目的 研究苦參水提物（WSF）抑制脂多糖（LPS）誘導(dǎo)大鼠巨噬細(xì)胞RAW264.7炎癥和氧化應(yīng)激的分子機(jī)制。方法 采用CCK-8法檢測(cè)WSF對(duì)RAW264.7細(xì)胞的最佳給藥質(zhì)量濃度；以LPS刺激RAW264.7細(xì)胞制備體外炎癥模型，隨機(jī)分為對(duì)照組、模型組、WSF組和WSF對(duì)照組，流式細(xì)胞術(shù)檢測(cè)活性氧（ROS）及一氧化氮（NO）水平，通過(guò)熒光定量PCR（qRT-PCR）和Western blotting檢測(cè)誘導(dǎo)型一氧化氮合酶（iNOS）、環(huán)氧合酶-2（COX-2）、核轉(zhuǎn)錄因子E2相關(guān)因子2（Nrf2）和血紅素加氧酶-1（HO-1）在分子水平上的變化；最后檢測(cè)細(xì)胞培養(yǎng)上清中細(xì)胞因子白細(xì)胞介素-6（IL-6）、腫瘤壞死因子-α（TNF-α）和IL-10的含量。結(jié)果 通過(guò)CCK-8實(shí)驗(yàn)確定WSF質(zhì)量濃度為0.01 mg/mL對(duì)細(xì)胞無(wú)毒性。與對(duì)照組比較，LPS刺激能夠顯著地增加細(xì)胞中NO和ROS產(chǎn)生，IL-6和TNF-α水平也明顯升高，iNOS和COX-2蛋白表達(dá)水平明顯提高（P<0.01、0.001），而Nrf2和HO-1蛋白表達(dá)水平降低（P<0.05）。與模型組比較，WSF干預(yù)后細(xì)胞NO、ROS、IL-6和TNF-α水平顯著降低，iNOS和COX-2蛋白表達(dá)水平顯著降低（P<0.05、0.01、0.001），Nrf2和HO-1蛋白表達(dá)水平和IL-10水平顯著升高（P<0.05、0.01、0.001）。結(jié)論 WSF可通過(guò)激活Nrf2/HO-1通路在LPS誘導(dǎo)的RAW264.7細(xì)胞炎癥和氧化應(yīng)激反應(yīng)中發(fā)揮保護(hù)效應(yīng)。

Objective To study the molecular mechanisms of antioxidant effect and anti-inflammatory of water extract of Sophora flavescens (WSF) in lipopolysaccharide (LPS)-induced RAW264.7 cells. Methods The optimum concentration of WSF was evaluated by CCK-8 assay. The inflammatory model was established with LPS by stimulating RAW264.7 cells in vitro. Then all cells were divided into control group, model group, WSF group and WSF control group. The levels of ROS and NO were analyzed with flow cytometry. Subsequently, the expression of iNOS, COX-2, Nrf2, and HO-1 was detected with qRT-PCR and Western blotting. Finally, the pro-inflammatory cytokines IL-6, TNF-α and anti-inflammatory cytokine IL-10 were detected by ELISA. Results The CCK-8 assay revealed that 0.01 mg/mL WSF did not affect the cell viability. Compared with control group, the LPS-induced inflammatory response could significantly increase the production of NO and ROS, and the IL-6 and TNF-α were also significantly increased (P<0.05, 0.01, and 0.001). Furthermore, the expression of iNOS and COX-2 were significantly increased (P<0.01, 0.001), but the expression of Nrf2 and HO-1 were inhibited (P<0.05). However, compared with model group, the WSF group not only significantly decreased the levels of NO, ROS, IL-6, and TNF-α, but also decreased the expression of iNOS and COX-2 (P<0.05, 0.01, and 0.001). In contrast, the the level of IL-10 and the expression of Nrf2 and HO-1 were significantly increased (P<0.05, 0.01, and 0.001). Conclusion These results suggested that SF exerted protective effect against LPS-induced inflammatory and oxidative responses in RAW 264.7 cells by the activation of the Nrf2/HO-1 pathway.

國(guó)家自然科學(xué)基金資助項(xiàng)目（81872832）；廣東省科技計(jì)劃項(xiàng)目（2016A020226004）；廣東省基礎(chǔ)與應(yīng)用基礎(chǔ)研究基金自然科學(xué)基金項(xiàng)目（2019A1515010806）；廣東省普通高校特色創(chuàng)新類項(xiàng)目（2017GXJK184）；廣東省中醫(yī)藥建設(shè)專項(xiàng)資金項(xiàng)目（20161068）；佛山科學(xué)技術(shù)學(xué)院博士啟動(dòng)項(xiàng)目（gg040952）；大學(xué)生創(chuàng)新訓(xùn)練項(xiàng)目