Ju Woo Hyun, Jun Do Youn, Lee Ji Young, Park Hae Sun, Woo Mi Hee, Park Sook Jahr, Kim Sang Chan, Yang Chae Ha, Kim Young Ho
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea.
Institute of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, South Korea.
J Ethnopharmacol. 2017 Jun 9;205:103-115. doi: 10.1016/j.jep.2017.04.029. Epub 2017 Apr 30.
The roots of Rubia cordifolia L. have been widely used as a traditional herbal medicine in Northeast Asia for treating inflammatory diseases.
To elucidate the anti-inflammatory mechanism of 2-carbomethoxy-2,3-epoxy-3- prenyl-1,4-naphthoquinone (CMEP-NQ), purified from the roots of R. cordifolia L. as the major anti-inflammatory component, in LPS-treated RAW264.7 murine macrophage cells.
Anti-inflammatory activity of CMEP-NQ was investigated in LPS-treated RAW264.7 cells by measuring the levels of NO, PGE, and cytokines (IL1β, IL-6, TNF-α) in the culture supernatants and the TLR4-mediated intracellular events including association of MyD88 with IRAK1, activation of IRAK1, TAK1, MAPKs, NF-κB/AP-1, and IRF3, and generation of ROS.
Pretreatment of RAW264.7 cells with CMEP-NQ reduced LPS-induced production of NO and PGE by suppressing iNOS and COX-2 gene expression. CMEP-NQ also reduced the secretion of IL-1β, IL-6, and TNF-α by down-regulating mRNA levels. Under these conditions, TLR4-mediated MyD88-dependent events were inhibited by CMEP-NQ, including the association of MyD88 with IRAK1, phosphorylation of IRAK1, TAK1, and MAPKs (ERK, JNK and p38 MAPK), and activation of NF-κB and AP-1. As TRIF-dependent events of TLR4 signaling, phosphorylation of IRF3 and induction of iNOS protein expression were also inhibited by CMEP-NQ. However, the binding of FITC-conjugated LPS to cell surface TLR4 was not affected by CMEP-NQ. Following LPS stimulation, intracellular ROS production was first detected by DCFH-DA staining at 1h; then it continuously increased until 16h. Although CMEP-NQ failed to exhibit DPPH radical- or ABTS radical-scavenging activity in vitro, LPS-induced ROS production in RAW264.7 cells was more efficiently blocked by CMEP-NQ than by NAC.
These results demonstrate that the suppressive effect of CMEP-NQ on LPS-induced inflammatory responses in RAW264.7 cells was mainly exerted via its inhibition of TLR4-mediated proximal events, such as MyD88-dependent NF-κB/AP-1 activation and ROS production, and TRIF-dependent IRF3 activation.
茜草的根在东北亚地区作为治疗炎症性疾病的传统草药被广泛使用。
阐明从茜草的根中纯化得到的主要抗炎成分2-甲氧羰基-2,3-环氧-3-异戊烯基-1,4-萘醌（CMEP-NQ）在脂多糖（LPS）处理的RAW264.7小鼠巨噬细胞中的抗炎机制。
通过测量培养上清液中一氧化氮（NO）、前列腺素E（PGE）和细胞因子（IL-1β、IL-6、肿瘤坏死因子-α（TNF-α））的水平以及TLR4介导的细胞内事件，包括髓样分化因子88（MyD88）与白细胞介素-1受体相关激酶1（IRAK1）的结合、IRAK1、转化生长因子β激活激酶1（TAK1）、丝裂原活化蛋白激酶（MAPKs）、核因子κB/激活蛋白-1（NF-κB/AP-1）和干扰素调节因子3（IRF3）的激活以及活性氧（ROS）的产生，研究CMEP-NQ在LPS处理的RAW264.7细胞中的抗炎活性。
用CMEP-NQ预处理RAW264.7细胞可通过抑制诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）基因表达来降低LPS诱导的NO和PGE产生。CMEP-NQ还通过下调mRNA水平来减少IL-1β、IL-6和TNF-α的分泌。在这些条件下，CMEP-NQ抑制了TLR4介导的MyD88依赖性事件，包括MyD88与IRAK1的结合、IRAK1、TAK1和MAPKs（细胞外信号调节激酶（ERK）、c-Jun氨基末端激酶（JNK）和p38 MAPK）的磷酸化以及NF-κB和AP-1的激活。作为TLR4信号传导的TRIF依赖性事件，IRF3的磷酸化和iNOS蛋白表达的诱导也被CMEP-NQ抑制。然而，异硫氰酸荧光素（FITC）偶联的LPS与细胞表面TLR4的结合不受CMEP-NQ影响。LPS刺激后，通过2′,7′-二氯二氢荧光素二乙酸酯（DCFH-DA）染色在1小时首次检测到细胞内ROS产生；然后持续增加直至16小时。尽管CMEP-NQ在体外未表现出清除二苯基苦味酰基自由基（DPPH自由基）或2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸自由基（ABTS自由基）的活性，但CMEP-NQ比N-乙酰半胱氨酸（NAC）更有效地阻断了LPS诱导的RAW264.7细胞中的ROS产生。
这些结果表明，CMEP-NQ对LPS诱导的RAW264.7细胞炎症反应的抑制作用主要是通过抑制TLR4介导的近端事件，如MyD88依赖性NF-κB/AP-1激活和ROS产生以及TRIF依赖性IRF3激活来实现的。
