沙蟾毒精对人肝癌细胞HepG2增殖凋亡的调控作用及其机制

Regulatory effects of arenobufagin on proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells and the mechanism

目的观察沙蟾毒精对人肝癌细胞株HepG2增殖、凋亡的调控作用,并探讨其机制。方法培养HepG2细胞并分为对照组和实验组,实验组分别加入5、10、20、40、80 nmol/L的沙蟾毒精,对照组正常培养,分别于培养24、48、72 h后,采用CCK-8法检测并计算细胞存活率。将HepG2细胞分为对照组和沙蟾毒精低、中、高剂量组,沙蟾毒精低、中、高剂量组分别加入1.25、2.5、5 nmol/L的沙蟾毒精,对照组正常培养。采用流式细胞术测算细胞凋亡率,ELISA法检测细胞培养上清液中的白细胞介素(IL)-6、IL-8、转化生长因子β1(TGF-β1)、血管内皮生长因子(VEGF)。通过分子对接预测沙蟾毒精与JAK1、JAK2、STAT3的结合度,Westernblotting法检测沙蟾毒精作用后HepG2细胞中的p-JAK1、p-JAK2、p-STAT3蛋白。结果5、10、20、40、80 nmol/L的沙蟾毒精作用24、48、72 h后HepG2细胞存活率均低于对照组(P均<0.01)。沙蟾毒精低、中、高剂量组细胞凋亡率依次增高,且均高于对照组(P均<0.05)。对照组和沙蟾毒精低、中、高剂量组细胞上清液IL-6、TGF-β1、VEGF水平依次降低(P均<0.05);沙蟾毒精中、高剂量组细胞上清液IL-8水平低于对照组,沙蟾毒精低、中、高剂量组细胞上清液IL-8水平依次降低(P均<0.05)。沙蟾毒精与JAK1、JAK2、STAT3有良好的亲和力;沙蟾毒精中、高剂量组细胞中p-JAK1、p-JAK2、p-STAT3蛋白相对表达量低于沙蟾毒精低剂量组和对照组,沙蟾毒精高剂量组p-JAK1、p-JAK2、p-STAT3蛋白相对表达量低于沙蟾毒精中剂量组(P均<0.05)。结论沙蟾毒精可抑制HepG2细胞增殖并诱导其凋亡,作用呈剂量依赖性;沙蟾毒精的作用机制可能与调控JAK/STAT3信号通路有关。

Objective To observe the regulatory effects of arenobufagin on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells,and to explore their mechanism.Methods HepG2 cells were cultured and divid-ed into the control group and experimental group.Arenobufagin of 5,10,20,40 and 80 nmol/L was added to the experi-mental group,respectively.The cells in the control group were cultured normally.After 24,48 and 72 h of culture,the cell survival rate was detected and calculated by CCK-8 method.HepG2 cells were then divided into the control group and low-dose,medium-dose and high-dose arenobufagin groups.Cells in the low-dose,medium-dose and high-dose arenobufa-gin groups were added with 1.25,2.5,and 5 nmol/L arenobufagin,respectively.The cells in the control group were cul-tured normally.The apoptosis rate was measured by flow cytometry.The levels of interleukin-6(IL-6),IL-8,transform-ing growth factor-β1(TGF-β1),and vascular endothelial growth factor(VEGF)in the supernatant of cell culture were de-tected by ELISA.The binding degree of arenobufagin to JAK1,JAK2 and STAT3 was detected by molecular docking,and the protein expression levels of p-JAK1,p-JAK2 and p-STAT3 in HepG2 cells treated with arenobufagin were detected by Western blotting.Results The survival rates of HepG2 cells treated with arenobufagin at 5,10,20,40 and 80 nmol/L for 24,48 and 72 h were lower than those of the control group(all P<0.01).The apoptosis rates of the low-dose,medium-dose and high-dose arenobufagin groups increased successively,which were higher than those of the control group(all P<0.05).The levels of IL-6,TGF-β1 and VEGF in the cell supernatant of the control group and the low-dose,medium-dose and high-dose areobufagin groups decreased in turn(all P<0.05).The levels of IL-8 in the cell supernatant of the medium-dose and high-dose arenobufagin groups were lower than that of the control group,and the levels of IL-8 in the cell superna-tant of the low-dose,medium-dose and high-dose arenobufagin groups decreased successively(all P<0.05).Arenobufagin had good affinity with JAK1,JAK2 and STAT3.The relative protein expression levels of p-JAK1,p-JAK2 and p-STAT3 in the medium-dose and high-dose arenobufagin groups were lower than those in the low-dose arenobufagin group and control group,and the relative protein expression levels of p-JAK1,p-JAK2 and p-STAT3 in the high-dose arenobufagin group were lower than that in the medium-dose arenobufagin group(all P<0.05).Conclusion Arenobufagin can inhibit the proliferation and induce apoptosis of HepG2 cells in a dose-dependant manner,and the mechanism may be related to the regulation of JAK/STAT3 signaling pathway.