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摘要：
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摘要：目的 研究榭皮黄酮（quercetin，Qu）影响肝癌细胞HepG2对5-氟尿嘧啶
（5-fluorouracil，5-FU）的敏感性及Traf6/NF-κB信号转导通路。方法 体外培养HepG2
细胞，取对数期细胞进行后续实验，将细胞分为对照组（不含任何药物的培养基孵
育细胞24 h）、单独Qu组（含40 μg/ml Qu的培养基孵育细胞24 h）、单独5-FU组（含
50 μmol/L 5-FU的培养基孵育细胞24 h）、Qu + 5-FU联合处理组（含40 μg/ml Qu和
50 μmol/L 5-FU的培养基共同孵育细胞24 h）、单独Traf6抑制剂C25-140组（2 μmol/L
C25-140的培养基孵育细胞8 h）、C25-140 + Qu + 5-FU组（C25-140预处理细胞8 h，
然后含40 μg/ml Qu和50 μmol/L 5-FU的培养基共同处理细胞24 h）。采用CCK8法检测
细胞活力，采用倒置显微镜记录细胞克隆数，采用流式细胞术检测细胞凋亡率，采用
Western blot检测剪切的半胱氨酸蛋白酶-7（cleaved-caspase 7，Cle-caspase 7）、Clecaspase 3、剪切的聚腺苷二磷酸-核糖聚合酶（cleaved poly ADP-ribose polymerase，
Cle-PARP）、肿瘤坏死因子受体相关蛋白6（tumor necrosis factor receptor-associated
factor-6，Traf6）、磷酸化转化生长因子-β活化激酶1（phosphorylation transforming
growth factor-β-activated kinase 1，p-TAK1）和磷酸化核转录因子（phosphorylation nuclear
factor kappa-B，p-NF-κB）蛋白的表达水平。结果 Qu（40 μg/ml）、5-FU（50 μmol/L）
和Qu + 5-FU组细胞相对活力分别为（82.3 ± 3.1）%、（53.7 ± 4.1）%和（42.4 ±
4.4）%，均显著低于对照组的（100.0 ± 3.4）%，差异有统计学意义（t = 5.83、9.54、
14.65，P均＜ 0.05）；Qu（40 μg/ml）组、5-FU（50 μmol/L）组和Qu + 5-FU组HepG2
细胞克隆数分别为534 ± 26、236 ± 25、115 ± 42，均显著低于对照组的701 ± 32（P
均＜ 0.05），且Qu + 5-FU组显著低于Qu（40 μg/ml）组和5-FU（50 μmol/L）组（t =
31.74，P ＜ 0.001；t = 11.34，P = 0.008）。Qu（40 μg/ml）组、5-FU（50 μmol/L）组
和Qu + 5-FU组HepG2细胞凋亡率 [（18.9 ± 4.2）% vs（21.4 ± 4.1）% vs（35.7 ± 3.6）%
vs（4.6 ± 1.5）%]、Cle-caspase 7（0.11 ± 0.02 vs 0.22 ± 0.03 vs 0.32 ± 0.03 vs 0.05 ± 0.02）、
Cle-caspase 3（0.13 ± 0.02 vs 0.18 ± 0.03 vs 0.28 ± 0.03）和Cle-PARP（0.15 ± 0.02 vs 0.24 ±
0.03 vs 0.41 ± 0.03 vs 0.08 ± 0.02）表达水平均显著高于对照组，且Qu + 5-FU组显著高
于Qu（40 μg/ml）组和5-FU（50 μmol/L）组（P均＜ 0.05）。Qu（40 μg/ml）组、5-FU
（50 μmol/L）组、Qu + 5-FU组HepG2细胞Traf6（0.28 ± 0.02 vs 0.19 ± 0.03 vs 0.11 ± 0.03
vs 0.38 ± 0.02）、p-TAK1（0.23 ± 0.02 vs 0.11 ± 0.03 vs 0.04 ± 0.03 vs 0.38 ± 0.02）和
p-NF-κB（0.28 ± 0.02 vs 0.13 ± 0.03 vs 0.05 ± 0.02 vs 0.44 ± 0.03）蛋白相对表达量均显著
低于对照组，且Qu + 5-FU组均显著低于Qu（40 μg/ml）组和5-FU（50 μmol/L）组（P
均＜ 0.05）。与对照组相比，C25-140组、Qu + 5-FU组和Qu + 5-FU + C25-140组HepG2细
胞相对活力 [（73.4 ± 4.1）% vs（65.8 ± 3.6）% vs（47.7 ± 3.9）% vs（100 ± 3.1）%] 和
细胞克隆数（456 ± 26 vs 413 ± 25 vs 305 ± 42 vs 763 ± 32）显著降低，细胞凋亡率 [（24.4 ±
4.1）% vs（29.9 ± 3.7）% vs（51.2 ± 3.7）% vs（3.9 ± 5.2）%] 显著升高（P均＜ 0.05）；
与Qu + 5-FU组比较，Qu + 5-FU + C25-140组HepG2细胞活力和克隆数显著降低，细胞
凋亡率显著升高（P均＜ 0.05）。与对照组相比，Traf6 mimics组HepG2细胞相对活力
[（152.4 ± 6.3）% vs（100 ± 3.5）%]、细胞凋亡率 [（5.3 ± 3.2）% vs（3.8 ± 2.1）%] 和
细胞克隆数（978 ± 26 vs 783 ± 32）均显著升高（P均＜ 0.05），Qu + 5-FU组和Traf6
mimics + Qu + 5-FU组HepG2细胞相对活力 [（65.8 ± 4.3）% vs（100 ± 3.5）%；（79.4 ±
4.9）% vs（100 ± 3.5）%] 和细胞克隆数（454 ± 25 vs 783 ± 32；623 ± 42 vs 783 ± 32）
显著降低，细胞凋亡率 [（34.7 ± 4.4）% vs（3.8 ± 2.1）%；（24.4 ± 3.5）% vs（3.8 ±
2.1）%] 显著升高（P均＜ 0.05）。与Qu + 5-FU组相比，Traf6 mimics + Qu + 5-FU组
HepG2细胞活力和克隆数显著升高，细胞凋亡率显著降低（P均＜ 0.05）。结论 Qu通
过抑制Traf6/NF-κB信号转导通路协同诱导5-FU对肝细胞癌的化学治疗作用。
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Abstract: Objective To observe the effects of quercetin (Qu) on the chemotherapeutic
sensitivity of 5-fluorouracil (5-FU) and Traf6/NF-κB signaling transduction pathway in
HepG2. Methods HepG2 cells were cultured in vitro and log-phase cells were harvested for
subsequent experiments. Cells were divided into control group (cells were incubated in a
drug-free medium for 24 h), Qu group (cells were incubated in a medium containing 40 μg/ml
of Qu for 24 h), 5-FU group (cells were incubated in a medium containing 50 μmol/L of 5-FU
for 24 h), Qu + 5-FU group (cells were incubated in a medium containing 40 μg/ml of Qu and
50 μmol/L of 5-FU for 24 h), C25-140 group (cells were incubated in a medium containing
2 μmol/L of C25-140 for 8 h) and C25-140 + Qu + 5-FU group (cells were firstly incubated
in a medium containing 2 μmol/L of C25-140 for 8 h, then treated with 40 μg/ml of Qu and
50 μmol/L of 5-FU for 24 h). CCK8 assay was performed to detect the cell viability. Cell
colonies were measured by inverted microscope. HepG2 cell apoptotic rate was determined
by flow cytometry. The expression levels of cleaved-caspase 7 (Cle-caspase 7), Cle-caspase 3,
cleaved poly ADP-ribose polymerase (Cle-PARP), tumor necrosis factor receptor-associated
factor-6 (Traf6), phosphorylation transforming growth factor-β-activated kinase 1 (p-TAK1)
and phosphorylation nuclear factor kappa-B (p-NF-κB) protein were detected by Western blot.
Results The relative cell viability in Qu (40 μg/ml) group, 5-FU (50 μmol/L) group and Qu +
5-FU group were (82.3 ± 3.1)%, (53.7 ± 4.1)% and (42.4 ± 4.4)%, respectively, which were
significantly lower than that in control group [(100.0 ± 3.4)%, t = 5.83, 9.54, 14.65, all P ＜
0.05]. The HepG2 cell colonies number in Qu (40 μg/ml) group, 5-FU (50 μmol/L) group and Qu + 5-FU
group were 534 ± 26, 236 ± 25 and 115 ± 42, respectively, which were significantly lower
than that in control group (701 ± 32; all P ＜ 0.05). The HepG2 cell colonies number in Qu +
5-FU group was significantly lower than that in Qu (40 μg/ml) group and 5-FU (50 μmol/L)
group (t = 31.74, P ＜ 0.001; t = 11.34, P = 0.008). The HepG2 cell apoptotic rates [(18.9 ±
4.2)% vs (21.4 ± 4.1)% vs (35.7 ± 3.6)% vs (4.6 ± 1.5)%], Cle-caspase 7 (0.11 ± 0.02 vs 0.22 ± 0.03
vs 0.32 ± 0.03 vs 0.05 ± 0.02), Cle-caspase 3 (0.13 ± 0.02 vs 0.18 ± 0.03 vs 0.28 ± 0.03) and
Cle-PARP (0.15 ± 0.02 vs 0.24 ± 0.03 vs 0.41 ± 0.03 vs 0.08 ± 0.02) in Qu (40 μg/ml) group,
5-FU (50 μmol/L) group and Qu + 5-FU group were significantly higher than those in control
group, and the above indexes in Qu + 5-FU group were significantly higher than those in Qu
(40 μg/ml) group and 5-FU (50 μmol/L) group (all P ＜ 0.05). The relative expression levels
of Traf6 (0.28 ± 0.02 vs 0.19 ± 0.03 vs 0.11 ± 0.03 vs 0.38 ± 0.02), p-TAK1 (0.23 ± 0.02 vs 0.11 ±
0.03 vs 0.04 ± 0.03 vs 0.38 ± 0.02) and p-NF-κB (0.28 ± 0.02 vs 0.13 ± 0.03 vs 0.05 ± 0.02
vs 0.44 ± 0.03) in Qu (40 μg/ml) group, 5-FU (50 μmol/L) group and Qu + 5-FU group were
significantly lower than those in control group, and the above indexes in Qu + 5-FU group
were significantly lower than those in Qu (40 μg/ml) group and 5-FU (50 μmol/L) group (all
P ＜ 0.05). Compared with control group, the cell viability [(73.4 ± 4.1)% vs (65.8 ± 3.6)%
vs (47.7 ± 3.9)% vs (100 ± 3.1)%] and colonies number (456 ± 26 vs 413 ± 25 vs 305 ± 42 vs
763 ± 32) in C25-140 group, Qu + 5-FU group and Qu + 5-FU + C25-140 group decreased
significantly and the apoptosis rate [(24.4 ± 4.1)% vs (29.9 ± 3.7)% vs (51.2 ± 3.7)% vs (3.9 ± 5.2)%]
increased significantly (all P ＜ 0.05); compared with Qu + 5-FU group, the cell viability and
colonies number in Qu + 5-FU + C25-140 group decreased significantly and the apoptosis
rate increased significantly (all P ＜ 0.05). Compared with control group, the cell viability [(152.4 ±
6.3)% vs (100 ± 3.5)%], apoptosis rate [(5.3 ± 3.2)% vs (3.8 ± 2.1)%] and colonies number
(978 ± 26 vs 783 ± 32) in Traf6 mimics group increased significantly, the cell viability [(65.8 ±
4.3)% vs (100 ± 3.5)%; (79.4 ± 4.9)% vs (100 ± 3.5)%] and colonies number (454 ± 25 vs
783 ± 32; 623 ± 42 vs 783 ± 32) in Qu + 5-FU group and Traf6 mimics + Qu + 5-FU group
decreased significantly and the apoptosis rate [(34.7 ± 4.4)% vs (3.8 ± 2.1)%; (24.4 ± 3.5)%
vs (3.8 ± 2.1)%] increased significantly (all P ＜ 0.05). Compared with Qu + 5-FU group, the
cell viability and colonies number in Traf6 mimics + Qu + 5-FU group increased significantly,
and the apoptosis rate decreased significantly (all P ＜ 0.05). Conclusions Qu synergized
the chemotherapeutic effect of 5-FU on hepatocellular carcinoma through the suppression of
Traf6/NF-κB signaling transduction pathway.
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