牧野菌素通过抑制TLR1/TLR2介导的NF-κB激活，抑制PamCSK诱导的RAW 264.7巨噬细胞炎症反应。

Makino inhibits PamCSK-induced inflammation in RAW 264.7 macrophages through suppressing TLR1/TLR2-mediated NF-κB activation.

作者信息

Sang Wei, Zhong Zhangfeng, Linghu Kegang, Xiong Wei, Tse Anfernee Kai Wing, Cheang Wai San, Yu Hua, Wang Yitao

机构信息

Institute of Chinese Medical Sciences, State Key Laboratory of Quality Research in Chinese Medicine, University of Macau, Macao, China.

4Guangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical University, Zhanjiang, China.

出版信息

Chin Med. 2018 Jul 5;13:37. doi: 10.1186/s13020-018-0193-x. eCollection 2018.

BACKGROUND

Makino (SP) is one of the important plant origins for the anti-inflammatory Chinese herbal medicine of Siegesbeckiae Herba. The current investigations indicated that the anti-inflammatory effects of SP were associated with the toll-like receptors (TLRs)-mediated nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) signaling pathways.

METHODS

Raw 264.7 macrophages were pretreated with the 50% ethanol extract of SP (SPE, 50-200 µg/mL) and then co-treated with PamCSK (200 ng/mL) for another 12 h. The inhibitory effect of SPE on PamCSK-stimulated NO release and post-inflammatory cytokines secretions were determined using Griess reagent and Elisa kits, respectively. The influence of SPE on NF-κB and MAPKs signaling relevant proteins was measured by Western blotting analysis, while the intracellular nitric oxide (NO) generation and NF-κB/p65 nuclear translocation were determined using Leica TCS SP8 laser scanning confocal microscope. Moreover, the effect of SPE on luciferase reporter gene in NF-κB-luc DNA transfected raw 264.7 cells was determined using the Dual-Glo luciferase assay system kit.

RESULTS

SPE dose-dependently (50-200 µg/mL) attenuated PamCSK-induced NO release, post-inflammatory cytokines (IL-6, TNF-α and MCP-1) secretions and intracellular NO generation in raw 264.7 cells. Biologically, SPE suppressed PamCSK-induced expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), phosphorylation of NF-κB/p65 and IκBα, but did not significantly show effect on the proteins involved in MAPKs signaling (p38, ERK and JNK). The results were further confirmed by NF-κB-luc reporter gene assay and p65 nuclear translocation assay.

CONCLUSIONS

In conclusion, SPE ameliorated PamCSK-induced inflammation in raw 264.7 cells through suppressing TLR 1/2-mediated NF-κB activation.

背景

牧野变种（SP）是中药豨莶草抗炎作用的重要植物来源之一。目前的研究表明，SP的抗炎作用与Toll样受体（TLRs）介导的核因子κB（NF-κB）和丝裂原活化蛋白激酶（MAPK）信号通路有关。

方法

用SP的50%乙醇提取物（SPE，50 - 200μg/mL）预处理Raw 264.7巨噬细胞，然后与PamCSK（200 ng/mL）共同处理12小时。分别使用Griess试剂和酶联免疫吸附测定（ELISA）试剂盒测定SPE对PamCSK刺激的一氧化氮（NO）释放和炎症后细胞因子分泌的抑制作用。通过蛋白质免疫印迹分析测定SPE对NF-κB和MAPKs信号相关蛋白的影响，同时使用徕卡TCS SP8激光扫描共聚焦显微镜测定细胞内一氧化氮（NO）生成和NF-κB/p65核转位。此外，使用双荧光素酶检测系统试剂盒测定SPE对NF-κB-luc DNA转染的Raw 264.7细胞中荧光素酶报告基因的影响。

结果

SPE以剂量依赖性方式（50 - 200μg/mL）减弱PamCSK诱导Raw 264.7细胞中NO释放、炎症后细胞因子（IL-6、TNF-α和MCP-1）分泌及细胞内NO生成。从生物学角度看，SPE抑制PamCSK诱导的环氧化酶-2（COX-2）、诱导型一氧化氮合酶（iNOS）表达、NF-κB/p65和IκBα磷酸化，但对MAPKs信号通路相关蛋白（p38、ERK和JNK）未显示出显著影响。NF-κB-luc报告基因检测和p65核转位检测进一步证实了上述结果。