汉黄芩苷通过抑制PI3K/Akt/Nrf2/ARE通路逆转SGC7901/cDDP细胞耐药性
王丽君^1,*, 王世广¹, 邓同兴^2
1郑州工业应用技术学院医学院，新郑 451150；²漯河医学高等专科学校解剖学教研室，漯河 462002
摘要
本研究旨在观察汉黄芩苷对人胃癌顺铂(cisplatin, cDDP)耐药细胞株SGC7901/cDDP耐药性的影响，并探讨其分子机制。采用浓度梯度递增法构建SGC7901/cDDP细胞；CCK-8法检测汉黄芩苷和cDDP对SGC7901/cDDP细胞活性的影响；Chou-Talalay中效分析法对两药的联合效应进行评价；流式细胞术检测活性氧(reactive oxygen species, ROS)含量和细胞凋亡；shRNA干扰技术抑制Nrf2基因表达；Western blot检测Nrf2、NQO1、GST-π、MRP1、cleaved Caspase-3、p-Akt和Akt的蛋白水平。结果显示，汉黄芩苷与顺铂单独作用或二者联用48 h均可剂量依赖性地抑制SGC7901/cDDP细胞活力(P < 0.05)；当抑制率超过16%时，二者联用呈协同效应；SGC7901/cDDP细胞的p-Akt、Nrf2和MRP1蛋白水平显著高于其亲代SGC7901细胞(P < 0.05)；汉黄芩苷与PI3K抑制剂LY294002均可抑制SGC7901/cDDP细胞Akt磷酸化(P < 0.05)，增加ROS含量(P < 0.05)，下调Nrf2蛋白水平(P < 0.05)，上调cleaved Caspase-3蛋白水平(P < 0.05)，最终导致细胞凋亡(P < 0.05)；但抗氧化剂NAC可减轻汉黄芩苷诱导的氧化应激和细胞凋亡(P < 0.05)；汉黄芩苷与沉默Nrf2基因均可使SGC7901/cDDP细胞NQO1、GST-π和MRP1蛋白水平下降(P < 0.05)。以上结果表明，汉黄芩苷可在体外增强SGC7901/cDDP细胞对cDDP的敏感性，这可能与其抑制PI3K/Akt/Nrf2/ARE通路，从而增强cDDP的毒性作用并触发氧化应激损伤有关。

关键词： 汉黄芩苷; 胃癌; 顺铂耐药性; Nrf2; 活性氧; Akt
分类号：R735.2

Wogonoside reverses cisplatin resistance in SGC7901/cDDP cells through inhibition of PI3K/Akt/Nrf2/ARE signaling pathway
WANG Li-Jun^1,*, WANG Shi-Guang¹, DENG Tong-Xing^2
1Medical College, Zhengzhou University of Industrial Technology, Xinzheng 451150, China；²Department of Anatomy, Luohe Medical College, Luohe 462002, China
Abstract
The aim of this study was to investigate the effect of wogonoside (WGS) on the cisplatin (cDDP) resistance in human gastric carcinoma SGC7901/cDDP cells and its possible mechanism. The drug-resistant SGC7901/cDDP cells were established by stepwise exposure to cDDP. CCK-8 assay was employed to detect the cytotoxic effect of WGS and cDDP on SGC7901/cDDP cells, and the combined effect of WGS and cDDP was analyzed by Chou-Talalay method. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). Nuclear factor erythroid-2 related factor 2 (Nrf2) gene was knocked down by using the short hairpin RNA (shRNA) approach. The protein levels of Nrf2, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S transferase-π (GST-π), multidrug resistance-associated protein 1 (MRP1), cleaved Capase-3, p-Akt and Akt were detected by Western blotting. The result showed that after various concentrations of WGS and/or cDDP treatment for 48 h, the cell viability was remarkably reduced in a dose-dependent manner (P < 0.05). When the inhibition rate exceeded 16%, the combination of WGS and cDDP produced a synergistic effect. The protein levels of p-Akt, Nrf2 and MRP1 in SGC7901/cDDP cells were higher than those in SGC7901 cells (P < 0.05). WGS and LY294002 (PI3K inhibitor) both remarkably decreased the phosphorylation level of Akt (P < 0.05), down-regulated the protein level of Nrf2 (P < 0.05), increased the content of ROS (P < 0.05), up-regulated the protein level of cleaved Caspase-3 (P < 0.05), and induced apoptosis (P < 0.05). Meanwhile, N-acetyl-L-cysteine (NAC) decreased apoptosis and oxidative stress reaction induced by WGS (P < 0.05). WGS and Nrf2 gene silencing both down-regulated the protein levels of NQO1, GST-π and MRP1 (P < 0.05). These results suggest that WGS may reverse cDDP resistance in SGC7901/cDDP cells through blocking the PI3K/Akt/Nrf2/ARE signaling pathway, thus enhancing the cytotoxicity of cDDP and inducing oxidative stress reaction and apoptosis.
Key words: wogonoside; gastric carcinoma; cisplatin resistance; Nrf2; reactive oxygen species; Akt

收稿日期：2018-01-18　　录用日期：2018-05-16
通讯作者：王丽君　　E-mail: wljwanglijun@163.com
DOI: 10.13294/j.aps.2018.0038
引用本文：
王丽君, 王世广, 邓同兴. 汉黄芩苷通过抑制PI3K/Akt/Nrf2/ARE通路逆转SGC7901/cDDP细胞耐药性[J]. 生理学报 2018; 70 (4): 397-405.
WANG Li-Jun, WANG Shi-Guang, DENG Tong-Xing. Wogonoside reverses cisplatin resistance in SGC7901/cDDP cells through inhibition of PI3K/Akt/Nrf2/ARE signaling pathway. Acta Physiol Sin 2018; 70 (4): 397-405 (in Chinese with English abstract).



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