molecules Article
Pterostilbene and 40 -Methoxyresveratrol Inhibited Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages
Yun Yao 1 , Kehai Liu 1,2 , Yueliang Zhao 1, *, Xiaoqian Hu 1,2, * and Mingfu Wang 1 1 2
*
College of Food Science and Technology, Shanghai Ocean University, No.999 Hu-Cheng-Huan Road, Shanghai 201306, China;
[email protected] (Y.Y.);
[email protected] (K.L.);
[email protected] (M.W.) Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306, China Correspondence:
[email protected] (Y.Z.);
[email protected](X.H.); Tel.: +86-21-6190-0514 (X.H.)
Academic Editor: Isabel C. F. R. Ferreira Received: 11 April 2018; Accepted: 8 May 2018; Published: 11 May 2018
Abstract: Pterostilbene (Pte) and 40 -Methoxyresveratrol (4MR) are methylated derivatives of resveratrol. We investigated the anti-inflammatory effect of Pte and 4MR in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. Both Pte and 4MR significantly reduced LPS-induced nitric oxide release by inhibiting the inducible nitric oxide synthase mRNA expression. Moreover, both of them inhibited LPS-induced mRNA expression of inflammatory cytokines including monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6 and IL-1β, and tumor necrosis factor α (TNF-α), and attenuated LPS-induced nuclear factor-κB (NF-κB) activation by decreasing p65 phosphorylation. In addition, 4MR but not Pte inhibited LPS-induced the activator protein (AP)-1 pathway in RAW 264.7 macrophages. Further study suggested that Pte had an inhibitory effect on extracellular regulated protein kinases (ERK) and p38 activation, but not on c-Jun N-terminal kinase (JNK), while 4MR had an inhibitory effect on JNK and p38 activation, but not on ERK. Taken together, our data suggested that Pte induced anti-inflammatory activity by blocking mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, while 4MR showed anti-inflammatory activity through suppression of MAPK, AP-1, and NF-κB signaling pathways in LPS-treated RAW 264.7 macrophages. Keywords: pterostilbene; 40 -methoxyresveratrol; inflammation; NF-κB; MAPK; AP-1
1. Introduction Inflammation is a kind of body defensive response to external stimuli. It can destroy and remove the detrimental agents and injured tissues, thereby benefiting tissue repair [1]. However, when this protective response is out of control, excessive cell and tissue damage will occur and lead to many diseases [2], such as diabetes [3], colitis [4], rheumatism [5], and atherosclerosis [6]. Thus, anti-inflammatory agents may be therapeutically useful. Macrophages play an essential role in the inflammatory response. The RAW264.7 cell line is well accepted as a suitable macrophage model [7]. Lipopolysaccharide (LPS) is commonly used for the induction of inflammatory models for its ability to regulate the expression of inflammatory-related enzymes and release of inflammatory mediators by activating multiple signaling pathways [8]. In LPS-induced RAW264.7 cells, toll-like receptor 4 (TLR4) recognizes and binds to LPS, promoting the association of TLR4 with the adaptor myeloid differentiation factor 88. Subsequently, progressive inflammatory signaling induces activation of mitogen-activated protein kinases (MAPKs), AP-1, and NF-κB protein kinases, eventually contributing to the inflammatory response [9,10]. Molecules 2018, 23, 1148; doi:10.3390/molecules23051148
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inflammatory signaling induces activation of mitogen‐activated protein kinases (MAPKs), AP‐1, and 2 of 11 NF‐κB protein kinases, eventually contributing to the inflammatory response [9,10]. AP‐1 is a crucial transcription factor regulating inflammation response [11]. LPS activates AP‐1 proteins (Fos and Jun heterodimers), which generally regulate their transcriptional activity through AP-1 is a crucial transcription factor regulating inflammation response [11]. LPS activates AP-1 interactions with adjacent proteins or with transcriptional coactivators at active domains to regulate proteins (Fos and Jun heterodimers), which generally regulate their transcriptional activity through the generation of inflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 [12,13]. The MAPKs consist interactions with adjacent proteins or with transcriptional coactivators at active domains to regulate of three main of members: the extracellular signal‐regulating kinase c‐Jun N‐terminal the generation inflammatory cytokines such as TNF-α, IL-1β, and (ERK), IL-6 [12,13]. The MAPKs protein consist kinase (JNK), and p38, and are an important family of serine/threonine protein kinases implicated in of three main members: the extracellular signal-regulating kinase (ERK), c-Jun N-terminal protein inflammation [14]. MAPKs are known to be AP‐1 regulators. LPS can activate MAPKs and cause AP‐ kinase (JNK), and p38, and are an important family of serine/threonine protein kinases implicated 1 phosphorylation, gene expression [8]. NF‐κB is and a nuclear in inflammation [14].eventually MAPKs areinitiating known toinflammatory be AP-1 regulators. LPS can activate MAPKs cause transcription factor regulating the transcription of various genes involved in inflammation [15]. AP-1 phosphorylation, eventually initiating inflammatory gene expression [8]. NF-κB is a nuclear Activation of NF‐κB promotes the expression of its downstream inflammatory cytokines such as transcription factor regulating the transcription of various genes involved in inflammation [15]. MCP‐1, IL‐6, IL‐1, and TNF‐α [16–18], and eventually induces an inflammatory response [19]. Thus, Activation of NF-κB promotes the expression of its downstream inflammatory cytokines such as the AP‐1, MAPK, and TNF-α NF‐κB pathways therapeutic and anpreventive targets in inflammatory MCP-1, IL-6, IL-1, and [16–18], and are eventually induces inflammatory response [19]. Thus, diseases. the AP-1, MAPK, and NF-κB pathways are therapeutic and preventive targets in inflammatory diseases. Pterostilbene (trans‐3,5‐dimethoxy‐4‐hydroxystilbene, Pte) (Figure 1A), a dimethylated analog Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, Pte) (Figure 1A), a dimethylated analog of resveratrol, is rich in in small small berries berries such such asas grapes grapes and and blueberries blueberries [20]. [20]. Pte Pte shows shows higher higher of resveratrol, is rich lipophilicity than resveratrol due to its methoxy substitution and therefore has a higher oral lipophilicity than resveratrol due to its methoxy substitution and therefore has a higher oral bioavailability compared compared with with resveratrol resveratrol [21]. [21]. Pte Pte has has been been reported reported to to have have anti-inflammatory anti‐inflammatory bioavailability 0 0 activity by others [22,23]; 4′‐methoxyresveratrol (3,5‐dihydroxy‐4′‐methoxystilbene, 4MR) (Figure 1B) activity by others [22,23]; 4 -methoxyresveratrol (3,5-dihydroxy-4 -methoxystilbene, 4MR) (Figure 1B) is a monomethylated analogue of resveratrol found in the Dipterocarpaceae and Gnetaceae, the anti‐ is a monomethylated analogue of resveratrol found in the Dipterocarpaceae and Gnetaceae, inflammatory activity of which has never been studied so far. the anti-inflammatory activity of which has never been studied so far. Molecules 2018, 23, 1148
Figure 1. Chemical structure of (A) pterostilbene (Pte) and (B) 4′‐methoxyresveratrol (4MR). Figure 1. Chemical structure of (A) pterostilbene (Pte) and (B) 40 -methoxyresveratrol (4MR). In the present study, we measured the anti‐inflammatory activities of Pte and 4MR using a In the present study, we measured the anti-inflammatory activities of Pte and 4MR using RAW264.7 macrophage model of inflammation. The concentrations of NO in the culture medium of a RAW264.7 macrophage model of inflammation. The concentrations of NO in the culture medium of Pte‐ and 4MR‐treated inflammatory cells were measured. The mRNA expression levels of inducible Pte- and 4MR-treated inflammatory cells were measured. The mRNA expression levels of inducible nitric oxide synthase (iNOS) iNOS as well as the proinflammatory cytokines MCP‐1, IL‐6, IL‐1β, and nitric oxide synthase (iNOS) iNOS as well as the proinflammatory cytokines MCP-1, IL-6, IL-1β, TNF‐α were evaluated by quantitative real‐time PCR (qPCR) after Pte and 4MR treatment. In and TNF-α were evaluated by quantitative real-time PCR (qPCR) after Pte and 4MR treatment. addition, the effects of Pte and 4MR on MAPK, AP‐1, and NF‐κB pathways were examined to explore In addition, the effects of Pte and 4MR on MAPK, AP-1, and NF-κB pathways were examined to their mechanism of action in LPS‐induced macrophages. explore their mechanism of action in LPS-induced macrophages. 2. Results 2. Results 2.1. Effects of Pte and 4MR on Cell Viability 2.1. Effects of Pte and 4MR on Cell Viability The effects of Pte and 4MR on cell viability was examined in RAW264.7 cells. As shown in Figure The effects of Pte and 4MR on cell viability was examined in RAW264.7 cells. As shown in Figure 2, 2, cells treated with Pte at 0–10 μM or 4MR at 0–30 μM for 24 h showed no significant cytotoxicity (p cells treated with Pte at 0–10 µM or 4MR at 0–30 µM for 24 h showed no significant cytotoxicity ≥ 0.05). Thus, 5 μM of Pte and 4MR was used for the subsequent experiments to exclude the possibility (p ≥ 0.05). Thus, 5 µM of Pte and 4MR was used for the subsequent experiments to exclude the that the inhibitory effect of Pte and 4MR on LPS‐induced inflammation was a result of cytotoxicity possibility that the inhibitory effect of Pte and 4MR on LPS-induced inflammation was a result of caused by cell viability reduction. cytotoxicity caused by cell viability reduction.
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Figure 2. Crystal violet assay showed the effect of (A) Pte and (B) 4MR on cell viability. Cells were Figure 2. Crystal violet assay showed the effect of (A) Pte and (B) 4MR on cell viability. Cells were Figure 2. Crystal violet assay showed the effect of (A) Pte and (B) 4MR on cell viability. Cells were treated with 0–20 μM of Pte and 0–40 μM 4MR of for 24 h. The data were representative of the three treated with 0–20 μM of Pte and 0–40 μM 4MR of for 24 h. The data were representative of the three treated with 0–20 µM of Pte and 0–40 µM 4MR of for 24 h. The data were representative of the three independent experiments and presented as the mean ± SEM. ### p
