葛根芩连汤抑制TNF-α诱导的Caco-2细胞氧化损伤配伍规律研究  

Combination rule of Gegen Qinlian Decoction on inhibiting oxidative damage stimulated by TNF-αin Caco-2 cells

作　　者：林川 董文敏 张倩芸 刘王振祖 梁琨[1] 王新宏[1] 安叡[1] LIN Chuan;DONG Wen-min;ZHANG Qian-yun;LIU Wang-zhenzu;LIANG Kun;WANG Xin-hong;AN Rui(School of Pharmacy,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Pudong Hospital Affiliated to Fudan University,Shanghai 201200,China)

机构地区：[1]上海中医药大学中药学院,上海201203 [2]复旦大学附属浦东医院,上海201200

出　　处：《中草药》2022年第20期6492-6499,共8页Chinese Traditional and Herbal Drugs

基　　金：上海市科技创新行动计划自然科学基金资助项目(19ZR1451900)。

摘　　要：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(80μg/L)组、小檗碱(6.73μg/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,18.75μg/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)m RNA和蛋白表达;荧光显微镜观察Nrf2蛋白入核情况。结果5.0~25.0μg/mL的含药培养基和10~80μg/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(80μg/L)group,berberine(6.73μg/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),18.75μg/mL].Except for the control group,cells in other groups were pretreated with TNF-αfor 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—25.0μg/mL drug-containing medium and 10—80μg/L TNF-αhad no significant effect on the viability of Caco-2 cells.Compared with TNF-αgroup,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 we

关 键 词：葛根芩连汤 葛根 配伍规律 氧化应激 结肠癌细胞 

分 类 号：R285.5[医药卫生—中药学]