目的 研究苦參水提物（WSF）抑制脂多糖（LPS）誘導大鼠巨噬細胞RAW264.7炎癥和氧化應(yīng)激的分子機制。方法 采用CCK-8法檢測WSF對RAW264.7細胞的最佳給藥質(zhì)量濃度；以LPS刺激RAW264.7細胞制備體外炎癥模型，隨機分為對照組、模型組、WSF組和WSF對照組，流式細胞術(shù)檢測活性氧（ROS）及一氧化氮（NO）水平，通過熒光定量PCR（qRT-PCR）和Western blotting檢測誘導型一氧化氮合酶（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；最后檢測細胞培養(yǎng)上清中細胞因子白細胞介素-6（IL-6）、腫瘤壞死因子-α（TNF-α）和IL-10的含量。結(jié)果 通過CCK-8實驗確定WSF質(zhì)量濃度為0.01 mg/mL對細胞無毒性。與對照組比較，LPS刺激能夠顯著地增加細胞中NO和ROS產(chǎn)生，IL-6和TNF-α水平也明顯升高，iNOS和COX-2蛋白表達水平明顯提高（P<0.01、0.001），而Nrf2和HO-1蛋白表達水平降低（P<0.05）。與模型組比較，WSF干預后細胞NO、ROS、IL-6和TNF-α水平顯著降低，iNOS和COX-2蛋白表達水平顯著降低（P<0.05、0.01、0.001），Nrf2和HO-1蛋白表達水平和IL-10水平顯著升高（P<0.05、0.01、0.001）。結(jié)論 WSF可通過激活Nrf2/HO-1通路在LPS誘導的RAW264.7細胞炎癥和氧化應(yīng)激反應(yīng)中發(fā)揮保護效應(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.

國家自然科學基金資助項目（81872832）；廣東省科技計劃項目（2016A020226004）；廣東省基礎(chǔ)與應(yīng)用基礎(chǔ)研究基金自然科學基金項目（2019A1515010806）；廣東省普通高校特色創(chuàng)新類項目（2017GXJK184）；廣東省中醫(yī)藥建設(shè)專項資金項目（20161068）；佛山科學技術(shù)學院博士啟動項目（gg040952）；大學生創(chuàng)新訓練項目