虎七散含药血清对食管癌细胞Ec9706糖酵解的影响及AMPK/FOXO3a通路的调节作用研究

Effect of Huqi San Contained Serum on Glycolysis of Esophageal Cancer Cells Ec9706 and Regulation of AMPK/FOXO3a Pathway

目的研究虎七散含药血清对食管癌细胞Ec9706糖酵解的影响及对磷酸腺苷激活的蛋白激酶(AMPK)/叉头框蛋白O3a(FOXO3a)通路的调节作用。方法收集Ec9706细胞,接种于细胞培养皿中,分别设置空白对照组、虎七散低、中、高剂量组及顺铂组,虎七散各剂量组分别加入虎七散含药血清,顺铂组加入2μg·mL^(-1)的顺铂,空白对照组加入新鲜DMEM培养液,四噻唑蓝(MTT)法测定Ec9706细胞增殖率,酶联免疫吸附法(ELISA)测定Ec9706细胞葡萄糖摄取量、乳酸生成量、己糖激酶(HK)及乳酸脱氢酶(LDH)活性,划痕实验测定Ec9706细胞24h迁移率,Transwell实验测定Ec9706细胞侵袭率,流式细胞术测定Ec9706细胞凋亡率,实时定量聚合酶链式反应(RT-qPCR)测定Ec9706细胞FOXO3a、B细胞淋巴瘤-2相关X蛋白(Bax)、B淋巴细胞瘤2(Bcl-2)及乳酸脱氢酶A(LDHA)mRNA水平,免疫印迹法(Western blot)测定Ec9706细胞p-AMPK、FOXO3a、Bax、Bcl-2及LDHA蛋白水平。结果与空白对照组比较,虎七散各剂量组及顺铂组Ec9706细胞24 h、48 h及72 h增殖率、葡萄糖摄取量、乳酸生成量、HK活性及LDH活性,Ec9706细胞24 h迁移率、侵袭率、Bcl-2及LDHA mRNA及蛋白水平、p-AMPK/AMPK水平显著降低(P<0.05),Ec9706细胞凋亡率、FOXO3a、Bax mRNA及蛋白水平显著升高(P<0.05),且随着虎七散剂量的增加,Ec9706细胞各项指标变化更优。结论虎七散能够显著抑制食管癌Ec9706细胞糖酵解过程,进而抑制Ec9706细胞增殖、迁移及侵袭过程,促进Ec9706细胞凋亡,其机制可能与调节AMPK/FOXO3a通路有关。

Objective To study the effects of Huqi San contained serum on glycolysis of esophageal cancer cells Ec9706and the regulation of AMPK/FOXO3a pathway.Methods Ec9706 cells were collected and inoculated in cell culture dishes.Blank control group,Huqi San low-dose,medium-dose and high-dose groups and cisplatin groups were set up,respectively.Each dose group of Huqi San was added Huqi San contained serum,cisplatin group was added 2μg·mL^(-1)cisplatin,and blank control group was added fresh DMEM culture medium.The proliferation rate of Ec9706 cells was determined by MTT assay,the glucose intake,lactic acid production,HK and LDH activities of Ec9706 cells were determined by ELISA assay,the 24 h mobility of Ec9706 cells was determined by scratch assay,and the invasion rate of Ec9706 cells was determined by Transwell assay.The apoptosis rate of Ec9706 cells was determined by flow cytometry,and the mRNA levels of FOXO3a,Bax,Bcl-2 and LDHA in Ec9706 cells were determined by RT-qPCR.The protein levels of p-AMPK,FOXO3a,Bax,Bcl-2 and LDHA in Ec9706 cells were determined by Western blot.Results Compared with blank control group,the proliferation rate,glucose intake,lactate production,HK activity and LDH activity of Ec9706 cells at 24 h,48 h and 72 h in Huqi San different doses groups and cisplatin group,the migration rate,invasion rate,Bcl-2 and LDHA mRNA and protein levels,p-AMPK/AMPK levels of Ec9706 cells at 24 h were significantly decreased(P<0.05).Apoptosis rate of Ec9706 cells,the mRNA and protein levels of FOXO3a and Bax were significantly increased(P<0.05),and with the increase of the dose of Huqi San,the changes of various indexes of Ec9706cells were better.Conclusion Huqi San can significantly inhibit the glycolysis of esophageal cancer Ec9706 cells,and inhibit Ec9706 cells proliferation,and promote the apoptosis of Ec9706 cells.The mechanism may be related to the regulation of AMPK/FOXO3a pathway.