目的 探討漢黃芩素 7-O-β-D-乙基葡萄糖醛酸苷（WODE）對脂多糖（LPS）誘導(dǎo)的小鼠巨噬細胞（RAW264.7）的抗氧化應(yīng)激作用及機制。方法 MTS法檢測WODE（2.5、5.0、10.0、20.0、40.0、80.0、160.0 μmol·L^-1）對RAW264.7細胞活力的影響；體外培養(yǎng)RAW264.7細胞，WODE（10、20、40 μmol·L^-1）或地塞米松（1 μmol·L^-1，陽性藥）預(yù)處理1 h，再給予LPS刺激24 h（造模過程不再加藥），對照組不加 LPS 和受試物，模型組只給予 LPS 刺激；熒光探針檢測胞內(nèi)活性氧（ROS）水平；Griess反應(yīng)測定細胞上清液中 NO 生成量；ELISA 檢測細胞上清液中腫瘤壞死因子-α（TNF-α）和白細胞介素-6（IL-6）的分泌；實時熒光定量 PCR（qRT-PCR）法檢測細胞內(nèi)誘導(dǎo)型一氧化氮合成酶（iNOS）、環(huán)氧化酶-2（COX-2）、白細胞介素-1β（IL-1β）、醌氧化還原酶1（NQO-1）、超氧化物歧化酶-1（SOD-1）的mRNA表達水平；Western blotting法檢測細胞核因子E2相關(guān)因子2（Nrf2）和血紅素加氧酶-（1 HO-1）蛋白表達水平；免疫熒光染色法檢測細胞內(nèi)Kelch ECH相關(guān)蛋白（1 Keap1）表達水平。結(jié)果 與對照組比較，WODE濃度小于40 μmol·L^-1時，細胞存活率沒有明顯變化；濃度大于80 μmol·L^-1時，細胞存活率下降，但未見統(tǒng)計學(xué)差異。與模型組比較，WODE 10、20、40 μmol·L^-1組 ROS水平顯著降低（P＜0.01）；20、40 μmol·L^-1 組 NO 釋放顯著降低（P＜0.05、0.01）；40 μmol·L^-1 組 iNOS mRNA 表 達 水 平 顯 著 降 低（P＜0.01）；10、20、40 μmol·L^-1 組 COX-2 和20、40 μmol·L^-1組IL-1β mRNA表達水平顯著降低（P＜0.05、0.01）；10、20、40 μmol·L^-1組TNF-α和IL-6的釋放受到顯著抑制（P＜0.01）；10、20、40 μmol·L^-1組NQO-1 mRNA表達水平顯著升高（P＜0.01），40 μmol·L^-1組SOD-1 mRNA表達水平顯著升高（P＜0.01）；10、20、40 μmol·L^-1組Keap1蛋白表達水平顯著降低（P＜0.01）；10、20、40 μmol·L^-1組HO-1蛋白和mRNA表達水平顯著提高（P＜0.05、0.01）；20、40 μmol·L^-1 組 Nrf2 蛋白和 40 μmol·L^-1 組 Nrf2 mRNA 表達水平顯著增加（P＜0.01）。結(jié)論 WODE 對 LPS 誘導(dǎo)的RAW264.7細胞的氧化應(yīng)激具有抑制作用，其作用機制可能與調(diào)控Keap1/Nrf2/HO-1信號通路相關(guān)。

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.

R285.5

“十三五”南京市衛(wèi)生青年人才培養(yǎng)第三層次項目（QRX17132）；2022年度“南京市中醫(yī)藥科技專項”（ZYYB202203）