Research ArticleAstaxanthin exerts anti-inflammatory and antioxidant effects in macrophages in NRF2-dependent and independent manners
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.
Section snippets
Cell culture and treatment
Murine RAW 264.7 macrophages (ATCC, Manassas, VA) were cultured in RPMI 1640 media as we previously described [25]. Bone marrow-derived macrophages (BMDM) were prepared from tibia and femur of wild-type (WT) and Nrf2 knockout (KO) mice or C57BL/6J mice and cultured as previously described [25]. All cells were maintained in 5% CO₂ at 37 °C.
ASTX powder was a generous gift from Fuji Chemical Industry (Toyama, Japan) and ASTX was prepared for in vitro experiments as we previously described [18].
ASTX repressed the expression of pro-inflammatory genes in RAW 264.7 macrophages
Studies have shown anti-inflammatory properties of ASTX in macrophages [21], [29], [30]. Consistently, we found that mRNA levels of Il-6 and Il-1β were significantly decreased by ASTX with no changes in Tnfα mRNA when RAW 264.7 macrophages were stimulated with LPS for 3 h (Fig. 1A). Our time-course experiment demonstrated that the induction of Il-6 and Il-1β expression by LPS reached the maximal levels at 12 h of LPS stimulation, and ASTX significantly decreased the pro-inflammatory gene
Discussion
Chronic inflammation and oxidative stress contribute to the pathogenesis of obesity-associated chronic diseases [12], [34]. Macrophages generate ROS that trigger oxidative stress and inflammation, which are critical underlying causes of chronic diseases [35], [36], [37]. In macrophages, while NFκB induced the production of pro-inflammatory mediators [2], [7], [8], [9], NRF2 counteracts NFκB by enhancing endogenous antioxidant defense [13], [38]. Studies have shown that ASTX exerts an
Acknowledgments
Authors declare no conflicts of interest. This work was supported by NIH 1R01DK108254, USDA HATCH CONS00972, and USDA multi-state HATCH CONS00916 to J. Lee; and USDA AFRI 2016-08864 to Y. Park.
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