苦参碱诱导肝癌HepG2细胞自噬的作用与机制

Induction of Autophagy by Matrine in Liver Cancer HepG2 Cells and Its Mechanism of Action

目的探讨苦参碱对肝癌HepG2细胞自噬的作用,进一步研究苦参碱诱导HepG2细胞自噬的机制。方法将0.0(对照组)、0.4、0.8、1.6g·L^(-1)苦参碱及0.4g·L~(^(-1))苦参碱+10mmol·L^(-1)自噬特异抑制剂(3-MA)分别作用于肝癌HepG2细胞,构建体外模型。采用免疫印迹法(Western blotting)检测自噬基因LC3-Ⅱ的表达,实时荧光定量PCR(Realtime-PCR)检测自噬基因WIPI1的表达,Annexin V FITC-PI流式细胞术检测HepG2细胞凋亡率,电镜观察HepG2细胞自噬体变化与凋亡情况。结果 0.4、0.8、1.6g·L^(-1)苦参碱作用的HepG2细胞LC3-Ⅱ表达相比对照组均显著增加(P<0.05),其中0.4g·L^(-1)苦参碱作用的HepG2细胞LC3-Ⅱ表达水平最高;0.4g·L^(-1)苦参碱作用的HepG2细胞自噬体、WIPI1mRNA表达水平较对照组明显增加(P<0.05)。0.4g·L^(-1)苦参碱联合3-MA作用的HepG2细胞凋亡率较单纯0.4g·L^(-1)苦参碱作用的凋亡率显著增加(P<0.05)。结论苦参碱诱导HepG2自噬且减少细胞凋亡,与WIPI1的表达相关。自噬抑制剂可抑制苦参碱诱导的自噬,促进肝癌细胞凋亡,二者联合可成为临床治疗肝癌的新措施。

Objective To investigate the effect of matrine on autophagy in liver cancer HepG2 cells and its mechanism of action.Methods HepG2 cells were treated with different doses of matrine（0.0,0.4,0.8 and 1.6 g·L-1）and 0.4 g·L-1 matrine＋10 mmol·L-1 3-MA,building an in vitro model.In addition,0.0 g·L-1 matrine treated was control group.The expression of LC3-Ⅱand WIPI1 was detected by Western blotting and real-time PCR,respectively.The apoptosis of HepG2 cells was measured by annexin V FITC-PI flow cytometry.The autophagosomes were observed using the electron microscope.Results Compared with the control group,matrine（0.4,0.8 and 1.6 g·L-1）treatment significantly increased the expression of LC3-Ⅱin HepG2 cells（P〈0.05）,the expression level was highest with 0.4 g·L-1 matrine.Furthermore,compared with control group,the treatment with 0.4 g·L-1 matrine obviously increased the autophagosomes and WIPI1 mRNA expression（P〈0.05）,with treatment with 0.4 g·L-1 matrine alone,the apoptosis of HepG2 cells was markedly increased by combined treatment with matrine and 3-MA（P〈0.05）.Conclusion Matrine induces autophagy and inhibits apoptosis in HepG2 cells through increasing WIPI1 expression.Autophagy inhibitor can suppress matrine-induced autophagy and promote apoptosis in HepG2 cells.Therefore,their combination may be a new method for clinical treatment of liver cancer.