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、200μg/mL)、顺铂20μg/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、200μg/mL组和顺铂20μg/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20μg/mL组比较,葛根素200μg/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。
[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(50,100,200μg/mL)and cisplatin 20μg/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 byWestern blot.[Results]Compared with blank group,the proliferation inhibition rate and apoptosis rate of puerarin 100μg/mL group.Puerarine 200μg/mL group and cisplatin 20μg/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-Ⅱwere 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-Ⅰratio were increased(P<0.01).Compared with Cisplatin 20μg/mL group,the proliferation inhibition rate and apoptosis rate of puerarin 200μg/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-Ⅱwere 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.
作者
胡亚丽
杨杰
庞茜茜
HU Yali;YANG Jie;PANG Qianqian(The First Affiliated Hospital of Hebei North University,Zhangjiakou 075000,China)
出处
《天津中医药》
CAS
2021年第11期1472-1477,共6页
Tianjin Journal of Traditional Chinese Medicine
基金
张家口市科技计划自筹经费项目(1621041D)。
