The Antioxidant and Anti-Inflammatory Activities of 8-Hydroxydaidzein (8-HD) in Activated Macrophage-Like RAW264.7 Cells

8-Hydroxydaidzein (8-HD) is a daidzein metabolite isolated from soybeans. This compound has been studied for its anti-proliferation, depigmentation, and antioxidant activities. However, the anti-inflammatory activities of 8-HD are not well-understood. Through its antioxidant effects in ABTS and DPPH assays, 8-HD reduces the production of sodium nitroprusside (SNP)-induced radical oxygen species (ROS). By triggering various Toll-like receptors (TLRs), 8-HD suppresses the inflammatory mediator nitric oxide (NO) without cytotoxicity. We examined the regulatory mechanism of 8-HD in lipopolysaccharide (LPS)-induced conditions. We found that 8-HD diminishes inflammatory gene expression (e.g., inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α) by regulating the transcriptional activities of nuclear factor (NF)-κB and activator protein 1 (AP-1). To find the potential targets of 8-HD, signaling pathways were investigated by immunoblotting analyses. These analyses revealed that 8-HD inhibits the activation of TAK1 and that phosphorylated levels of downstream molecules decrease in sequence. Together, our results demonstrate the antioxidant and anti-inflammatory actions of 8-HD and suggest its potential use in cosmetics or anti-inflammatory drugs.


Introduction
The human immune system comprises two arms: innate and adaptive immunity. Innate immunity is the first barrier faced by invading pathogens. It recognizes pattern-associated molecular patterns (PAMPs). Adaptive immunity removes pathogens in the late phases of infection and is accompanied by immunological memory [1,2]. PAMPs are recognized by specific pattern-recognition receptors (PRRs). These include the NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and Toll-like receptors (TLRs). In particular, TLRs recognize a wide range of PAMPs, including lipids, proteins, glycans, and nucleic acids. TLRs therefore serve a pivotal role in inflammation. Each TLR recognizes specific ligands [2,3].

Antioxidant Effects of 8-HD
We evaluated the antioxidant effects of 8-HD using 2,2′-azino-bis(3-ethylbenzothiazoline-6sulphonic acid) diammonium salt (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. In the ABTS assays, 8-HD cleared ABTS radicals, even at low concentrations ( Figure 2a). In the DPPH assays, free radicals were scavenged in a dose-dependent manner (Figure 2b). The IC50 values for each experiment were 2.19 μM and 58.93 μM, respectively. Additionally, we treated a keratinocyte cell line (HaCaT cells) with SNP, a NO-releasing reagent. We then examined the effect of 8-HD on NO production. 8-HD slightly decreased the amount of NO without affecting cell viability (Figure 2c,d). Figure 2 shows the antioxidant effects of 8-HD, both in cells and in cell-free systems.

Effect of 8-HD on Nitric Oxide Production
Next, we investigated the immunomodulatory effects of 8-HD in a macrophage cell line (RAW264.7 cells). We induced RAW264.7 cells using different stimuli: lipopolysaccharide (LPS, a TLR4 inducer); polyinosinic-polycytidylic acid (poly[I:C], a TLR3 inducer); and peptidoglycan (PGN, a TLR2 inducer). 8-HD suppressed nitric oxide (NO) production without cytotoxicity when cells were stimulated by each inducer (Figure 3a,c). To increase the reliability of the NO assay, we used L-NAME as a positive control. L-NAME also decreased NO production without cytotoxicity (Figure 3b,d). These results suggest that 8-HD suppresses various TLR agonists.

Effect of 8-HD on Nitric Oxide Production
Next, we investigated the immunomodulatory effects of 8-HD in a macrophage cell line (RAW264.7 cells). We induced RAW264.7 cells using different stimuli: lipopolysaccharide (LPS, a TLR4 inducer); polyinosinic-polycytidylic acid (poly[I:C], a TLR3 inducer); and peptidoglycan (PGN, a TLR2 inducer). 8-HD suppressed nitric oxide (NO) production without cytotoxicity when cells were stimulated by each inducer (Figure 3a,c). To increase the reliability of the NO assay, we used L-NAME as a positive control. L-NAME also decreased NO production without cytotoxicity (Figure 3b,d). These results suggest that 8-HD suppresses various TLR agonists.  Figure 3 shows that 8-HD suppresses the inflammatory mediator production controlled by multiple TLR agonists. We deciphered the regulatory mechanism of 8-HD in controlling TLR4mediated inflammatory responses. To better understand the regulation of inflammation by 8-HD at the transcriptional level, we isolated mRNA and conducted semi-quantitative PCR to determine the presence of representative pro-inflammatory mediators (iNOS, COX-2, and TNF-α). Under the LPS challenge, 8-HD significantly reduced the expression of iNOS and TNF-α; COX-2 was only slightly affected by 8-HD ( Figure 4a). Next, we examined levels of phosphorylated, inflammation-related transcription factors over time. Phosphorylated transcription factors translocate to the nucleus [22][23][24]. Figure 4b shows that phosphorylation of the NF-κB subunits (p65 and p50) was regulated by 8-HD at 15 and 30 min, respectively. Regarding the AP-1 transcription factor, it was found that 8-HD can decrease the phosphorylation level of c-Fos but not c-Jun at 15 min ( Figure 4c). These results suggest that 8-HD can regulate inflammation by suppressing the transcriptional activities of NF-κB and AP-1. (c,d) The cytotoxic effects of 8-HD and L-NAME on RAW264.7 and HEK293T cells were tested by MTT assay; ## p < 0.01 versus a normal group (untreated group); ** p < 0.01 versus a control group (induced group). Figure 3 shows that 8-HD suppresses the inflammatory mediator production controlled by multiple TLR agonists. We deciphered the regulatory mechanism of 8-HD in controlling TLR4-mediated inflammatory responses. To better understand the regulation of inflammation by 8-HD at the transcriptional level, we isolated mRNA and conducted semi-quantitative PCR to determine the presence of representative pro-inflammatory mediators (iNOS, COX-2, and TNF-α). Under the LPS challenge, 8-HD significantly reduced the expression of iNOS and TNF-α; COX-2 was only slightly affected by 8-HD (Figure 4a). Next, we examined levels of phosphorylated, inflammation-related transcription factors over time. Phosphorylated transcription factors translocate to the nucleus [22][23][24]. Figure 4b shows that phosphorylation of the NF-κB subunits (p65 and p50) was regulated by 8-HD at 15 and 30 min, respectively. Regarding the AP-1 transcription factor, it was found that 8-HD can decrease the phosphorylation level of c-Fos but not c-Jun at 15 min (Figure 4c). These results suggest that 8-HD can regulate inflammation by suppressing the transcriptional activities of NF-κB and AP-1.

Anti-Inflammatory Effects of 8-HD on NF-κB and AP-1 Signaling
To investigate the way in which 8-HD inhibits NF-κB and AP-1 transcriptional activities, various signaling pathways were analyzed by immunoblotting. First, we assessed the NF-κB signaling molecules IκBα and IKKα/β, which are important for the nuclear translocation of NF-κB via the degradation of IκBα. Interestingly, the NF-κB inhibitory protein IκBα was not regulated by 8-HD (Figure 5a). This result indicates that 8-HD is not able to suppress the transcriptional activities of NF-κB (Figures 4b and 5a), due the absence of IκBα degradation. For the AP-1 signaling pathway, however, 8-HD inhibited the activation of ERK and JNK at 5 min (Figure 5b). We therefore analyzed certain upstream molecules of ERK and JNK at earlier time points. The phosphorylated form of MEK1/2 was clearly decreased at 2 and 3 min. 8-HD blocked the activity of MKK4 at 3 min, and also blocked the activity of MKK7 at 2, 3, and 5 min. The activity of TAK1, a common upstream molecule of the MAPKKs, was also inhibited by 8-HD (Figure 5c).

Anti-Inflammatory Effects of 8-HD on NF-κB and AP-1 Signaling
To investigate the way in which 8-HD inhibits NF-κB and AP-1 transcriptional activities, various signaling pathways were analyzed by immunoblotting. First, we assessed the NF-κB signaling molecules IκBα and IKKα/β, which are important for the nuclear translocation of NF-κB via the degradation of IκBα. Interestingly, the NF-κB inhibitory protein IκBα was not regulated by 8-HD (Figure 5a). This result indicates that 8-HD is not able to suppress the transcriptional activities of NF-κB (Figures 4b and 5a), due the absence of IκBα degradation. For the AP-1 signaling pathway, however, 8-HD inhibited the activation of ERK and JNK at 5 min (Figure 5b). We therefore analyzed certain upstream molecules of ERK and JNK at earlier time points. The phosphorylated form of MEK1/2 was clearly decreased at 2 and 3 min. 8-HD blocked the activity of MKK4 at 3 min, and also blocked the activity of MKK7 at 2, 3, and 5 min. The activity of TAK1, a common upstream molecule of the MAPKKs, was also inhibited by 8-HD (Figure 5c). To confirm the inhibitory effect of 8-HD on TAK1, we transfected a TAK1 construct into HEK293T cells. We then screened for the downstream molecules MEK1/2, MKK4, and MKK7. Consistent with our previous results, the activities of MEK1/2, MKK4, and MKK7 were downregulated (Figure 5d). The immunoblotting results showed that 8-HD targets TAK1 and NF- To confirm the inhibitory effect of 8-HD on TAK1, we transfected a TAK1 construct into HEK293T cells. We then screened for the downstream molecules MEK1/2, MKK4, and MKK7. Consistent with our previous results, the activities of MEK1/2, MKK4, and MKK7 were downregulated (Figure 5d).

8-HD is known to have antioxidant, anti-proliferation, and depigmentation bioactivities
The antioxidant effects of 8-HD were evaluated by various methods. Using the Fenton system, the xanthine oxidase system, and FRAP assays, G. Rimbach et al., and JS Park et al. observed that 8-HD scavenges hydroxyl radicals and superoxide, and reduces Fe 3+ to Fe 2+ [17,19]. We also confirmed the antioxidant effects of 8-HD using DPPH and ABTS assays. We observed that 8-HD significantly scavenges free radicals (Figure 2a,b). Moreover, we proved that 8-HD reduces cell-mediated radical production [17,19]. In fact, 8-HD has a potent ability to decrease various kinds of free radicals. It is worth noting that 8-HD is absorbed well during oral administration, showing antioxidant effects in vivo [18]. For these reasons, 8-HD has the potential to be used in drugs and health supplements.

Discussion
8-HD is known to have antioxidant, anti-proliferation, and depigmentation bioactivities [16,19,21]. Here, we further confirmed the antioxidant and anti-inflammatory effects of 8-HD. 8-HD suppresses the inflammatory responses triggered by different TLR ligands (Figure 3a). We explored the regulatory mechanisms of 8-HD, specifically in terms of the NF-κB and AP-1 inflammatory signaling pathways. 8-HD downregulates c-Fos transcriptional activity (Figure 4b,c). TAK1 was revealed as the target protein of 8-HD (Figure 5c,d).
The antioxidant effects of 8-HD were evaluated by various methods. Using the Fenton system, the xanthine oxidase system, and FRAP assays, G. Rimbach et al., and JS Park et al. observed that 8-HD scavenges hydroxyl radicals and superoxide, and reduces Fe 3+ to Fe 2+ [17,19]. We also confirmed the antioxidant effects of 8-HD using DPPH and ABTS assays. We observed that 8-HD significantly scavenges free radicals (Figure 2a,b). Moreover, we proved that 8-HD reduces cell-mediated radical production [17,19]. In fact, 8-HD has a potent ability to decrease various kinds of free radicals. It is worth noting that 8-HD is absorbed well during oral administration, showing antioxidant effects in vivo [18]. For these reasons, 8-HD has the potential to be used in drugs and health supplements.
We determined that 8-HD regulates TAK1 activity. Consequently, the transcriptional activities of the MAPKs and AP-1 are blocked (Figure 4c,d). In the case of NF-κB signaling, 8-HD inhibited the transcriptional activities of p65 and p50 (Figure 4b), but we did not observe a change in the phosphorylated forms of IκBα or IKKα/β (Figure 5a). In addition to the classical IKK/IκBα/NF-κB pathway, alternative pathways have been proposed-MAPK cascades could regulate the activation of NF-κB, or other kinases could phosphorylate the NF-κB subunits. ERK could act upstream of NF-κB, regulating its DNA-binding affinity. It is known that JNK and p38 are involved in cytoplasmic NF-κB activation and control its activity in the nucleus [27,28]. This suggests that 8-HD could regulate the transcriptional activation of NF-κB and AP-1 by inhibiting TAK1 and/or the MAPKs, as summarized in Figure 6. Apart from the IKKs, there are reports of other kinases that phosphorylate NF-κB. For example, p50 could be phosphorylated by protein kinase A (PKA) or Chk1, affecting its DNA-binding affinity. In the case of p65, GSK3β can phosphorylate the serine at position 468 [29]. The ability of 8-HD to inhibit the activities of these kinases should be addressed in future studies. Nonetheless, it is expected that the inhibitory activity of 8-HD on NF-κB activity is marginal, since the phosphorylation and degradation of IκBα are major pathways to activate NF-κB. Therefore, related points will be further studied to clarify this issue.
The industrial development of natural products is currently receiving much attention. Natural products have traditionally been a source of new drugs [30]. For example, eupatilin (sold as Stillen ® , a 95% ethanol extract of Artemisia asiatica Nakai) is widely prescribed for gastritis and peptic ulcers in Korea [31,32]. In addition, the demand for natural and eco-friendly cosmetics is increasing [33,34]. Cosmetic ingredients with anti-inflammatory and antioxidant effects have been shown to reduce irritation [35,36]. Researchers have, in fact, tried to develop natural products for use across many industries [34,37,38]. In light of this trend, it is worth noting that 8-HD originates from soybeans. Like soybeans, 8-HD has potent antioxidant and anti-inflammatory effects. Used synthetically, 8-HD has the potential to be incorporated into many new products, including drugs, cosmetics, and health supplements. In addition, since our team has focused on its development as an immunomodulator targeted at skin inflammation, it is expected that this strategy will overcome various limits caused by liver metabolism and marginal in vivo absorption. Therefore, related studies regarding the pharmacokinetic and pharmacodynamics properties of 8-HD under topical and oral administration conditions will continuous investigate this possibility.

DPPH Assays
DPPH decolorimetric assays were performed to examine the scavenging effect of 8-HD [39,40]. A mixture of 8-HD (0-50 µM) and 250 µM DPPH was incubated at 37 • C for 30 min. Ascorbic acid (500 µM) was used as a positive control. After incubation, the absorbance at 517 nm of each sample was measured by spectrophotometry. The DPPH scavenging effect was expressed as the percent inhibition: where A 0 is the absorbance of DPPH, and A 1 is the absorbance of the sample.

ABTS Assays
ABTS scavenging assays were performed with modifications [41]. A mixture of 7.4 mM ABTS and 2.4 mM potassium persulfate (at a ratio of 1:1) was incubated at room temperature overnight to generate ABTS radical cations (ABTS•+). Solutions of 8-HD and ABTS were loaded into 96-well plates at a ratio of 1:1. Ascorbic acid (50 µM) was used as a positive control. After 30 min of incubation at 37 • C, the absorbance of each fraction was measured at 730 nm. The ABTS scavenging effect was expressed as a percentage: where A 0 is the absorbance of ABTS, and A 1 is the absorbance of the sample.

Preparation of mRNA and Semi-Quantitative PCR
To quantify the expression of pro-inflammatory cytokines, RAW264.7 cells were pre-treated with 8-HD for 30 min. Cells were then exposed to LPS for 6 h. Total RNA was isolated with TRIzol reagent according to the manufacturer's instructions. Semi-quantitative PCR was conducted as previously described [43].

Statistical Analyses
The results were analyzed using an ANOVA/Scheffe's post hoc test or the Kruskal-Wallis/Mann-Whitney tests. A p-value < 0.05 was considered statistically significant. All of the statistical tests were carried out using the computer program SPSS (SPSS Inc., Chicago, IL, USA).