大车前苷增强二甲双胍对HepG2细胞增殖、凋亡和自噬的作用研究

Effects of Procyanoside on Proliferation,Apoptosis and Autophagy of HepG2 Cells Enhanced by Metformin

目的:探究大车前苷(plantamajoside,PMS)联合二甲双胍(metformin,MET)对人肝癌HepG2细胞增殖、凋亡和自噬的影响。方法:将HepG2细胞分为Control组、PMS组、MET组和MET+PMS组,分别采用15mmol·L^(-1)MET及250mg·L^(-1)PMS单独或联合干预24h后,采用溴脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,BrdU)染色检测HepG2细胞增殖情况;赫斯特(Hoechst)染色检测HepG2细胞凋亡情况;RT-qPCR检测Beclin1mRNA、p62mRNA及LC3IImRNA的表达;蛋白免疫印迹(Western Blot)法检测肿瘤增殖抗原Ki67、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、半胱氨酸蛋白酶3(Caspase-3)、Caspase-9、Beclin1、p62、微管相关蛋白1轻链3II(microtubule-assaiated pro-tein 1 light chain 3 II,LC3II)/LC3I、磷脂酰肌醇-3-羟激酶(phosphatidylinositol-3-hydroxykinase,PI3K)、蛋白激酶B(protein kinasesB,AKT)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)及p-PI3K、p-AKT、p-mTOR的表达;免疫荧光法检测LC3阳性的细胞数及加入自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)后LC3阳性的细胞数。结果:与Control组比较,MET+PMS组细胞的增殖率、Ki67、PCNA、p62、p-PI3K/PI3K、p-AKT/AKT及p-mTOR/mTOR的表达显著降低(P<0.05);细胞凋亡率、LC3阳性细胞数及Caspase-3、Caspase-9、Beclin1、LC3II/LC3I的表达显著升高(P<0.05);且加入自噬抑制剂3-MA后,3-MA+MET+PMS组LC3阳性细胞数显著低于MET+PMS组。结论:大车前苷可增强二甲双胍对人肝癌HepG2细胞增殖的抑制作用,促进HepG2细胞凋亡和自噬。

Objective:To investigate the effects of procyanoside combined with metformin on proliferation,apoptosis and autophagy of human hepatoma HepG2 cells.Methods:HepG2 cells were divided into Control group,PMS group,MET group and MET+PMS group.After intervention with 15 mmol·L^(-1) metformin(MET)and 250 mg·L^(-1) plantamaxide(PMS)alone or in combination for 24 hours,the proliferation of HepG2 cells was detected by bromodeoxyuridine(BrdU)staining;the apoptosis of HepG2 cells was detected by Hoechst staining;the expressions of Beclin1 mRNA,p62 mRNA and LC3 II mRNA were detected by fluorescence quantitative polymerase chain reaction(qPCR);Western blot method was used to detect the expression of tumor proliferating antigen Ki67,proliferating cell nuclear antigen(PCNA),caspase-3,caspase-9,autophagy genes Beclin1,p62,microtubule-assaiated protein 1 light chain 3 II(LC3 II)/LC3 I,Phosphatidylinositol-3-hydroxykinase(AKT),rapamycin target of rapamycin(mTOR),p-PI3 K,p-AKT and p-mTOR.The number of LC3 positive cells and the number of LC3 positive cells after adding autophagy inhibitor 3-methyladenine(3-MA)were detected by immunofluorescence.Results:Compared with the Control group,the cell proliferation rate,the expression of Ki67,PCNA,p62,p-PI3 K/PI3 K,p-AKT/AKT,and p-mTOR/mTOR in each treatment group decreased significantly(P<0.05);the apoptosis rate,the number of LC3 positive cells and the expression of caspase-3,caspase-9,Beclin1 and LC3 II/LC3 I increased significantly(P<0.05);after adding autophagy inhibitor 3-MA,the number of LC3 positive cells in 3-MA+MET+PMS group was significantly lower than that in MET+PMS group;the effect of met combined with PMS is more significant.Conclusion:Procyanoside can enhance the inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and promote apoptosis and autophagy.