目的 探讨葛根芩连汤缓解肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）诱导的人结肠癌Caco-2细胞氧化应激损伤作用及其配伍规律。方法 将Caco-2细胞随机分为对照组、TNF-α（80 μg/L）组、小檗碱（6.73 μg/mL）组及葛根芩连汤配伍组（全方组、去甘草组、去葛根组、葛根甘草组和黄芩黄连组，18.75 μg/mL）。除对照组外，其余各组细胞给予TNF-α预处理2 h，再给予相应药物处理24 h。MTT检测Caco-2细胞活力；流式细胞术检测细胞内活性氧（reactive oxygen species，ROS）水平；比色法检测细胞中总超氧化物歧化酶（total superoxide dismutase，T-SOD）活性和丙二醛（malondialdehyde，MDA）、还原型谷胱甘肽（glutathione，GSH）水平；qRT-PCR和Western blotting分别检测核因子E2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）、还原型辅酶I/II醌氧化还原酶1[NAD(P)H quinine oxidoreductase 1，NQO1]和血红素氧合酶-1（heme oxygenase 1，HO-1）mRNA和蛋白表达；荧光显微镜观察Nrf2蛋白入核情况。结果 5.0～25.0 μg/mL的含药培养基和10～80 μg/L的TNF-α对Caco-2细胞活性无明显影响。与TNF-α组比较，各给药组Caco-2细胞ROS水平显著降低（P＜0.01）；除去葛根组和黄芩黄连组外，其余各组MDA水平显著降低（P＜0.01），GSH水平明显升高（P＜0.01）；除去葛根组外，其余各给药组Nrf2基因表达明显升高（P＜0.01），全方组和去甘草组NQO1基因表达明显升高（P＜0.05、0.01），全方组和葛根甘草组HO-1基因表达明显升高（P＜0.05、0.01）；除黄芩黄连组外，其余各组Nrf2和HO-1蛋白表达显著升高（P＜0.01）；除去葛根组和黄芩黄连组外，其余各组NQO1蛋白水平显著升高（P＜0.05、0.01），Nrf2蛋白入核明显增加（P＜0.05、0.01）。结论 缺少葛根对葛根芩连汤抑制Caco-2细胞氧化应激损伤有较大影响。

Objective To investigate the effect of Gegen Qinlian Decoction (葛根芩连汤) on relieving tumor necrosis factor-α (TNF-α)-induced oxidative stress injury in human colon cancer Caco-2 cells and its compatibility. Methods Caco-2 cells were randomly divided into control group, TNF-α (80 μg/L) group, berberine (6.73 μg/mL) group and Gegen Qinlian Decoction compatibility group [Gegen Qinlian group (GD), Gancao (Glycyrrhizae Radix et Rhizoma) absent group (WGC), Gegen (Puerariae Lobatae Radix) absent group (WG), Puerariae Lobatae Radix-Glycyrrhizae Radix et Rhizoma group (GG), Huangqin (Scutellariae Radix)-Huanglian (Coptidis Rhizoma) group (QL), 18.75 μg/mL]. Except for the control group, cells in other groups were pretreated with TNF-α for 2 h, and then treated with corresponding drugs for 24 h. Caco-2 cell viability was detected by MTT; Intracellular reactive oxygen species (ROS) level were detected by flow cytometry; Total superoxide dismutase (T-SOD) activity and malondialdehyde (MDA), glutathione (GSH) levels were detected by colorimetry; qRT-PCR and Western blotting were used to detect nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H quinine oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) mRNA and protein expressions; Fluorescence microscope was used to observe nuclear penetration of Nrf2 protein. Results 5.0—25.0 μg/mL drug-containing medium and 10—80 μg/L TNF-α had no significant effect on the viability of Caco-2 cells. Compared with TNF-α group, ROS level in Caco-2 cells of each administration group were significantly decreased (P < 0.01). Except for WG group and QL group, MDA level in the other groups were significantly decreased (P < 0.01), and GSH level was significantly decreased (P < 0.01). Nrf2 gene expression was significantly increased in all drug-administered groups except WG group (P < 0.01), and NQO1 gene expression was significantly increased in GD group and WGC group (P < 0.05, 0.01), HO-1 gene expression was significantly increased in GD group and GG group (P < 0.05, 0.01). Except for QL group, protein expressions of Nrf2 and HO-1 in other groups were significantly increased (P < 0.01); Except for WG group and QL group, NQO1 protein expression was significantly increased in the other groups (P < 0.05, 0.01), and Nrf2 protein into the nucleus was significantly increased (P < 0.05, 0.01). Conclusion The lack of Puerariae Lobatae Radix has a negative effect on the inhibition of oxidative stress injury of Caco-2 cells by Gegen Qinlian Decoction.

上海市科技创新行动计划自然科学基金资助项目(19ZR1451900)