山楂原花青素下调Traf6/NF-κB信号通路诱导肝癌细胞凋亡

Hawthorn Proanthocyanidins Induce the Apoptosis of Huh-7 Cells by Down-Regulating the Traf6/NF-κB Signaling Pathway

目的:探讨山楂原花青素(hawthorn procyanidins,HPC)诱导肝癌细胞Huh-7增殖和促进其凋亡的机制。方法:体外常规培养Huh-7细胞株和过表达Traf6基因Huh-7细胞株,配制0、5、10、20、40、80μg/mL不同质量浓度的HPC,采用细胞计数8(cell counting kit-8,CCK-8)试剂盒检测HPC对Huh-7细胞和过表达Traf6基因Huh-7细胞增殖的抑制作用,并选40μg/mL作为HPC实验质量浓度;利用实时定量聚合酶链反应检测Huh-7细胞中Traf6、NF-κB?p65、Bax和Caspase-3的mRNA表达水平;利用蛋白质免疫印迹法(Western blot)检测Huh-7细胞中Traf6、NF-κB p65、Bax和Caspase-3蛋白表达水平;利用Annexin V-FITC/PI凋亡检测试剂盒检测HPC对Huh-7细胞凋亡的影响。结果:HPC质量浓度大于20μg/mL时可以明显抑制Huh-7细胞增殖,并随HPC质量浓度升高Huh-7细胞的相对存活率显著降低(P<0.05);Traf6蛋白过表达可以促进Huh-7细胞的增殖。与空白对照组比较,40μg/mL的HPC可以显著抑制肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor associated factor 6,Traf 6)和核转录因子-κB(nuclear factor kappa B,NF-κB) p65 mRNA和蛋白表达(P<0.05),显著增强Bax和Caspase-3 mRNA和蛋白表达(P<0.05),显著促进Huh-7细胞的凋亡(P<0.05)。结论:HPC可以通过下调Traf6/NF-κB信号通路诱导肝癌细胞凋亡。

The purpose of this study was to explore the mechanism by which hawthorn proanthocyanidins(HPC)inhibit cell proliferation and promote apoptosis in Huh-7 cells.Huh-7 cells and Traf6-overexpressed Huh-7 cells were cultured routinely at HPC concentrations of 0,5,10,20,40 and 80μg/mL.Cell counting kit-8(CCK-8)was used to detect the inhibitory effect of HPC on the proliferation of Huh-7 cells and Traf6-overexpressed Huh-7 cells.The mRNA expression of the tumor necrosis factor receptor associated factor 6(Traf6),nuclear factor kappa-B p65(NF-κB p65),Bax and caspase-3 genes were evaluated by real-time polymerase chain reaction(PCR).The protein expression of Traf6,NF-κB p65,Bax and caspase-3 were tested by Western blotting.Double staining with Annexin V-FITC and propidium iodide(PI)was used to test and calculate the apoptosis of Huh-7 cells.Treatment with HPC at concentrations higher than 20μg/mL could inhibit Huh-7 cell proliferation in a dose-dependent way(P<0.05).The over-expression of Traf6 promoted the proliferation of Huh-7 cells.Compared with the control group,HPC at 40μg/mL significantly decreased the relative mRNA and protein expression of Traf6 and NF-κB p65(P<0.05),increased the relative mRNA and protein expression of Bax and caspase-3(P<0.05),and promoted Huh-7 cell apoptosis(P<0.05).We concluded that HPC induce apoptosis in Huh-7 cells by down-regulating the Traf6/NF-κB signaling pathway.