Vaccaria hypaphorine alleviates lipopolysaccharide-induced inflammation via inactivation of NFκB and ERK pathways in Raw 264.7 cells

Sun et al. BMC Complementary and Alternative Medicine (2017) 17:120 DOI 10.1186/s12906-017-1635-1 RESEARCH ARTICLE Open Access Vaccaria hypaphorine alleviates lipopolysaccharide-induced inflammation via inactivation of NFκB and ERK pathways in Raw 264.7 cells Haijian Sun1†, Weiwei Cai1†, Xu Wang1†, Yanling Liu2, Bao Hou1, Xuexue Zhu1 and Liying Qiu1* Abstract Background: Activation of macrophage is involved in many inflammation diseases. Lipopolysaccharide (LPS) is a powerful inflammatory signal contributing to monocytes/macrophages activation associated with increased proinflammatory cytokines expressions. We recently identified that vaccarin was expected to protect endothelial cells from injury. Hypaphorine was abundantly found in vaccaria semen. However, the potential roles and underlying mechanisms of vaccaria hypaphorine on macrophage inflammation have been poorly defined. Methods: This study was designed to determine the effects of vaccaria hypaphorine on LPS-mediated inflammation in RAW 264.7 cells. Results: In this study, we demonstrated that vaccaria hypaphorine dramatically ameliorated LPS-induced nitric oxide (NO) release and productions of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, monocyte chemoattractant protein 1 (MCP-1) and prostaglandin E2 (PGE2) in RAW 264.7 cells. LPS-stimulated expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were down-regulated by vaccaria hypaphorine. Furthermore, vaccaria hypaphorine retarded LPS-induced phosphorylation of ERK, nuclear factor kappa beta (NFκB), NFκB inhibitor IκBα, and IKKβ. Immunofluorescence staining revealed that vaccaria hypaphorine eliminated the nuclear translocation of NFκB in LPS-treated RAW 264.7 cells. Conclusion: It was seen that vaccaria hypaphorine counteracted inflammation via inhibition of ERK or/and NFκB signaling pathways. Collectively, we concluded that vaccaria hypaphorine can be served as an anti-inflammatory candidate. Keywords: COX-2, iNOS, ERK, NFκB, Inflammation, Hypaphorine Background Inflammation is considered as a tissue protective immune response against injurious stimuli including damaged cells, irritants and bacteria [1]. The process of inflammation is regulated by initiating, maintaining and shutting signals [2]. However, the imbalanced inflammation may induce cellular and tissue damage in different diseases such as atherosclerosis [3], hypertension [4], diabetes [5], cancer [6], and neurodegenerative disorders [7]. * Correspondence: † Equal contributors 1 Department of Basic Medicine, Wuxi Medical School, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China Full list of author information is available at the end of the article The immune system may produce inflammation mediators response to chemical, physical, or infectious stress [1]. Macrophages are critical determinants for multiple biological processes during the immune response [7]. Macrophages can release various cytokines and growth factors to exert three major functions including antigen presentation, phagocytosis, and immunomodulation during the process of inflammation [8]. Injured or activated macrophages may coordinate inflammatory responses through releasing various inflammatory mediators [8]. Overproduction of pro-inflammatory cytokine by macrophages leads to destructive inflammation in the body [9]. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sun et al. BMC Complementary and Alternative Medicine (2017) 17:120 Page 2 of 11 Lipopolysaccharide (LPS) acts as a switch for macrophages activation as evidenced by excessive expressions in nitric oxide (NO), tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), IL-6, IL-10, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein 1 (MCP-1) [10, 11]. Pro-inflammatory stimuli mediated-upregualtion of cyclooxygenase-2 (COX-2) is a major contributor to PGE2 synthesis [12]. Mitogen-activated protein kinases (MAPKs) and nuclear factor kappa beta (NFκB) signaling pathways may be two important intracellular molecular pathways involving inflammatory cascade response to LPS stimulation in RAW264.7 cells [13–15]. Vaccarin is the main component of Vaccaria segetalis seeds [16]. Vaccarin is recently identified to be a major flavonoid glycoside [17]. The emergence of vaccarin has attracted considerable attention due to its diverse biological activities [18]. Vaccarin dose-relatedly promoted the proliferation, migration, tube formation and neovascularization of human microvascular endothelial cells through activation of Akt and ERK signals [19]. The construction of bacterial cellulose-vaccarin membranes exhibited no cytotoxicity for cell growth, which was found to be a potential candidate for wound healing in rat skin models [20]. We recently demonstrated that vaccarin may protect endothelial cells from oxidative stress-induced injury via negatively regulation of Notch signaling [21]. We further established that vaccarin may obviously ameliorate high glucose-mediated endothelial cell injury by reversing cell viability and migratory ability [22]. The existing evidence suggested that vaccarin may function as novel therapeutic agent for endothelium dysfunction. The hypaphorine is an indole alkaloid from Erythrina velutina that exhibits sleep promoting effects in normal mice [23]. The hypaphorine from different marine sources is shown to possess anti-inflammatory properties [24]. Fungal auxin antagonist hypaphorine obviously inhibited indole-3-acetic acid-dependent superoxide production by competitively binding to the putative binding site of indole-3-acetic acid [25]. Hypaphorine was also a key component of Vaccaria segetalis. However, the potential roles and mechanisms of vaccaria hypaphorine on macrophages inflammation were largely unknown. In this study, we investigated that the possible mechanism by which vaccaria hypaphorine protected RAW 264.7 murine macrophages from LPS-induced inflammation response in vitro. lung adenocarcinoma cell line A549 and mouse lung cancer cell Lewis were purchased from American Type Culture Collection (Rockville, MD, USA). Human microvascular endothelial cells HMEC-1 was obtained from the Health and Medicine Research of French National Institute. Human umbilical vein endothelial cells EA · hy926, mouse fibroblast cells L929, human brea (...truncated)