Neochlorogenic acid inhibits against LPS-activated inflammatory responses through up-regulation of Nrf2/HO-1 and involving AMPK pathway

Abstract

Acute and chronic inflammatory diseases are associated with excessive inflammation due to the accumulation of pro-inflammatory mediators and cytokines produced by macrophages. In the present study, we investigated the anti-inflammatory properties of neochlorogenic acid (nCGA) from Lonicera japonica on lipopolysaccharide (LPS)-activated inflammation in macrophages and participation of the AMPK/Nrf2 pathway. nCGA pretreatment significantly reduced the production of nitric oxide, prostaglandin E₂, TNF-α, reactive oxygen species, IL-1β, and IL-6 by LPS-activated macrophages. Moreover, both transcript and protein levels of inducible nitric oxide synthase and cyclooxygenase-2 were reduced by nCGA in LPS-activated macrophages. nCGA inhibited NF-κB activation by attenuating IKKα/β and IκBα phosphorylation in LPS-stimulated macrophages. Moreover, nCGA attenuated LPS-elevated JAK-1, STAT-1, and MAPK phosphorylation. We further evaluated the possible role of nCGA in the induction of AMPK/Nrf2 signal pathways required for the protein expression of HO-1 and NQO-1. nCGA induced AMPK activation via phosphorylation of LKB1 and CaMKII and by the inhibitory phosphorylation of GSK3β. It stimulated the overexpression of Nrf2/ARE-regulated downstream proteins, such as NQO-1 and HO-1. Furthermore, the anti-inflammatory effects of nCGA were attenuated in macrophages subjected to siRNAs specific for HO-1, NQO-1, Nrf2, and AMPK. Accordingly, these results indicate that nCGA, as an AMPK/Nrf2 signal activator, prevents excessive macrophage-mediated responses associated with acute and chronic inflammatory disorders.

Introduction

Excessive inflammatory response to pathogen infection may result in the development of acute and chronic diseases and is known to be partly caused by macrophage activation. Macrophages are a major factor of the immune system and are critical for cellular function, removal of cellular debris, and repair and regeneration of tissue via alternative activation (Dushkin, 2012; Hansel and Barnes, 2009). Macrophages produce pro-inflammatory mediators, including nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E₂ (PGE₂), and cytokines, such as IL-1β, IL-6, and TNF-α. Although the limited production of inflammatory cytokines and mediators is crucial for immune defenses and tissue regeneration, excessive accumulation following their release by macrophages is injurious to neighboring cells and tissues and is involved in the development or progression of numerous diseases, including systemic respiratory diseases, autoimmune diseases, chronic obstructive pulmonary disease, type I diabetes, and cancer (Cheng et al., 2015; Chiou et al., 2016).

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) and plays an essential role in activating host defenses by modulating inflammatory responses. In macrophages, TLR4 activation is regulated throughout inflammation progression and in response to pathogens. TLR4 triggers intracellular pathways that include different adaptor proteins, including the myeloid differentiation primary response gene 88/interleukin-1 receptor-associated kinase (IRAK)-1/IRAK-4/TNF receptor-associated factor-6 complex (He et al., 2017). This complex facilitates the activation of a large number of intracellular signaling pathways, such as NF-κB, JAK/STAT, ERK-1/2, p38 MAPK, and JNK signaling pathways (Hsu et al., 2013). These signaling pathways regulate the induction of genes encoding pro-inflammatory enzymes and cytokines, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), via NF-κB activation. Therefore, components of these signaling pathways represent possible therapeutic targets for the treatment of acute and chronic inflammatory diseases (Khajuria et al., 2017; Kim et al., 2016).

Acute and chronic inflammatory responses can involve the induction of the macrophage M1 phenotype, triggering the production of pro-inflammatory mediators and cytokines. Macrophages with an M2-like phenotype release anti-inflammatory mediators, including AMPK, Nrf2, heme oxygenase-1 (HO-1), and NADPH dehydrogenase quinone-1 (NQO-1) (Ahn et al., 2017; Akram et al., 2016). These multifunctional proteins are associated with host cell defense and repair systems in acute and chronic inflammatory disorders. The significance of AMPK, Nrf2, HO-1, and NQO-1 in mediating anti-inflammatory properties has been well-verified in vitro and in vivo, and in HO-1, NQO-1, Nrf2, and AMPK knock-out mice. AMPK regulates cellular energy status, including glucose and protein metabolism, and is a principal regulator of oxidative stress responses and anti-inflammatory processes (Ci et al., 2017; Kaufmann et al., 2016). AMPK promotes anti-inflammatory responses by activating antioxidant defense systems via downstream signaling pathways including Nrf2. AMPK also regulates the inhibitory phosphorylation of GSK3β to accelerate the nuclear translocation and activation of Nrf2. Antioxidant-responsive element (ARE) regulates anti-inflammatory genes via the transcription factor, Nrf2. Nrf2/ARE-dependent genes include phase II detoxification enzymes such as HO-1 and NQO-1. In vitro experiments have demonstrated that LPS-induced inflammatory responses can be inhibited by activating Nrf2, HO-1, and NQO-1. AMPK/Nrf2/ARE signaling regulates the induction of LPS-induced inflammatory mediators, including inducible iNOS, COX-2, TNF-α, and IL-6, in macrophages. AMPK/Nrf2/ARE activation also suppresses the NF-κB signaling pathway in LPS-induced macrophages (Lee et al., 2016). Many phenolic compounds target AMPK/Nrf2 pathways to exert anti-inflammatory effects. AMPK/Nrf2 activation by these phenolic compounds has also been stated to induce HO-1- and NQO-1-mediated anti-inflammatory effects by inhibiting oxidative stress. Therefore, the development of novel therapeutics that modulate the functional interaction between Nrf2 and AMPK may have significant value in the treatment of acute and chronic inflammatory diseases (Mo et al., 2014; Chai et al., 2016).

Although nonsteroidal anti-inflammatory drugs are used extensively to treat acute and chronic inflammatory disorders, these drugs can cause drug-related morbidities, such as heart attack, gastrointestinal bleeding, and stroke. Therefore, naturally occurring phenolic compounds capable of ameliorating acute and chronic inflammatory diseases have received much attention. Phenolic compounds can play valuable roles in preventing and treating acute and chronic inflammatory diseases as many of them have strong antioxidant properties (Nam et al., 2017). Phenolic compounds are considered the main bioactive components produced by Lonicera japonica, a plant used since centuries in eastern Asia for its antioxidant, antitumor, anti-inflammatory, and anti-aggregative effects (Yoo et al., 2008; Ryu et al., 2010). Neochlorogenic acid (nCGA), a phenolic compound produced by L. japonica capable of inducing antioxidant and anti-inflammatory signaling pathways, is thought to exert neuroprotective effects by reducing neuroinflammation. nCGA has also been found to be helpful in the prevention and treatment of rheumatoid arthritis and associated complications (Cheng et al., 2015; Kwon et al., 2015; Lin et al., 2016). Although nCGA-induced anti-inflammatory effects have been well-established, the mechanisms underlying these effects are not fully understood. In the current study, we investigated whether nCGA alleviates LPS-induced inflammatory responses by up-regulating Nrf2-related genes and whether AMPK signaling is involved in this mechanism.