紫草素对HepG2细胞增殖、凋亡和PI3K/Akt/NF-κB信号通路蛋白表达的影响

Effects of shikonin on proliferation,apoptosis and PI3K/Akt/NF-κB signaling pathway protein expression in HepG2 cells in vitro

目的研究紫草素对肝癌HepG2细胞增殖、凋亡和磷脂酰肌醇3-激酶(PI3K)/蛋白激酶(Akt)/核转录因子-κB(NF-κB)信号通路的影响。方法以0(对照)、1、2.5和5μmol/L紫草素分别处理对数生长期的HepG2细胞48 h,分别采用MTT法检测细胞增殖,使用流式细胞仪检测细胞凋亡,采用Western blot法检测凋亡相关蛋白(Bax、Bcl-2、Caspase-3)、细胞自噬相关蛋白(LC3-Ⅰ、LC3-Ⅱ、p62)和PI3K/Akt/NF-κB通路蛋白表达情况。结果自小剂量、中剂量到大剂量,紫草素处理HepG2细胞增殖抑制率分别为(23.7±3.5)%、(36.2±6.1)%和(56.9±8.3)%,均显著高于对照组[(0.0±0.0)%,P<0.05],细胞凋亡率分别为(19.2±5.3)%、(37.4±7.6)%和(58.6±8.8)%,均显著高于对照组[(2.5±1.2)%,P<0.05];细胞凋亡相关蛋白表达Bax/Bcl-2比值和Caspase-3相对表达量分别为(1.3±0.2)和(2.7±0.3)、(8.2±0.6)和(0.45±0.10)和(0.78±0.16)和(0.95±0.21),显著高于对照组[分别为(0.6±0.1)和(0.18±0.06),P<0.05];细胞自噬相关蛋白表达LC3-Ⅱ/LC3-Ⅰ比值为(1.25±0.08)、(1.43±0.10)和(1.76±0.22),显著高于对照组[(0.96±0.08),P<0.05],p62相对表达水平分别为(0.81±0.09)、(0.62±0.15)和(0.43±0.08),显著低于对照组[(1.06±0.05),P<0.05];PI3K、Akt和p65蛋白表达水平分别为[(0.64±0.16)、(0.51±0.12)和(0.32±0.06)]、[(0.54±0.17)、(0.37±0.05)和(0.05±0.01)]和[(0.63±0.15)、(0.52±0.10)和(0.36±0.09)],均显著低于对照组[分别为(0.84±0.13)、(0.76±0.15)和(0.89±0.11),P<0.05]。结论紫草素可能通过抑制PI3K/Akt/NF-κB信号通路关键蛋白表达促进HepG2细胞凋亡和自噬,从而具有抑瘤作用。

Objective The aim of this study was to investigate the effects of shikonin,a herbal medicine,on proliferation,apoptosis and phosphatidylinositol 3-kinase(PI3K)/protein kinase(Akt)/nuclear transcription factor-κB(NF-κB)signaling pathway protein expression in HepG2 cells in vitro.Methods HepG2 cells in logarithmic growth phase were treated with different concentration[0(control),1,2.5 and 5μmol/L of shikonin for 48h.The proliferation inhibition rate of HepG2 cells was detected by MTT assay,the apoptosis by flow cytometry and the expression of apoptosis-related proteins(Bax,Bcl-2,Caspase-3),autophagy-related proteins(LC3-I,LC3-II,p62)and PI3K/Akt/NF-κB proteins were detected by Western bloting.Results After intervention for 48 h,the proliferation inhibition rates of cells from low,middle and high dose of shikonin were(23.7±3.5)%,(36.2±6.1)%and(56.9±8.3)%,all significantly higher than[(0.0±0.0)%,P<0.05]in the control,and theapoptosis rates were(19.2±5.3)%,(37.4±7.6)%and(58.6±8.8)%,also significantly higher than[(2.5±1.2)%,P<0.05]in the control;the apoptosis-related protein expression Bax/Bcl-2 ratio and relative expression of Caspase-3 were(1.3±0.2)and(2.7±0.3),(8.2±0.6)and(0.45±0.10),and(0.78±0.16)and(0.95±0.21),all significantly higher than[(0.6±0.1)and(0.18±0.06),respectively,P<0.05]in the control;the autophagy-related protein expression LC3-II/LC3-I ratio were(1.25±0.08),(1.43±0.10)and(1.76±0.22),significantly higher than[(0.96±0.08),P<0.05]in the control,while the relative expression of p62 were(0.81±0.09),(0.62±0.15),(0.43±0.08),significantly lower than that[(1.06±0.05),P<0.05]in the control,and the PI3K,Akt and p65 protein expression were[(0.64±0.16),(0.51±0.12)and(0.32±0.06)],[(0.54±0.17),(0.37±0.05)and(0.05±0.01)],and[(0.63±0.15),(0.52±0.10)and(0.36±0.09)],all significantly lower than[(0.84±0.13),(0.76±0.15)and(0.89±0.11)],respectively,P<0.05]in the control.Conclusion Shikonin promotes apoptosis and autophagy of HepG2 cells in vitro,which might be related to the inhibition of PI3K/Akt/NF-κB signaling pathway protein expression.