葛根素诱导人肝癌HepG2细胞凋亡与自噬及其分子机制研究

Study of puerarin induce apoptosis and autophagy in human liver cancer HepG2 cells and its molecular mechanism

关键词:  葛根素  肝癌  凋亡  自噬  Akt/mTOR/Beclin1通路

Key words:  puerarin  liver cancer  apoptosis  autophagy  Akt/mTOR/Beclin1 pathway

摘要:

[目的] 探索葛根素（Puerarin）诱导人肝癌HepG2细胞凋亡与自噬及其可能的分子机制。[方法] 以二甲基亚砜（DMSO，空白组）、葛根素（50、100、200 μg/mL）、顺铂20 μg/mL分别干预对数生长期人肝癌HepG2细胞。48 h后，CCK-8法检测细胞增殖抑制率，Annexin V-FITC/PI双染法检测细胞凋亡水平，吖啶橙染色法观察细胞自噬状况，蛋白免疫印迹法（Western blot）检测蛋白激酶B（Akt）、磷酸化Akt（p-Akt）、激活型半胱氨酸蛋白酶（Cleaved Caspase-3）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、酵母ATG6同源物（Beclin1）、微管相关蛋白1轻链3（LC3）蛋白表达。[结果] 与空白组比较，葛根素100、200 μg/mL组和顺铂20 μg/mL组HepG2细胞增殖抑制率和凋亡率显著升高（P<0.01），自噬溶酶体数量明显增多（P<0.01），p-Akt、p-mTOR、bcl-2表达明显下调而Cleaved Caspase-3、Bax、Beclin1、LC3-I、LC3-Ⅱ表达明显上调（P<0.05或P<0.01），Akt磷酸化（p-Akt/Akt比值）降低且Bax/bcl-2、LC3-Ⅱ/LC3-Ⅰ比值升高（P<0.01）。与顺铂20 μg/mL组比较，葛根素200 μg/mL组细胞增殖抑制率和凋亡率显著升高（P<0.05或P<0.01），自噬溶酶体数量明显增多（P<0.05），p-Akt、p-mTOR表达下调而Cleaved Caspase-3、Bax、Beclin1、LC3-Ⅱ明显上调（P<0.05或P<0.01），Akt磷酸化降低且Bax/Bcl-2、LC3-Ⅱ/LC3-I比值升高（P<0.01）。[结论] 葛根素具有诱导人肝癌HepG2细胞凋亡与自噬的作用，该作用具有一定的剂量依赖性；抑制Akt/mTOR/Beclin1通路进而诱导促凋亡、促自噬相关蛋白表达和活化可能是其重要的分子机制。

Abstract:

[Objective] To study the effects of puerarin induce apoptosis and autophagy in human liver cancer HepG2 cells and its molecular mechanism. [Methods] The DMSO (blank control group),puerarin (50,100,200 μg/mL) and cisplatin 20 μg/mL were used to interfere with human liver cancer HepG2 cells in logarithmic growth respectively. 48h later,the proliferation inhibition rate of HepG2 cells was detected by CCK-8 method,the apoptosis level was detected by Annexin V-FITC/PI double staining;the autophagy of cells was observed by acridine orange staining;the expression of Akt,p-Akt,Cleaved Caspase-3,bcl-2,Bax,p-mTOR,Beclin1,LC3 proteins were detected by Western blot. [Results] Compared with blank group,the proliferation inhibition rate and apoptosis rate of puerarin 100 μg/mL group. Puerarine 200 μg/mL group and cisplatin 20 μg/mL group were increased (P<0.01),the number of autophagolysosomes was significantly increased (P<0.01);the expression of p-Akt,p-mTOR,bcl-2 were down-regulated and the expression of Cleaved Caspase-3,Bax,Beclin1,LC3-Ⅰ,LC3-Ⅱ were up-regulated (P<0.05 or P<0.01),the Akt phosphorylation ratio (p-Akt/Akt) was decreased and the Bax/bcl-2,LC3-Ⅱ/LC3-Ⅰ ratio were increased (P<0.01). Compared with Cisplatin 20 μg/mL group,the proliferation inhibition rate and apoptosis rate of puerarin 200 μg/mL group were increased(P<0.01 or P<0.01),the number of autophagolysosomes was significantly increased (P<0.05);the expression of p-Akt,p-mTOR were down-regulated and the expression of Cleaved Caspase-3,Bax,Beclin1,LC3-Ⅱ were up-regulated (P<0.05 or P<0.01),the Akt phosphorylation ratio was decreased and the Bax/bcl-2,LC3-Ⅱ/LC3-Ⅰratio were increased (P<0.01). [Conclusion] Puerarin can induce apoptosis and autophagy in human liver cancer HepG2 cells,which has a dose-dependent effect;inhibiting Akt/mTOR signaling pathway to induce pro-apoptosis,autophagy-related protein expression and activation may be its important molecular mechanism.