Astaxanthin exerts anti-inflammatory and antioxidant effects in macrophages in NRF2-dependent and independent manners

Abstract

Although anti-inflammatory effects of astaxanthin (ASTX) have been suggested, the underlying mechanisms have not been fully understood. Particularly, the modulatory action of ASTX in the interplay between nuclear factor E2-related factor 2 (NRF2) and nuclear factor κB (NFκB) to exert its anti-inflammatory effect in macrophages is unknown. The effect of ASTX on mRNA and protein expression of pro-inflammatory and antioxidant genes and/or cellular reactive oxygen species (ROS) accumulation were determined in RAW 264.7 macrophages, bone marrow-derived macrophages (BMDM) from wild-type (WT) and Nrf2-deficient mice, and/or splenocytes and peritoneal macrophages of obese mice fed ASTX. The effect of ASTX on M1 and M2 macrophage polarization was evaluated in BMDM. ASTX significantly decreased LPS-induced mRNA expression of interleukin 6 (Il-6) and Il-1β by inhibiting nuclear translocation of NFκB p65; and attenuated LPS-induced ROS with an increase in NRF2 nuclear translocation, concomitantly decreasing NADPH oxidase 2 expression in RAW 264.7 macrophages. In BMDM of WT and Nrf2-deficient mice, ASTX decreased basal and LPS-induced ROS accumulation. The induction of Il-6 mRNA by LPS was repressed by ASTX in both types of BMDM while Il-1β mRNA was decreased only in WT BMDM. Furthermore, ASTX consumption lowered LPS sensitivity of splenocytes in obese mice. ASTX decreased M1 polarization of BMDM while increasing M2 polarization. ASTX exerts its anti-inflammatory effect by inhibiting nuclear translocation of NFκB p65 and by preventing ROS accumulation in NRF2-dependent and -independent mechanisms. Thus, ASTX is an agent with anti-inflammatory and antioxidant properties that may be used for the prevention of inflammatory conditions.

Introduction

Inflammation and oxidative stress are implicated in the pathogenesis of chronic diseases [1], [2]. Macrophages play an important role in body's inflammatory responses and they are generally divided into two phenotypes, i.e., M1 and M2 macrophage [3], [4]. M1 macrophages are classically activated by interferon γ (IFNγ) and lipopolysaccharide (LPS); and they produce reactive oxygen species (ROS) and pro-inflammatory cytokines including tumor necrosis factor α (TNFα), interleukin (IL)-1β, and IL-6 [4], [5]. M2 macrophages, alternatively activated by IL-4 and IL-13, modulate inflammatory reactions by producing anti-inflammatory cytokines, such as IL-10 [4], [5]. As M1 and M2 macrophages have somewhat opposing actions, the balance of M1/M2 phenotype is important for regulating immune and inflammatory processes [4], [6].

Nuclear factor κB (NFκB) is the transcription factor that promotes inflammation by increasing the production of reactive oxygen species (ROS) and pro-inflammatory cytokines in macrophages. In the cytoplasm, NFκB binds to the inhibitor protein, i.e., IκB, from which it is released for nuclear translocation to induce the expression of pro-inflammatory mediators when it is activated by inflammatory insults [7], [8], [9]. Nuclear factor E2-related factor 2 (NRF2) plays a critical role in endogenous antioxidant defense mechanism and prevents oxidative stress by stimulating the production of antioxidant enzymes [10], [11]. In addition, the activation of NRF2 inhibits inflammatory responses associated with NFκB in macrophages [12], [13]. Furthermore, a reduction in intracellular ROS levels inhibits pro-inflammatory signals in macrophages [1]. Thus, bioactive compounds that can activate NRF2 and possess antioxidant properties may have therapeutic potentials to prevent the development of inflammation-related diseases.

Astaxanthin (ASTX) is a xanthophyll carotenoid that has anti-inflammatory and antioxidant properties [14]. We demonstrated that ASTX exerts hypolipidemic effects in apolipoprotein E knockout mice [15] and diet-induced obesity mice [16]. Furthermore, recently, we showed that ASTX inhibits inflammation and fibrosis in the liver of diet-induced nonalcoholic steatohepatitis [17]. Our in vitro studies also support anti-fibrogenic effects of ASTX [18], [19], [20]. Studies have shown that ASTX exerts its anti-inflammatory function by reducing the expression of pro-inflammatory cytokines via the inhibition of NFκB activation in alveolar macrophage, neutrophils, and lymphocytes [21], [22], [23], [24]. However, although NRF2 pathway plays a critical role in the prevention of ROS accumulation and oxidative stress, the effects of ASTX on NRF2-mediated inhibition of pro-inflammatory cytokine production and ROS accumulation in macrophages have yet to be determined. Therefore, in the present study, we sought to determine the effect of ASTX on the interplay of NRF2 and NFκB for its anti-inflammatory and antioxidant properties in macrophages.