INTRODUCTION

Inflammation is a complex biological response of body tissues against harmful stimulations. However, an excessive inflammatory response dysregulates tissue functions and eventually leads to severe tissue damages. Macrophages are major inflammatory and immune effector cells, which can be activated by exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines [1, 2]. During inflammation, pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, and inflammatory mediators, such as nitric oxide (NO) and prostaglandin (PG) E2, are largely produced by activated macrophages [3, 4]. Excessive production of these molecules can lead to the development of chronic diseases, such as inflammatory arthritis, cancer, and atherosclerosis [5,6,7]. Hence, substances inhibiting the production of these molecules would be developed as therapeutic agents for inflammatory diseases.

Nuclear factor-kappaB (NF-κB) is a critical regulator of inflammation as well as cell survival [8]. In response to inflammatory stimuli, inhibitory-κB kinase (IKK) is activated and initiates the phosphorylation and degradation of inhibitor of κB (IκB), which is bound to NF-κB. After dissociating from IκB, NF-kB translocates into the nucleus and promotes the transcriptions of pro-inflammatory mediators [9,10,11]. NF-κB is alternatively activated by mitogen-activated protein kinases (MAPKs), including p38 kinase, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) [12]. Recent studies have demonstrated that the phosphatidyl inositol 3-kinase/Akt (PI3K/Akt) pathway is also responsible for the activation of NF-κB and subsequent expression of NF-κB-regulated inflammatory genes [8].

Heme oxygenase-1 (HO-1) is one major antioxidative enzyme, and it inhibits the secretion of pro-inflammatory mediators, including NO, PGE2, TNF-α, IL-6, and IL-1β as well as reactive oxygen species (ROS) by activated macrophages [13,14,15,16,17,18]. Nuclear transcription factor erythroid 2-related factor 2 (Nrf2) regulates the translation of HO-1. Upon oxidative stress challenge, Nrf2 translocates into the nucleus and binds to the antioxidant response element (ARE) promoter region of target genes such as HO-1 [19]. The anti-inflammatory reaction of HO-1 gene is exerted by suppressing the expression of pro-inflammatory mediators via NF-κB inactivation [20].

During our recent studies aiming at developing anti-inflammatory compounds from brown algae, we found sargahydroquinoic acid (SHQA) is a major component in one marine brown alga species, Sargassum macrocarpum, which was comprised to be 37.6% of its ethanolic extract. SHQA selectively accelerated cerebral blood flow through dilatation of the basilar artery without lowering systemic blood pressure [21]. It also inhibited TNF-α-induced MMP expression and age-related inflammation by suppressing the NF-kB pathway [22]. In this study, we found that SHQA showed strong anti-inflammatory activities at physiological concentrations (0.4-0.8 μM). This led us to elucidate the anti-inflammatory actions of SHQA and its molecular mechanism using LPS-activated macrophages.

MATERIALS AND METHODS

Reagents

Bovine serum albumin (BSA), 4,6′-diamidino-2-phenylindole (DAPI), 2′,7′-dichlorofluorescein diacetate (DCF-DA), dimethyl sulfoxide (DMSO), LPS, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). CellTiter96 AQueous One Solution Cell Proliferation assay kit, dual luciferase assay kit, pRL-TK DNA, and superscript reverse transcriptase were obtained from Promega (Madison, WI, USA). Murine NF-κB promoter/luciferase DNA was purchased from Stratagene (La Jolla, CA, USA). Primary antibodies were purchased from Abcam (Danvers, MA, USA). Alexa Fluor 488-conjugated secondary antibody, TRIzol, and Lipofectamine/Plus from Life Technology (Carlsbad, CA, USA) were used. Enhanced chemiluminescence detection kit was purchased from GE Healthcare Bio-Sciences (Piscataway, NJ, USA).

Isolation of SHQA

Sargassum macrocarpum was collected along the coast of Busan, South Korea in April 2018. Specimen identity was confirmed by an algologist (N.G. Kim), at the Department of Marine Biology and Aquaculture, Gyeongsang National University, South Korea. The isolation of SHQA had been described before [23]. In short, dried seaweed (1.0 kg) was extracted twice with 80% (v/v) ethanol (6 L/each). The ethanolic extract (102 g) was obtained by concentration under reduced pressure. The separation and elution conditions can be referred to [23]. The structure and chromatogram of SHQA are shown in Fig. 1.

Fig. 1
figure 1

HPLC chromatogram and structure of sargahydroquinoic acid.

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Spectrometric Analysis of SHQA

1H- and 13C-NMR spectra were determined on a JNM ECP-400 spectrometer (JEOL, Japan), using CD3OD with tetramethylsilane as an internal standard. Heteronuclear multiple quantum correlation and heteronuclear multiple bond correlation spectra were recorded using pulsed-field gradients.

Structure Identification of SHQA

C27H36O4 (MW = 424): 1H-NMR (CD3OD, 400 MHz); 3.25 (2H, d, J = 7.4 Hz, H-1), 5.29 (1H, dt, J = 7.3 and 1.4 Hz, H-2), 2.08 (2H, m, H-4), 2.12 (2H, m, H-5), 5.14 (1H, t, J = 7.0 Hz, H-6), 2.08 (2H, m, H-8), 2.50 (2H, dt, J = 7.0 and 7.0, H-9), 5.83 (1H, t, J = 7.3 Hz, H-10), 2.21 (2H, t, J = 7.7 Hz, H-12), 2.12 (2H, m, H-13), 5.07 (1H, tt, J = 7.3 and 1.4 Hz, H-14), 1.65 (3H, s, CH3-16), 1.56 (3H, s, CH3-17), 1.59 (3H, s, CH3-19), 1.70 (3H, s, H-20), 6.38 (2H, brs, H-3′ and H-5′), 2.14 (3H, s, aromatic-CH3). 13C-NMR (CD3OD, 100 MHz); 29.6 (C-1), 124.1 (C-2), 136.8 (C-3), 40.9 (C-4), 27.6 (C-5), 125.96 (C-6), 135.5 (C-7), 40.3 (C-8), 28.9 (C-9), 142.7 (C-10), 132.9 (C-11), 36.0 (C-12), 29.0 (C-13), 124.8 (C-14), 133.3 (C-15), 25.9 (C-16), 17.8 (C-17), 171.7 (C-18), 15.96 (C-19), 16.2 (C-20), 146.5 (C-1′), 131.3 (C-2′), 114.5 (C-3′), 151.4 (C-4′), 115.7 (C-5′), 127.5 (C-6′), 16.9 (aromatic-CH3).

Cell Culture and Sample Treatment

Murine macrophages cell line RAW 264.7 cells (ATCC, Rockville, MD, USA) were maintained in Dulbecco’s modified Eagle medium containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin sulfate in a humidified incubator with 5% CO2 air condition. Cells were pretreated with SHQA at indicated concentrations for 1 h followed by stimulating with LPS (1 μg/mL).

Cytotoxicity Assay

Cell viability was determined using the CellTiter96® AQueous One Solution Cell Proliferation assay kit. Cells were seeded at a density of 5 × 104 cells/well into 96-well. After 24 h, cells were pretreated with different concentrations of SHQA for 1 h then stimulated with LPS (1 μg/mL). Twenty-four hours later, the culture medium was replaced with a mixture of 95 μL fresh culture medium and 5 μL MTS solution. After 1 h, the absorbance reading at 490 nm was acquired using a microplate reader (Glomax Multi Detection System, Promega).

Cyclooxygenase-2 Activity

Cyclooxygenase (COX)-2 activity was analyzed using an assay kit from Cayman Chemical Company (Ann Arbor, MI, USA). Cell pellets were homogenized in cold buffer (0.1 M Tris-HCl, pH 7.8 containing 1 mM EDTA) and centrifuged at 10,000×g for 15 min. The supernatant was used for COX-2 enzyme solution. One hundred microliters of assay buffer, 10 μL of hemin, and 10 μL of supernatant were mixed and then 10 μL of different concentrations of SHQA was added and mixed. The reaction mixture was incubated for 5 min at 25°C, and then 20 μL N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and 20 μL of arachidonic acid solution were added. After incubating for 5 min at 25°C, the absorbance at 590 nm was measured. The values were normalized by total cell number

Intracellular ROS Level

The intracellular ROS level was measured using fluorescent probe DCF-DA. Cells were seeded into a black 96-well plate at a density of 5 × 104/well. On the second day, the cells were co-treated with SHQA and LPS. Two hours later, the cells were then treated with 20 μM DCF-DA. Thirty minutes later, the fluorescence intensity was acquired as described before [24].

Measurement of NO, PGE2, TNF-α, IL-1β, and IL-6

Cells (seeded at 10 × 105 cells/well) were pretreated with SHQA (0-0.8 μM) for 1 h followed by stimulation with LPS for 24 h. Culture media were collected after centrifuging at 2000×g for 10 min and stored at −75°C before analysis. The nitrite concentration was measured as an indicator of NO production, using the Griess reaction [17]. Levels of TNF-α, IL-1β, and IL-6 were quantitatively determined using ELISA kits from R&D Systems (Minneapolis, MN, USA). PGE2 concentrations in culture media were measured using an enzyme immunoassay (EIA) kit from Cayman Chemical, according to the manufacturer’s recommended protocol.

Reverse Transcription-Polymerase Chain Reaction

RAW 264.7 cells plated in a 6-well culture plate were pretreated with SHQA for 1 h and stimulated with LPS for 6 h. Total RNA was isolated using TRIzol reagent and cDNA was synthesized with oligo-dT primer and M-MLV reverse transcriptase. PCR was conducted with corresponding primers (Supplementary Table 1). Images were acquired through EZ-Capture II (ATTO, Tokyo, Japan) and densitometric analysis was conducted using the CS analyzer ver. 3.00 software (ATTO).

Immunocytochemical Analysis

RAW 264.7 cells were seeded on glass coverslips in 6-well plates and cultured for 24 h. After incubation with SHQA for 1 h, cells were stimulated with LPS (1 μg/mL) for 30 min. The specific procedure for immunofluorescence analysis of NF-kB and Nrf2 has been described in our previous paper [25].

Cytosolic and Nuclear Extract Preparation

RAW 264.7 cells in 60-mm cell culture plates were pretreated with SHQA for 1 h and then stimulated with LPS for 30 min. Cytosolic and nucleus fractions were separated as previously described [23].

Transfection and Luciferase Assay

pNF-κB promoter/luciferase DNA (2 μg) along with control pRL-TK DNA (40 ng) was transfected into RAW 264.7 cells using Lipofectamine Plus reagents. Thirty-six hours later, cells were treated with SHQA for 1 h then stimulated with LPS (1 μg/mL). After 6 h, cell wells were washed with ice-cold PBS twice and cell lysate was harvested with 70 μL of lysis buffer (0.5 mM HEPES pH 7.8, 1% Triton N-101, 1 mM CaCl2, and 1 mM MgCl2). Luciferase activity was determined with a luciferase assay kit. The luciferase activity was normalized with the expression of control pRL-TK.

Western Blot Analysis

Cells were pretreated with 0.4, 0.6, and 0.8 μM of SHQA for 1 h then challenged with LPS (1 μg/mL) for indicated time durations. The specific procedures for Western blot had been described before [25].

In Vivo Pro-inflammatory Cytokine Assay

Animal studies were conducted with the experimental protocols and procedures approved by the Animal Ethics Committee of the Pukyong National University. Mice were randomly grouped (n = 3). Mice were given SHQA (10 and 20 mg/kg of body weight) with oral administration for 4 consecutive days. Mice from the control group were given 20% propylene glycol at equal volume. One hour after the last oral administration, mice were intraperitoneally injected with LPS (2.0 mg/kg). After 2 h, blood was collected by cardiac puncture, and serum was obtained by centrifuging at 800×g for 20 min. Concentrations of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, MCP-1, and IL-17 in the serum samples were measured.

Statistical Analysis

Quantitative results were expressed as mean ± standard deviation (SD) from at least three independent experiments unless otherwise indicated. Data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s test for post hoc analysis. Differences with a P value less than 0.05 were considered statistically significant. All analyses were carried out using SPSS, version 10.07 (SPSS Inc., Chicago, IL, USA).

RESULTS

Effect of SHQA on Secretions of NO and PGE2

To evaluate the effect of SHQA on NO production in LPS-induced macrophages, we measured the nitrite concentrations in the culture media. As shown in Fig. 2a, SHQA dose-dependently reduced LPS-induced NO secretion, and IC50 was estimated to be 0.49 ± 0.03 μM (P < 0.05). SHQA also significantly inhibited PGE2 secretion (IC50 = 1.32 ± 0.12 μM, P < 0.05, Fig. 2b). MTS assay revealed that SHQA caused no cytotoxicity at concentration up to 1.6 μM in RAW 264.7 cells (Supplementary Fig. 1). Thus, the inhibited secretions of NO and PGE2 by SHQA were not due to cytotoxicity.

Fig. 2
figure 2

Effect of SHQA on secretions of NO and PGE2 in LPS-activated RAW 264.7 cells. Cells were pretreated with different concentrations of SHQA (0.4, 0.6, 0.8 μM) for 1 h then stimulated with LPS for 24 h. The cultured media were used to measure a) NO and b) PGE2 concentrations. Data are presented as mean ± SD. # P < 0.05, compared to the control group; * P < 0.05, compared to the LPS- treated group.

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Effect of SHQA on iNOS and COX-2 Expressions

To determine whether the suppression of NO and PGE2 production by SHQA was associated with reduced iNOS and COX-2 expressions, we assessed the protein and mRNA expression levels of iNOS and COX-2. As shown in Fig. 3a, SHQA markedly suppressed the LPS-mediated increase of the iNOS protein level. However, COX-2 protein expression was not altered by SHQA. Consistent with protein results, SHQA inhibited LPS-induced iNOS transcription, whereas COX-2 mRNA level was not reduced by SHQA (Fig. 3). Interestingly, SHQA did not inhibit COX-2 expression, despite the reduced PGE2 production. To clarify this discrepancy, we analyzed the effect of SHQA on COX-2 activity. As shown in Fig. 3b, SHQA strongly inhibited COX-2 activity in a dose-dependent manner.

Fig. 3
figure 3

Inhibitory effect of SHQA on expression of iNOS and COX-2 in LPS-activated RAW 264.7 cells. Cells were pretreated with SHQA for 1 h then stimulated with LPS for 16 and 24 h to determine mRNA and protein levels of a) iNOS and b) COX-2, respectively. c) Cells were pretreated with indicated concentrations of SHQA for 1 h then stimulated with LPS for 24 h. COX-2 activity was determined by COX-2 activity assay kit. COX-2 activity is normalized by total cell number. Data are presented as mean ± SD. #P < 0.05, compared to the control group; *P < 0.05, compared to the LPS- treated group.

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Effect of SHQA on the Production of Pro-inflammatory Cytokines

Pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, are secreted at the early stage of the inflammatory response and recognized as key biomarkers of inflammation. We quantified the concentrations of these cytokines in the media using ELISA kits. As shown in Fig. 4, LPS-induced TNF-α, IL-1β, and IL-6 secretions were markedly decreased by SHQA in a dose-dependent manner. These results showed that SHQA suppressed LPS-induced pro-inflammatory cytokine secretions, indicating that SHQA inhibits the initial phase of the LPS-stimulated inflammatory response.

Fig. 4
figure 4

Effect of SHQA on secretion of pro-inflammatory cytokines in LPS-stimulated RAW 264.7 cells. Cells were pretreated with SHQA for 1 h and then stimulated with LPS for 24 h. a) TNF-α, b) IL-1β, and c) IL-6 protein levels in the cultured media were determined by ELISA. The data are presented as mean ± SD. *P < 0.05 compared to the LPS- treated group.

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Effect of SHQA on LPS-Induced NF-κB Activation

NF-κB, as an important transcription factor, regulates the pro-inflammatory cytokines in LPS-activated macrophages. The effects of SHQA on the transcriptional activation of NF-κB in LPS-treated macrophages were analyzed. As shown in Fig. 5a, SHQA inhibited LPS-induced NF-κB promoter activity (P < 0.05). To further determine the effect of SHQA on NF-κB, cells were pretreated with SHQA, and proteins associated with NF-κB activation were determined using Western blot. After the LPS challenge, IKKβ was markedly phosphorylated and the phosphorylation was dose-dependently suppressed by SHQA (Fig. 5b). LPS-induced degradation of IκB-α was also attenuated by SHQA pretreatment. Moreover, the elevated nuclear NF-κB level, caused by LPS, was dose-dependently suppressed by SHQA. These results demonstrated that SHQA effectively inhibited the nuclear translocation and activation of NF-κB by suppressing the phosphorylation of IKKβ and the degradation of IκB-α.

Fig. 5
figure 5

Effect of SHQA on NF-κB activation in LPS-stimulated RAW 264.7 cells. a) Cells were transfected with NF-κB promoter-containing luciferase DNA along with control pRL-TK DNA for 40 h. Transfected cells were treated with LPS in the absence or presence of SHQA for 6 h. b) Cells pretreated with SHQA for 1 h were stimulated with LPS for 30 min. Cytosolic and nuclear fractions were prepared and used for Western blot. Data are presented as mean ± SD. #P < 0.05, compared to the control group. *P < 0.05, compared to the LPS-treated group.

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Effect of SHQA on the Phosphorylation of Signaling Proteins

To further elucidate the possible molecular mechanisms of SHQA on NF-κΒ inactivation in LPS-activated macrophages, we examined the effect of SHQA on the phosphorylation of Akt and MAPKs, which are implicated with NF-κΒ activation. As shown in Fig. 6, SHQA did not affect the phosphorylation of JNK, whereas SHQA dose-dependently suppressed the phosphorylation of Akt, ERK, and p38 MAPK in LPS-activated cells. These data additionally suggested that SHQA regulates the activity of NF-κB, at least partially, through inhibiting Akt, ERK, and p38 MAPK phosphorylation.

Fig. 6
figure 6

Effects of SHQA on phosphorylations of Akt and MAPKs in LPS-stimulated RAW 264.7 cells. Cells were pretreated with SHQA for 1 h then stimulated with LPS for 30 min. Cell lysates were separated with SDS-PAGE and detected by Western blot. #P < 0.05, compared to the control group. *P < 0.05, compared to the LPS- treated group.

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Effect of SHQA on the Regulation of HO-1 and Nrf2

Enhanced endogenous HO-1 leads to cellular protection against oxidative damage. Thus, we determined whether SHQA induces the protein and mRNA expressions of HO-1. As shown in Fig. 7a, the productions of HO-1 protein and mRNA were induced by LPS stimulation and further augmented dose-dependently by SHQA. Moreover, SHQA remarkably inhibited LPS-induced ROS production in a dose-dependent manner (Fig. 7b).

Fig. 7
figure 7

Effect of SHQA on regulation of HO-1 and Nrf2. a) Cells were pretreated with SHQA for 1 h then stimulated with LPS for 16 or 24 h to determine mRNA or protein levels, respectively. b) Cells were treated with SHQA and LPS simultaneously for 2 h. The cells were then treated with 20 μM DCF-DA for 30 min at 37°C. Data are presented as mean ± SD. #P < 0.05, compared to the control group. *P < 0.05, compared to the LPS-treated group.

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Anti-inflammatory Activity of SHQA in Mice

To investigate the in vivo anti-inflammatory activity of SHQA, the serum pro-inflammatory cytokine levels were determined by ELISA. Administration of SHQA for 4 days did not cause any changes in body weight, or serum AST and ALT levels in mice (data not shown). The LPS treatment group showed increased levels of pro-inflammatory cytokines such as TNF-α (Fig. 8a), IL-1β (Fig. 8b), IL-6 (Fig. 8c), and IL-17 (Fig. 8d) in their serum; however, SHQA administration for 4 days suppressed the elevation of these pro-inflammatory cytokines in serum.

Fig. 8
figure 8

Effect of SHQA on pro-inflammatory cytokines in serum of LPS-injected mice. Mice were administrated with indicated concentrations of SHQA for 4 days and intraperitoneally injected with LPS (2.0 mg/kg). The concentrations of a) TNF-α, b) IL-1β, c) IL-6, and d) IL-17 in the serum were measured by ELISA kits. Data are presented as mean ± SD (n = 3). #P < 0.05, compared to the control group. *P < 0.05, compared to the LPS-treated group.

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DISCUSSION

Health beneficial actions of S. macrocarpum have been associated with its high levels of meroterpenoids in the ethanolic extract. Recently we reported that ethanolic extract of S. macrocarpum showed anti-inflammatory activity in both in vivo and in vitro models [23, 26]. The quantitative analysis result showed that the content of SHQA was estimated to be 37.6% of the extract, suggesting that SHQA is a major compound in the extract of S. macrocarpum. Thus, we isolated SHQA from S. macrocarpum to investigate its anti-inflammatory mechanisms using RAW 264.7 macrophages. The anti-inflammatory effects of SHQA were, at least partly, attributed to the inhibition of the NF-κB signal pathway. Additionally, we found that Nrf2 and reduced cellular ROS level also play a critical role in inhibiting LPS-induced inflammatory response by SHQA.

SHQA selectively accelerated cerebral blood flow through dilatation of the basilar artery without lowering systemic blood pressure [21]. In a recent report, SHQA isolated from S. yezoense inhibited TNFα-induced MMP-2/-9 expression by suppressing AP-1 and NF-kB pathways via PPARα [22].

SHQA did not inhibit the production of COX-2, which is responsible for PGE2 production. Thus, we analyzed the inhibitory activity of SHQA on COX-2 activity and found that SHQA had strong COX-2 inhibitory activity (IC50, 0.46 ± 0.02 μM). Thus, we confirmed that the inhibition of PGE2 by SHQA was caused by its COX-2 inhibitory activity.

Adverse effects of excess NO are attributed to NO itself as an inflammatory factor and peroxynitrite that is produced by the reaction of NO and superoxide, leading to the oxidative damage of cellular molecules. Under pathophysiological conditions, a large amount of NO produced by iNOS provokes inflammatory progress and acts synergistically with pro-inflammatory cytokines [27]. Compounds capable of suppressing NO production may be attractive anti-inflammatory agents, and for this reason, the suppressive effects of natural compounds on NO production have been intensively studied for developing anti-inflammatory agents [23, 25, 28]. In this study, we found that SHQA remarkably inhibited iNOS protein and mRNA expressions in LPS-stimulated RAW 264.7 cells, leading to inhibition of NO production. PGE2 is also a key inflammatory mediator, whose production is primarily regulated by COX-2 [29]. Recent studies have shown that in vivo and in vitro treatments with natural compounds reduce inflammation by suppressing COX-2 [30]. In this study, SHQA directly inhibited the production of PGE2 through inhibiting COX-2 activity, but not COX-2 protein and mRNA expressions. LPS-injected mice are a classical animal model for evaluating potential anti-inflammatory agents because they show systemic inflammation [30]. Pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, are related to the systemic signal of inflammation [31]. Also, high levels of pro-inflammatory cytokines contribute to various inflammatory diseases such as atherosclerosis, liver disease, and inflammatory arthritis [7, 32]. SHQA suppressed the releases of TNF-α, IL-1β, and IL-6 in LPS-activated macrophages. Similarly, we found that SHQA suppressed the levels of IL-1β, IL-6, IL-17, and TNF-α in the serum of LPS-stimulated mice (Fig. 7), indicating SHQA would be a promising candidate of therapeutic agent for inflammatory diseases. This is, to our knowledge, the first study to address the inhibition of pro-inflammatory cytokines in vivo by SHQA.

NF-κB is a critical transcription factor in the regulation of inflammatory mediators and pro-inflammatory cytokines [33, 34]. In a normal condition, NF-κB is sequestered by IκB-α in the cytoplasm. LPS initiates the activation of NADPH oxidase in immune cells and provokes ROS production, leading to activation of NF-κB through phosphorylation of IKKβ and degradation of IκB-α. The free NF-κB moves from cytosol to the nucleus and controls the expression of inflammatory genes [35,36,37]. In this study, we demonstrated that SHQA strongly suppresses the protein levels of NF-κB in the nucleus and cytosol in LPS-treated cells, based on Western blot analysis. Moreover, SHQA effectively inhibits IKKβ phosphorylation and IκB-α degradation, suggesting that inhibition of NF-κB activation by SHQA is largely attributed by inhibition of phosphorylation of IKKβ and degradation of IκB-α.

Induction of HO-1 attenuates oxidative damage and shows anti-inflammatory properties, which was demonstrated by in vitro and in vivo inflammatory models [38, 39]. The anti-inflammatory mechanism of HO-1 can be explained by the enzymatic degradation of pro-inflammatory heme and the production of anti-inflammatory CO and biliverdin, which is subsequently converted into bilirubin, a strong antioxidant [38]. HO-1 is transcriptionally regulated by Nrf2, a basic transcription factor required for cell protection against oxidative stress. LPS-induced ROS activates Nrf2 and leads to the overproduction of HO-1 [40]. Nrf2-mediated enhanced HO-1 by lutein repressed inflammatory responses via downregulating NF-κB signaling in LPS-stimulated BV2 cells, indicating that HO-1 is associated with inhibition of inflammation [41, 42]. In the present study, we found that SHQA dose-dependently increased HO-1 expression via Nrf2 activation. This report is, to our best knowledge, the novel findings to address that SHQA accomplished anti-inflammatory exertion through Nrf2/HO-1 pathway in LPS-stimulated cells.

Cumulative information derived from experimental reports has proven that NF-κB and Nrf2 are key regulators for maintaining the balance of cellular redox status and responding to inflammation [43]. Both pathways share common effectors and regulatory proteins for their activation; however, they control opposite cellular processes that lead to either cytoprotection or pathological development [44]. As evidence of crosstalk between NF-κB and Nrf2, NF-κB activation is suppressed by various phytochemicals activating Nrf2; however, detailed mechanistic evidence of Nrf2 role on NF-κB signaling is still unclear [41]. PI3K/Akt and MAPKs are critical signaling proteins connected with the transcriptional regulation of NF-κB and Nrf2 by degrading their respective inhibitors, IκB-α and Keap1, respectively [45, 46]. Lutein suppressed inflammatory responses through attenuating NF-kB activation by inhibiting the phosphorylation of p38 MAPK, JNK, and Akt, while promoting Nrf2 activation by ERK phosphorylation in LPS-stimulated BV2 cells [42]. In this study, we found that the phosphorylation of Akt, ERK, and p38 was suppressed by SHQA, suggesting Akt and MAPK pathways are involved in the repressed LPS-induced NF-κB activation by SHQA.

In conclusion, we demonstrated that SHQA inhibited the production of NO, PGE2, and multiple cytokines, including TNF-α, IL-6, and IL-1β in LPS-activated RAW 264.7 cells. The anti-inflammatory action of SHQA is associated with the inhibition of the NF-κB pathway as well as the activation of the Nrf2 pathway. Moreover, SHQA suppressed PGE2 production by direct inhibition of COX-2 activity. Oral administration of SHQA alleviated systemic inflammation in mice via reducing circulatory pro-inflammatory cytokines. Verification of SHQA’s anti-inflammatory action in cellular and animal models will be beneficial to further the application of SHQA in the therapeutic agent for inflammatory diseases.