Li Yinghua, Cao Yan, Xu Jing, Qiu Lei, Xu Weiheng, Li Jie, Song Yunlong, Lu Bin, Hu Zhenlin, Zhang Junping
School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
School of Pharmacy, Second Military Medical University, Shanghai 200433, China.
J Ethnopharmacol. 2017 Feb 23;198:15-23. doi: 10.1016/j.jep.2016.12.041. Epub 2016 Dec 24.
Esculentoside A (EsA) is a saponin isolated from the root of Phytolacca esculenta, an herb which has long been used in Traditional Chinese Medicine for various inflammatory diseases. EsA has been reported to have potent anti-inflammatory properties both in vitro and in vivo.
The present study focused on the molecular mechanism of EsA for its anti-inflammatory effects in RAW264.7 cells stimulated with lipopolysaccharide (LPS).
Enzyme Linked Immunosorbent Assay (ELISA) showed EsA dose dependently inhibited the production of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide in RAW264.7 cells. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay further confirmed the suppression of LPS-induced TNF-α, IL-6 and iNOS gene expression by EsA on a transcriptional level. Moreover, EsA treatment markedly suppressed LPS-stimulated IκB phosphorylation and degradation as well as LPS-stimulated luciferase reporter construct driven by κB response elements in RAW264.7 cells. In addition, EsA significantly reduced LPS-induced stimulation of p38 and JNK, but not ERK1/2, phosphorylation. Furthermore, we used a computational method called "reverse docking" to search the possible binding proteins of EsA from the potential drug target database (PDTD), and focused on CK2 as the primary binding protein of EsA. Afterward, we further tested EsA directly interacts with recombinant CK2 using SPR assay. In CK2 kinase activity assay, EsA inhibited recombinant CK2 holoenzyme activity obviously in a dose-dependent manner. In addition, TBB (4, 5, 6, 7-tetrabromo-2-benzotriazole, a pharmacological inhibitor of CK2) blocked IL-6 release in a dose-dependent manner, whereas co-treatment of cells with EsA and TBB did not have an additive effect.
Taken together, these results indicate that EsA blocks the LPS-induced pro-inflammatory molecules expression, at least in part, by impediment of LPS-triggered activation of NF-κB and p38/JNK MAPK pathways in macrophages. Furthermore, we discovered for the first time EsA as a ligand for CK2, which was involved in the inhibition of EsA to the expression of inflammatory cytokines. These findings extended our understanding on the cellular and molecular mechanisms responsible for the anti-inflammatory activity of EsA.
商陆皂苷甲（EsA）是从商陆根中分离得到的一种皂苷，商陆作为一种草药，长期以来在传统中药中用于治疗各种炎症性疾病。据报道，EsA在体外和体内均具有强大的抗炎特性。
本研究聚焦于EsA对脂多糖（LPS）刺激的RAW264.7细胞产生抗炎作用的分子机制。
酶联免疫吸附测定（ELISA）显示，EsA可剂量依赖性地抑制RAW264.7细胞中肿瘤坏死因子α（TNF-α）、白细胞介素-6（IL-6）和一氧化氮的产生。实时定量逆转录聚合酶链反应（RT-PCR）分析进一步证实在转录水平上EsA可抑制LPS诱导的TNF-α、IL-6和诱导型一氧化氮合酶（iNOS）基因表达。此外，EsA处理显著抑制了LPS刺激的IκB磷酸化和降解，以及LPS刺激的RAW264.7细胞中由κB反应元件驱动的荧光素酶报告构建体。此外，EsA显著降低了LPS诱导的p38和JNK磷酸化，但对细胞外信号调节激酶1/2（ERK1/2）磷酸化无影响。此外，我们使用一种名为“反向对接”的计算方法从潜在药物靶点数据库（PDTD）中搜索EsA可能的结合蛋白，并将酪蛋白激酶2（CK2）作为EsA的主要结合蛋白。随后，我们使用表面等离子体共振（SPR）分析进一步检测EsA与重组CK2的直接相互作用。在CK2激酶活性测定中，EsA以剂量依赖性方式明显抑制重组CK2全酶活性。此外，4,5,6,7-四溴-2-苯并三唑（TBB，一种CK2的药理学抑制剂）以剂量依赖性方式阻断IL-6释放，而EsA与TBB共同处理细胞则没有相加效应。
综上所述，这些结果表明EsA至少部分地通过阻碍LPS触发的巨噬细胞中核因子κB（NF-κB）和p38/应激活化蛋白激酶（JNK）丝裂原活化蛋白激酶（MAPK）信号通路的激活，来阻断LPS诱导的促炎分子表达。此外，我们首次发现EsA是CK2的配体，其参与了EsA对炎性细胞因子表达的抑制作用。这些发现扩展了我们对EsA抗炎活性的细胞和分子机制的理解。
