摘要
目的观察中药雷公藤甲素(TPL)对肝癌细胞增殖、凋亡、代谢的影响,探讨其可能的作用机制。方法对细胞进行培养与转染,药物处理细胞24 h、48 h、72 h后,CCK-8法检测细胞存活率,确定药物对细胞的半抑制浓度(IC50)。野生型细胞分为对照组、给药组。转染细胞分为si-NC组、si-NC+TPL组、si-HIF+TPL组。流式细胞术检测细胞凋亡率,氟代脱氧葡萄糖(18F-FDG)摄取实验检测TPL对肝癌细胞糖代谢水平的影响。实时荧光定量PCR检测细胞糖酵解途径关键酶乏氧诱导因子-1α(HIF-1α)、己糖激酶2(HK2)、葡萄糖转运蛋白1(GLUT1)、丙酮酸激酶(PKM2)、乳酸脱氢酶A(LDHA)的mRNA水平。蛋白免疫印迹法检测HIF-1α、HK2、PKM2、LDHA蛋白的表达水平。结果(1)当TPL浓度达到40 mol/L以上时,细胞的存活率显著降低(P<0.05)。(2)与对照组相比,给药组(40 mol/L)细胞凋亡率明显增加(P<0.05)。(3)给药组(40 mol/L)细胞对18F-FDG的相对摄取率,与对照组比较,明显降低(P<0.05)。(4)相较于对照组,给药组HIF-1α、HK2、GLUT1、PKM2、LDHA等糖酵解途径关键基因mRNA表达水平降低(P<0.01)。(5)TPL干预细胞后,HIF-1α、HK2、PKM2、LDHA等蛋白表达水平降低(P<0.05)。敲减HIF-1α基因后,影响其下游蛋白的表达。结论中药TPL可抑制肝癌细胞增殖、侵袭,影响其代谢,诱导其凋亡,其机制可能与HIF-1α介导的糖酵解途径有关。
Objective To observe the effects of triptolide(TPL)on proliferation,apoptosis and metabolism of hepatocellular carcinoma cells and explore its possible mechanism.Methods The cells were cultured and transfected.After 24 h,48 h and 72 h of drug treatment,CCK-8 kit was used to detect the cell survival rate and make clear 50%inhibiting concentration(IC50)of the drug on cells.Wild-type cells were divided into two groups control group and TPL group.The transfected cells were divided into three groups:si-NC group,si-NC+TPL group and si-HIF+TPL group.Flow cytometry was used to detect the apoptotic rate of hepatocellular carcinoma cells.Fluorodeoxyglucose(18F-FDG)uptake assay was used to detect the effect of triptolide on glucose metabolism in hepatoma cells.Quantitative Real-time PCR was used to detect the mRNA expressive level of Bonding enzymes,such as hypoxia inducible factor-1α(HIF-1α),hexokinase 2(HK2),glucose transporter 1(GLUT1),pyruvate kinase 2(PKM2),lactate dehydrogenase A(LDHA)on glycolysis pathways.The protein expressive level of hypoxia inducible factor-1α(HIF-1α),hexokinase 2(HK2),pyruvate kinase(PKM2),lactate dehydrogenase A(LDHA)was detected by Western Blot.Results(1)When TPL concentration reached 40 mol/L,the cell survival rate decreased significantly(P<0.05).(2)Compared with the control group,the apoptosis rate of TPL group(40 mol/L)was significantly increased(P<0.05).(3)In TPL group(40 mol/L),the relative uptake rate of FDG decreased significantly(P<0.05).(4)Compared with the control group,the expression level of HIF-1α、HK2、GLUT1、PKM2、LDHA and other key genes in glycolysis pathway decreased significantly(P<0.01).(5)After TPL intervention,the expression of HIF-1α、HK2、PKM2、LDHA decreased(P<0.05).After knockdown of HIF-1αgene,the expression of downstream protein was affected.Conclusion Triptolide can significantly inhibit the proliferation and apoptosis ofhepatocellular carcinoma cells,and its mechanism may be related to the glycolysis pathway mediated by HIF-1α.
作者
李恬
靳明明
宋少莉
黄钢
LI Tian;JIN Ming-ming;SONG Shao-li;HUANG Gang(Shanghai University of Traditional Chinese Medicine,Shanghai 200120;Shanghai University of Medicine & Health Sciences,Shanghai 201318;Fudan University Shanghai Cancer Center,Shanghai 200032)
出处
《世界中西医结合杂志》
2020年第6期981-985,990,共6页
World Journal of Integrated Traditional and Western Medicine
基金
国家自然科学基金项目(81830052,81530053,81771861,81971648)
上海市分子影像学重点实验室项目(18DZ2260400)。
