[关键词]
[摘要]
目的 探讨三七总皂苷(PNS)对脂多糖(LPS)诱导的RAW264.7细胞iNOS-NO-NF-κB信号通路相关分子表达及活性的影响。方法 MTT比色法检测PNS对RAW264.7细胞增殖的抑制作用;不同浓度PNS(25、50、100 μg/mL)干预体外培养LPS诱导的RAW264.7细胞24、48 h后,Griess法检测NO变化;PNS干预24 h后,Western blotting检测iNOS、NF-κB、p-NF-κB、IKKα、p-IKKα、p-ΙκΒα蛋白表达量的变化;PNS干预4 h后,激光共聚焦显微镜检测NF-κB转位入核情况。结果 PNS浓度大于150 μg/mL时,才表现出显著抑制细胞增殖的作用。25、50、100 μg/mL PNS作用于RAW264.7细胞24和48 h后,与模型组比较,NO生成量均显著降低(P<0.001)。50、100 μg/mL PNS作用于RAW264.7 24 h后,iNOS、NF-κB蛋白表达量显著降低,磷酸化蛋白p-NF-κB、p-IKKα、p-ΙκΒα表达水平也显著降低(P<0.01、0.001)。与模型组比较,PNS 25、50、100 μg/mL组核因子p65入核荧光强度显著降低(P<0.01、0.001)。结论 PNS能够显著抑制LPS诱导的RAW264.7细胞iNOS-NO-NF-κB信号通路的活性,降低细胞炎症反应。
[Key word]
[Abstract]
Objective To investigate the effect of Panax notoginseng saponins (PNS) on the expression and activity of iNOS-NONF-κB signaling pathway related molecules in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in vitro. Methods RAW264.7 cells were treated with different concentrations (25-100 μg/mL) of PNS for different periods of time (24-48 h). Antiinflammation effects of PNS in LPS-stimulated RAW264.7 macrophages were measured based on the detection of nitric oxide (NO) overproduction (Griess method, DAF-FM DA fluorescence assay and NO2- scavenging assay), and the cell proliferation was measured by MTT assay, the expression of iNOS, NF-κB, p-NF-κB, IKKα, p-IKKα, p-IκBα by Western blot, the NF-κB p65 translocation by confocal laser scanning fluorescence microscopy. Results When the concentration of PNS was more than 150 μg/mL, the inhibition of cell proliferation was significant. 25, 50, 100 μg/mL PNS treated RAW264.7 cells for 24 and 48 h, compared with the model group, NO production was significantly reduced (P<0.001). After PNS (25, 50 and 100 μg/mL) was applied to RAW264.7 cells for 24 h, the expression of iNOS, NF-κB proteins and the expression of p-NF-κB, p-IKKα, p-IκBα proteins were all decreased in a dose-dependment manner (P<0.01, 0.001). Conclusion Taken together, PNS exerts anti0inflammatory effects, by inhibiting the activity of iNOS-NO-NF-κB signaling pathway in LPS-treated RAW264.7 macrophages.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(81760818、81160492);云南省应用基础研究计划项目-中医联合专项[2017FF11(-013)]