Src Is a Prime Target Inhibited by Celtis choseniana Methanol Extract in Its Anti-Inflammatory Action.

Han Gyung Kim,Gi-Ho Sung,Yo Han Hong,Subin Choi,Jong-Hoon Kim,Young-Su Yi,Keejung Yoon,Jongsung Lee,Deok Jeong,Deok Hyo Yoon,Jae Youl Cho,Seungihm Lee,Suntaek Hong

doi:10.1155/2018/3909038

Abstract

Celtis choseniana is the traditional plant used at Korea as a herbal medicine to ameliorate inflammatory responses. Although Celtis choseniana has been traditionally used as a herbal medicine at Korea, no systemic research has been conducted on its anti-inflammatory activity. Therefore, the present study explored an anti-inflammatory effect and its underlying molecular mechanism using Celtis choseniana methanol extract (Cc-ME) in macrophage-mediated inflammatory responses. In vitro anti-inflammatory activity of Cc-ME was evaluated using RAW264.7 cells and peritoneal macrophages stimulated by lipopolysaccharide (LPS), pam3CSK4 (Pam3), or poly(I:C). In vivo anti-inflammatory activity of Cc-ME was investigated using acute inflammatory disease mouse models, such as LPS-induced peritonitis and HCl/EtOH-induced gastritis. The molecular mechanism of Cc-ME-mediated anti-inflammatory activity was examined by Western blot analysis and immunoprecipitation using whole cell and nuclear fraction prepared from the LPS-stimulated RAW264.7 cells and HEK293 cells. Cc-ME inhibited NO production and mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and tumor necrosis factor-alpha (TNF-α) in the RAW264.7 cells and peritoneal macrophages induced by LPS, pam3, or poly(I:C) without cytotoxicity. High-performance liquid chromatography (HPLC) analysis showed that Cc-ME contained anti-inflammatory flavonoids quercetin, luteolin, and kaempferol. Among those, the content of luteolin, which showed an inhibitory effect on NO production, was highest. Cc-ME suppressed the NF-κB signaling pathway by targeting Src and interrupting molecular interactions between Src and p85, its downstream kinase. Moreover, Cc-ME ameliorated the morphological finding of peritonitis and gastritis in the mouse disease models. Therefore, these results suggest that Cc-ME exerted in vitro and in vivo anti-inflammatory activity in LPS-stimulated macrophages and mouse models of acute inflammatory diseases. This anti-inflammatory activity of Cc-ME was dominantly mediated by targeting Src in NF-κB signaling pathway during macrophage-mediated inflammatory responses.

Highlights

Inflammation is the defensive process that protects a human body from infection by various pathogens, including bacteria, viruses, fungi, and protozoans, and is mainly induced by innate immune cells, including phagocytic macrophages, neutrophils, and dendritic cells [1,2,3]
To examine an anti-inflammatory activity of Celtis choseniana methanol extract (Cc-ME) in macrophages, we first determined the level of nitric oxide (NO) production, a critical event in inflammatory reaction, in the Cc-ME-treated RAW264.7 cells and peritoneal macrophages treated with toll-like receptors (TLRs) ligands such as LPS derived from Gram (−) bacteria, Pam3CSK derived from Gram (+) bacterial, or poly(I:C) derived from virus
We explored an anti-inflammatory activity of Cc-ME in vitro using macrophages and in vivo using acute inflammatory disease models in mice

Summary

Introduction

Inflammation is the defensive process that protects a human body from infection by various pathogens, including bacteria, viruses, fungi, and protozoans, and is mainly induced by innate immune cells, including phagocytic macrophages, neutrophils, and dendritic cells [1,2,3]. Lipopolysaccharide (LPS) is one of the most powerful agonists for activating inflammatory responses in macrophages that bind to its molecular receptor, TLR4, which transduces inflammatory signal cascades via two major adaptor molecules, toll/ interleukin-1 receptor-domain-containing, adapter-inducing interferon-β (TRIF), and myeloid differentiation response gene 88 (MyD88) [8]. Sequential activation of these proteins induces the expression of proinflammatory cytokines and genes, including tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and the production of inflammatory mediators, including prostaglandin E2 (PGE2) and nitric oxide (NO) [4,5,6,7]. Increased expression and production of these genes and mediators promote inflammatory responses and induce phagocytosis activity among innate immune cells [9, 10]

Methods

Results

Conclusion