-1))decreased(P<0.05).In addition,JYXD(2,4,8 g·L^(-1))inhibited the glucose uptake and lactic acid production of HGC-27 cells.Western blot showed that compared with the control group,JYXD(2,4,8 g·L^(-1))downregulated the expression levels of SOX2,Nanog,OCT4,PKM2,LDH,GLUT1,and HK2(P<0.05,P<0.01)in a concentration-dependent manner.Conclusion:JYXD may inhibit the proliferation and reduce the stemness of HGC-

关 键 词：胃癌 健脾养正消癥汤 有氧糖酵解 肿瘤干细胞特性 增殖 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5