目的 探讨汉黄芩素 7-O-β-D-乙基葡萄糖醛酸苷（WODE）对脂多糖（LPS）诱导的小鼠巨噬细胞（RAW264.7）的抗氧化应激作用及机制。方法 MTS法检测WODE（2.5、5.0、10.0、20.0、40.0、80.0、160.0 μmol·L^-1）对RAW264.7细胞活力的影响；体外培养RAW264.7细胞，WODE（10、20、40 μmol·L^-1）或地塞米松（1 μmol·L^-1，阳性药）预处理1 h，再给予LPS刺激24 h（造模过程不再加药），对照组不加 LPS 和受试物，模型组只给予 LPS 刺激；荧光探针检测胞内活性氧（ROS）水平；Griess反应测定细胞上清液中 NO 生成量；ELISA 检测细胞上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）的分泌；实时荧光定量 PCR（qRT-PCR）法检测细胞内诱导型一氧化氮合成酶（iNOS）、环氧化酶-2（COX-2）、白细胞介素-1β（IL-1β）、醌氧化还原酶1（NQO-1）、超氧化物歧化酶-1（SOD-1）的mRNA表达水平；Western blotting法检测细胞核因子E2相关因子2（Nrf2）和血红素加氧酶-（1 HO-1）蛋白表达水平；免疫荧光染色法检测细胞内Kelch ECH相关蛋白（1 Keap1）表达水平。结果 与对照组比较，WODE浓度小于40 μmol·L^-1时，细胞存活率没有明显变化；浓度大于80 μmol·L^-1时，细胞存活率下降，但未见统计学差异。与模型组比较，WODE 10、20、40 μmol·L^-1组 ROS水平显著降低（P＜0.01）；20、40 μmol·L^-1 组 NO 释放显著降低（P＜0.05、0.01）；40 μmol·L^-1 组 iNOS mRNA 表 达 水 平 显 著 降 低（P＜0.01）；10、20、40 μmol·L^-1 组 COX-2 和20、40 μmol·L^-1组IL-1β mRNA表达水平显著降低（P＜0.05、0.01）；10、20、40 μmol·L^-1组TNF-α和IL-6的释放受到显著抑制（P＜0.01）；10、20、40 μmol·L^-1组NQO-1 mRNA表达水平显著升高（P＜0.01），40 μmol·L^-1组SOD-1 mRNA表达水平显著升高（P＜0.01）；10、20、40 μmol·L^-1组Keap1蛋白表达水平显著降低（P＜0.01）；10、20、40 μmol·L^-1组HO-1蛋白和mRNA表达水平显著提高（P＜0.05、0.01）；20、40 μmol·L^-1 组 Nrf2 蛋白和 40 μmol·L^-1 组 Nrf2 mRNA 表达水平显著增加（P＜0.01）。结论 WODE 对 LPS 诱导的RAW264.7细胞的氧化应激具有抑制作用，其作用机制可能与调控Keap1/Nrf2/HO-1信号通路相关。

Objective To investigate the anti-oxidative stress effect and mechanism of WODE on lipopolysaccharide (LPS) -induced mouse macrophages (RAW264.7 cells). Methods The effects of different concentrations of WODE (2.5, 5.0, 10.0, 20.0, 40.0, 80.0, and 160.0 μmol·L^-1) on the viability of RAW264.7 cells were detected by MTS assay. RAW264.7 cells were cultured in vitro, wogonin 7-O-β-D-ethyl glucuronide (10, 20, 40 μmol·L^-1) or dexamethasone (1 μmol·L^-1, positive drug) was pretreated for one hour, and then LPS was administered for 24 hours (no drug was added during the modeling process). The control group was not given LPS and the test substance, while the model group was only given LPS stimulation. Intracellular reactive oxygen species (ROS) levels were detected by fluorescent probe. The production of NO was determined by Griess reaction. The secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the cell supernatant was detected by ELISA. The mRNA expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, quinone oxidoreductase 1 (NQO-1), superoxide dismutase 1 (SOD-1) were detected by qRT-PCR. The protein expression levels of nuclear factor E2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were detected by Western blotting. The expression of Kelch ECH-related protein 1 (Keap1) protein was detected by immunofluorescence staining. Results Compared with control group, there was no significant change in cell survival rate when wogonin 7-O-β-D-ethyl glucuronide concentration was less than 40 μmol·L^-1, the cell survival rate decreased when the concentration greater than 80 μmol·L^-1, but there was no statistical difference. Compared with the model group, the ROS level in wogonin 7-O-β-Dethyl glucuronide 10, 20, 40 μmol·L^-1 group was significantly lower (P＜0.01), the NO release was significantly decreased in 20, 40 μmol·L^-1 group (P＜0.05, 0.01), the expression level of iNOS mRNA in 40 μmol·L^-1 group significantly decreased (P＜0.01), COX-2 in 10, 20, 40 μmol·L^-1 group and IL-1β mRNA expression level in 20, 40 μmol·L^-1 group significantly decreased (P＜0.05, 0.01), TNF-α and IL-6 release in 10, 20, 40 μmol·L^-1 group were significantly inhibited (P＜0.01), the expression level of NQO-1 mRNA in 10, 20, 40 μmol·L^-1 group was significantly increased (P＜0.01), the expression level of SOD-1 mRNA in 40 μmol·L^-1 group significantly increased (P＜0.01), the expression level of Keap1 protein in 10, 20, 40 μmol·L^-1 group significantly decreased (P＜0.01), the protein and mRNA expression levels of HO-1 protein in 10, 20, 40 μmol·L^-1 group were significantly increased (P＜0.05, 0.01), Nrf2 protein expression in 10, 20, 40 μmol·L^-1 group and Nrf2 mRNA expression in 40 μmol·L^-1 group was significantly increased (P＜0.01). Conclusion Wogonin 7-O-β-D-ethyl glucuronide inhibited LPS-induced oxidative stress in RAW264.7 cells, which might be related to the regulation of Keap1/Nrf2/HO-1 signaling pathway.

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“十三五”南京市卫生青年人才培养第三层次项目（QRX17132）；2022年度“南京市中医药科技专项”（ZYYB202203）