State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China.
College of Pharmaceutical Science, Soochow University, Suzhou, 215123, China.
Phytother Res. 2017 Jul;31(7):1072-1077. doi: 10.1002/ptr.5828. Epub 2017 May 15.
Previous reports showed that nepetoidin B (NTB), a natural product isolated from many herbs, has anti-fungal and anti-bacterial effects. In this study, the antiinflammatory effect of NTB was investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The cytotoxic effect of NTB and LPS was determined by MTT assay. The nitric oxide (NO) production was detected by Griess assay. The TNF-α and IL-6 levels were determined by enzyme-linked immunosorbent assay kits. Protein expressions were tested by western blotting. The transcription activity of inducible nitric oxide synthase (iNOS) was detected by luciferase assay. Immunofluorescence assay was used to observe the visualization of NF-κB/p65 nuclear translocation. NTB and LPS showed no obvious cytotoxic effect on RAW 264.7 cells. NTB remarkably inhibited LPS-induced NO and TNF-α secretion in a concentration-dependent manner while showed no significant effect on IL-6 secretion. NTB inhibited LPS-induced iNOS protein expression and transcription activity without affecting cyclooxygenase-2. Furthermore, NTB suppressed LPS-stimulated NF-κB/p65 phosphorylation and nuclear translocation. In addition, NTB significantly inhibited LPS-induced phosphorylation of JNK1/2 and p38MAPK without affecting ERK1/2. LPS-induced inhibition of mitogen-activated protein kinase phosphatase-5 (MKP-5) was completely reversed by NTB. In conclusion, these results suggested that NTB inhibited LPS-stimulated NO production possibly via modulation of iNOS mediated by MKP-5/NF-κB pathways in RAW 264.7 cells. Copyright © 2017 John Wiley & Sons, Ltd.
先前的报告表明，从许多草药中分离得到的天然产物nepetoidin B（NTB）具有抗真菌和抗菌作用。在这项研究中，研究了 NTB 在脂多糖（LPS）刺激的 RAW 264.7 巨噬细胞中的抗炎作用。通过 MTT 测定法测定 NTB 和 LPS 的细胞毒性作用。通过格里斯测定法检测一氧化氮（NO）的产生。通过酶联免疫吸附试剂盒测定 TNF-α和 IL-6 水平。通过蛋白质印迹法检测蛋白表达。通过荧光素酶测定法检测诱导型一氧化氮合酶（iNOS）的转录活性。通过免疫荧光法观察 NF-κB/p65 核易位的可视化。NTB 和 LPS 对 RAW 264.7 细胞没有明显的细胞毒性作用。NTB 以浓度依赖性方式显着抑制 LPS 诱导的 NO 和 TNF-α分泌，而对 IL-6 分泌没有显着影响。NTB 抑制 LPS 诱导的 iNOS 蛋白表达和转录活性，而不影响环加氧酶-2。此外，NTB 抑制 LPS 刺激的 NF-κB/p65 磷酸化和核易位。此外，NTB 显着抑制 LPS 诱导的 JNK1/2 和 p38MAPK 的磷酸化，而不影响 ERK1/2。NTB 完全逆转了 LPS 诱导的丝裂原活化蛋白激酶磷酸酶-5（MKP-5）的抑制。总之，这些结果表明，NTB 通过调节 MKP-5/NF-κB 途径介导的 iNOS 抑制 LPS 刺激的 NO 产生，在 RAW 264.7 细胞中。版权所有©2017 年 John Wiley＆Sons，Ltd.
