苦参水提物激活Nrf2/HO-1信号通路抑制炎症和氧化应激机制研究

Inhibitory effect of water extract of Sophora flavescens on inflammation and oxidative stress through activating Nrf2/HO-1 signaling pathway

目的研究苦参水提物(WSF)抑制脂多糖(LPS)诱导大鼠巨噬细胞RAW264.7炎症和氧化应激的分子机制。方法采用CCK-8法检测WSF对RAW264.7细胞的最佳给药质量浓度;以LPS刺激RAW264.7细胞制备体外炎症模型,随机分为对照组、模型组、WSF组和WSF对照组,流式细胞术检测活性氧(ROS)及一氧化氮(NO)水平,通过荧光定量PCR(qRT-PCR)和Western blotting检测诱导型一氧化氮合酶(iNOS)、环氧合酶-2(COX-2)、核转录因子E2相关因子2(Nrf2)和血红素加氧酶-1(HO-1)在分子水平上的变化;最后检测细胞培养上清中细胞因子白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和IL-10的含量。结果通过CCK-8实验确定WSF质量浓度为0.01 mg/mL对细胞无毒性。与对照组比较,LPS刺激能够显著地增加细胞中NO和ROS产生,IL-6和TNF-α水平也明显升高,iNOS和COX-2蛋白表达水平明显提高(P<0.01、0.001),而Nrf2和HO-1蛋白表达水平降低(P<0.05)。与模型组比较,WSF干预后细胞NO、ROS、IL-6和TNF-α水平显著降低,iNOS和COX-2蛋白表达水平显著降低(P<0.05、0.01、0.001),Nrf2和HO-1蛋白表达水平和IL-10水平显著升高(P<0.05、0.01、0.001)。结论WSF可通过激活Nrf2/HO-1通路在LPS诱导的RAW264.7细胞炎症和氧化应激反应中发挥保护效应。

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-αand 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-αwere 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.