Wistin Exerts an Anti-Inflammatory Effect via Nuclear Factor-κB and p38 Signaling Pathways in Lipopolysaccharide-Stimulated RAW264.7 Cells

Inflammation is an immune response to cellular damage caused by various stimuli (internal or external) and is essential to human health. However, excessive inflammatory responses may be detrimental to the host. Considering that the existing drugs for the treatment of inflammatory diseases have various side effects, such as allergic reactions, stomach ulcers, and cardiovascular problems, there is a need for research on new anti-inflammatory agents with low toxicity and fewer side effects. As 4′,6-dimethoxyisoflavone-7-O-β-d-glucopyranoside (wistin) is a phytochemical that belongs to an isoflavonoid family, we investigated whether wistin could potentially serve as a novel anti-inflammatory agent. In this study, we found that wistin significantly reduced the production of nitric oxide and intracellular reactive oxygen species in lipopolysaccharide-stimulated RAW 264.7 cells. Moreover, wistin reduced the mRNA levels of pro-inflammatory enzymes (inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2)) and cytokines (interleukin (IL)-1β and IL-6) and significantly reduced the protein expression of pro-inflammatory enzymes (iNOS and COX-2). Furthermore, wistin reduced the activation of the nuclear factor-κB and p38 signaling pathways. Together, these results suggest that wistin is a prospective candidate for the development of anti-inflammatory drugs.


Introduction
Inflammation is the body's immune response and defense mechanism against tissue damage caused by exposure to harmful or toxic external agents, infections, and physical injuries [1]. Inflammation resulting from infection or injury is associated with inflammatory responses such as immune cell recruitment and accumulation, release of inflammatory mediators, and changes in blood vessel permeability [2]. Generally, there are two types of inflammation: acute and chronic. Acute inflammation is a rapid process that repairs quickly to minimize damage and restore tissue homeostasis [3]. However, in chronic conditions, the inflammatory response continues, resulting in severe organ damage [4]. Chronic inflammation promotes the progression of several diseases, including cardiovascular disease, inflammatory bowel disease, rheumatoid arthritis and diabetes [5]. Crohn's disease (CD) is the most prevalent IBD syndrome treated using 6-mercaptopurine (6-MP) and its prodrug azathioprine (AZA) [6]. As immunosuppressive drugs, 6-MP and AZA inhibit inflammation by blocking the body's immune response; however, they may cause disease recurrence and side effects, such as hepatotoxicity [6]. Sulfasalazine, a medication for patients with rheumatoid arthritis, has immunomodulatory properties and suppresses pro-inflammatory cytokines; however, it can cause gastrointestinal side effects, such as headache, dizziness, activity both in vitro and in vivo [32]. Compound 4',6-dimethoxyisooflavone-7-O-β-d-glucopyranoside (wistin), belonging to the isoflavone family, is an agonist of PPARγ and PPARα in adipocytes and hepatocytes, respectively [33,34]. However, its anti-inflammatory role has not been investigated. Therefore, in this study, we aimed to identify its antiinflammatory effects and elucidate the molecular mechanisms underlying its anti-inflammatory effects in LPS-stimulated RAW264.7 cells.

Effects of Wistin on Cell Viability in LPS-Induced RAW 264.7 Cells
Because isoflavones have been reported to be cytotoxic at high doses, we attempted to confirm whether wistin is cytotoxic at high concentrations [35]. The cytotoxicity of wistin (Figure 1a) on the viability of RAW 264.7 cells was evaluated using the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (MTT) assay. Cotreatment of wistin (50 μM, 100 μM, and 150 μM) and LPS (0.1 μg/mL) did not show cytotoxicity in RAW 264.7 at 24 h (Figure 1b). The cells were treated with the indicated concentrations of wistin (0, 50, 100, 150 μM) for 30 min prior to treatment with LPS (0.1 μg/mL) for 24 h, and then cell viability was examined using MTT assay. The data are presented as the means ± SD; n = 3.

Effects of Wistin on the Production of Pro-Inflammatory Mediators in LPS-Induced RAW 264.7 Cells
Prolonged inflammatory processes increase the production of nitric oxide (NO) and reactive oxygen species (ROS), leading to tissue dysfunction [36]. ROS can regulate proinflammatory gene expression, and NO is an important pro-inflammatory mediator in inflammatory signaling [37]. To examine the anti-inflammatory effects of wistin, we investigated the production of NO and ROS. Wistin showed a significant decrease in LPSinduced NO production in a dose-dependent manner compared to that in the control group ( Figure 2a). In addition, dose-dependent inhibition of intracellular ROS generation by wistin was identified using a microplate reader, fluorescence-activated cell sorting (FACS), and fluorescence microscopy (Figure 2b-e). These data suggest that wistin can reduce the production of pro-inflammatory mediators (NO and ROS) induced by LPS. for 30 min prior to treatment with LPS (0.1 µg/mL) for 24 h, and then cell viability was examined using MTT assay. The data are presented as the means ± SD; n = 3.

Effects of Wistin on the Production of Pro-Inflammatory Mediators in LPS-Induced RAW 264.7 Cells
Prolonged inflammatory processes increase the production of nitric oxide (NO) and reactive oxygen species (ROS), leading to tissue dysfunction [36]. ROS can regulate proinflammatory gene expression, and NO is an important pro-inflammatory mediator in inflammatory signaling [37]. To examine the anti-inflammatory effects of wistin, we investigated the production of NO and ROS. Wistin showed a significant decrease in LPSinduced NO production in a dose-dependent manner compared to that in the control group ( Figure 2a). In addition, dose-dependent inhibition of intracellular ROS generation by wistin was identified using a microplate reader, fluorescence-activated cell sorting (FACS), and fluorescence microscopy (Figure 2b-e). These data suggest that wistin can reduce the production of pro-inflammatory mediators (NO and ROS) induced by LPS.

Effects of Wistin on Pro-Inflammatory Enzymes and Cytokine Gene Expression in LPS-Induced RAW 264.7 Cells
Next, we investigated the involvement of wistin in the modulation of mRNA levels of inflammatory enzymes (iNOS and COX-2) and pro-inflammatory cytokines (IL-1β and IL-6) at indicated time points after LPS treatment [38]. Wistin significantly decreased the mRNA expression levels of inflammatory enzymes (iNOS and COX-2) (Figure 3a,b). The expression levels of pro-inflammatory cytokines (IL-1β and IL-6) were not detected at 0 h after LPS treatment and were the highest at 12 h after LPS treatment. In addition, wistin-treated groups showed a significant decrease in the mRNA expression levels of proinflammatory cytokines at each time point compared to LPS treatment groups. Therefore, these results suggest that wistin could modulate pro-inflammatory enzymes and cytokine gene expression induced by LPS.

Effects of Wistin on Pro-Inflammatory Enzymes and Cytokine Gene Expression in LPS-Induced RAW 264.7 Cells
Next, we investigated the involvement of wistin in the modulation of mRNA levels of inflammatory enzymes (iNOS and COX-2) and pro-inflammatory cytokines (IL-1β and IL-6) at indicated time points after LPS treatment [38]. Wistin significantly decreased the mRNA expression levels of inflammatory enzymes (iNOS and COX-2) (Figure 3a,b). The expression levels of pro-inflammatory cytokines (IL-1β and IL-6) were not detected at 0 h after LPS treatment and were the highest at 12 h after LPS treatment. In addition, wistintreated groups showed a significant decrease in the mRNA expression levels of pro- (f) Quantitative analysis of ROS production using ImageJ software. The scale bar represents 100 µm. * p < 0.05 compared with the LPS-treated group. MFI: mean fluorescence intensity. The data are presented as the means ± SD; n = 3.

Effects of Wistin on the Protein Expression Level of Pro-Inflammatory Enzymes in LPS-Induced RAW 264.7 Cells
The expression of iNOS and COX-2 regulates key inflammatory mediators [39]. Therefore, we investigated whether wistin exhibits anti-inflammatory effects by inhibiting iNOS and COX-2 protein expression. As shown in Figure 4a,b, wistin significantly reduced the protein expression levels of iNOS and COX-2 compared to those in the LPS group. Therefore, these results suggest that wistin could also inhibit the expression of pro-inflammatory enzymes at the protein level.
inflammatory cytokines at each time point compared to LPS treatment groups. Therefore, these results suggest that wistin could modulate pro-inflammatory enzymes and cytokine gene expression induced by LPS.

Effects of Wistin on the Protein Expression Level of Pro-Inflammatory Enzymes in LPS-Induced RAW 264.7 Cells
The expression of iNOS and COX-2 regulates key inflammatory mediators [39]. Therefore, we investigated whether wistin exhibits anti-inflammatory effects by inhibiting iNOS and COX-2 protein expression. As shown in Figure 4a,b, wistin significantly reduced the protein expression levels of iNOS and COX-2 compared to those in the LPS group. Therefore, these results suggest that wistin could also inhibit the expression of proinflammatory enzymes at the protein level.

Effects of Wistin on the Activation of AKT/NF-кB Pathway in LPS-Induced RAW 264.7 Cells
The NF-κB pathway regulates the expression and production of pro-inflammatory enzymes and cytokines [40]. Therefore, we examined the phosphorylation level of LPSinduced AKT and NF-κB (p65 subunit) following wistin (150 μM) treatment for 2 h. Wistin significantly reduced the phosphorylation level of AKT and p65 compared to that in the

Effects of Wistin on the Activation of AKT/NF-κB Pathway in LPS-Induced RAW 264.7 Cells
The NF-κB pathway regulates the expression and production of pro-inflammatory enzymes and cytokines [40]. Therefore, we examined the phosphorylation level of LPSinduced AKT and NF-κB (p65 subunit) following wistin (150 µM) treatment for 2 h. Wistin significantly reduced the phosphorylation level of AKT and p65 compared to that in the LPS group (Figure 5a-d). Furthermore, in the absence of LPS stimulation, p65 (red in the merged image) was present in the cytoplasm. Upon LPS stimulation, p65 (pink in merged images) was translocated to the nucleus, whereas wistin treatment reduced the nuclear translocation of p65 (red and pink in merged images) (Figure 5e). These results suggest that wistin suppresses the AKT/NF-κB signaling pathway. The effects of wistin on the nuclear translocation of p65 (red) using fluorescence microscopy. The merged images were acquired by overlaying two channels (p65 (red) and DAPI (blue)). * Indicates translocation of p65 from the cytoplasm to the nucleus. The scale bar represents 10 μm. (f) Quantitative analysis of the nuclear translocation of p65 using ImageJ software. * p < 0.05 compared with the LPS-treated group. The data are presented as the means ± SD; n = 3.

Effects of Wistin on the Activation of MAPK Pathway in LPS-Induced RAW 264.7 Cells
The MAPK pathway is known for its role in the modulation of inflammatory responses [41]. We investigated the role of wistin in the phosphorylation of three MAPKs (p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)). Phosphorylation of p38 was decreased by wistin, but that of ERK and JNK was not af- The cells were treated with the indicated concentrations of wistin for 30 min prior to treatment with LPS (0.1 µg/mL) for 2 h. Then, the phosphorylation of AKT (a) and p65 (c) were measured using a Western blot. Quantitative analysis of the p-AKT/β-actin (b) and p-p65/β-actin (d) using image J. (e) The effects of wistin on the nuclear translocation of p65 (red) using fluorescence microscopy. The merged images were acquired by overlaying two channels (p65 (red) and DAPI (blue)). * Indicates translocation of p65 from the cytoplasm to the nucleus. The scale bar represents 10 µm. (f) Quantitative analysis of the nuclear translocation of p65 using ImageJ software. * p < 0.05 compared with the LPS-treated group. The data are presented as the means ± SD; n = 3.

Effects of Wistin on the Activation of MAPK Pathway in LPS-Induced RAW 264.7 Cells
The MAPK pathway is known for its role in the modulation of inflammatory responses [41]. We investigated the role of wistin in the phosphorylation of three MAPKs (p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)). Phosphorylation of p38 was decreased by wistin, but that of ERK and JNK was not affected compared to that in the LPS group ( Figure 6). Therefore, these results suggest that wistin could work by negatively regulating the p38 MAPK pathway.

Discussion
Inflammation is a protective response to harmful stimuli (PAMP and DAMP) by the immune system [42]. However, chronic inflammation can cause various diseases, such as cardiovascular disease, inflammatory bowel disease, rheumatoid arthritis, and diabetes [5]. Ibuprofen, naproxen, and other NSAIDs are conventional anti-inflammatory drugs used to treat rheumatism, osteoarthritis, arteriosclerosis, neuroinflammatory diseases, and other inflammatory diseases [8,10]. However, these drugs have several side effects, such as an increased risk of gastric mucosal injury, renal injury, and other medical complications [8,10]. Therefore, it is important to identify novel anti-inflammatory agents that

Discussion
Inflammation is a protective response to harmful stimuli (PAMP and DAMP) by the immune system [42]. However, chronic inflammation can cause various diseases, such as cardiovascular disease, inflammatory bowel disease, rheumatoid arthritis, and diabetes [5]. Ibuprofen, naproxen, and other NSAIDs are conventional anti-inflammatory drugs used to treat rheumatism, osteoarthritis, arteriosclerosis, neuroinflammatory diseases, and other inflammatory diseases [8,10]. However, these drugs have several side effects, such as an increased risk of gastric mucosal injury, renal injury, and other medical complications [8,10]. Therefore, it is important to identify novel anti-inflammatory agents that can overcome the shortcomings of conventional anti-inflammatory drugs. Several plants have been used as folk medicines for the treatment and prevention of diseases [43]. Currently, sinecatechin extracted from green tea and silymarin extracted from milk thistle seeds (Silybum marianum) are used as plant-derived anti-inflammatory drugs for treating HPV and HBV infections, respectively [24][25][26][27]. Furthermore, as plant-derived anti-inflammatory drugs, eupatilin and JOINS tablets are used to treat gastritis and knee osteoarthritis [44,45]. Isoflavones have antioxidant, anticancer, antibacterial, and anti-inflammatory properties [32]. Our study highlights the potential of wistin as an anti-inflammatory agent with fewer side effects via modulation of the inflammatory signaling pathway. However, further in vivo and clinical studies of wistin in this regard are warranted.
During inflammation, NO plays an important role in the regulation of immune and inflammatory responses [46]. Additionally, the high production of ROS during inflammation can lead to cell damage through the oxidation of DNA, RNA, and proteins [47]. iNOS and COX-2 are pro-inflammatory mediators that are regulated by pro-inflammatory transcription factors [48]. When we examined the effects of wistin on NO and ROS generation, we found that wistin reduced the levels of both NO and ROS. In addition, wistin significantly decreased the mRNA expression of inflammatory enzymes (iNOS and COX-2) and inflammatory cytokines (IL-1β and IL-6). Furthermore, wistin decreased the protein expression of iNOS and COX-2. Therefore, these results suggest that wistin exerts anti-inflammatory effects by modulating inflammatory enzymes, inflammatory mediators, and cytokines.
NF-κB signaling is a well-known inflammatory pathway that regulates the expression of pro-inflammatory cytokines (IL-1β and IL-6) and pro-inflammatory enzymes (iNOS and COX-2) [49]. p65 (RelA) is a component of NF-κB, and phosphorylation of p65 induces the expression of a variety of genes [50]. In addition, AKT regulates NF-κB by phosphorylating the IκB kinase (IKK) complex, which phosphorylates the p65 subunit [19]. Wistin reduced the phosphorylation of AKT and p65 in LPS-stimulated RAW264.7. In addition, wistin decreased the translocation of p65 from the cytosol to the nucleus. These results suggest that wistin exerts anti-inflammatory effects by inhibiting the AKT/NF-κBsignaling pathway.
In conclusion, we demonstrated that wistin exerts anti-inflammatory effects by downregulating pro-inflammatory mediators in LPS-mediated signaling. Wistin could regulate pro-inflammatory mediators, including NO, ROS, pro-inflammatory cytokines, and enzymes, by inhibiting the NF-κB and p38 signaling pathways (Figure 7). Similarly, genistein and daidzein inhibit inflammatory mediators via the NF-κB and MAPK signaling pathways [30,32]. They belong to the isoflavonoid family and exhibit versatile pharmacological activities [30,32]. Therefore, wistin may be developed as a plant-derived anti-inflammatory agent with fewer side effects than other conventional anti-inflammatory drugs.

Cell Culture
The RAW264.7 cell line (mouse origin) was obtained from Dr. Sung Ho Ryu's lab (POSTECH, Korea). The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin and incubated at 37 °C with 5% CO2.

Cell Culture
The RAW264.7 cell line (mouse origin) was obtained from Dr. Sung Ho Ryu's lab (POSTECH, Korea). The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin and incubated at 37 • C with 5% CO 2 .

Cell Viability
RAW 264.7 cells were plated in a 48-well plate at a density of 3 × 10 4 cells/well. After 24 h, RAW 264.7 cells were pre-treated with 50, 100, and 150 µM of wistin for 30 min, followed by LPS (0.1 µg/mL) treatment and incubation for 24 h. After that, the DMEM medium was removed, and 1mg/mL of MTT solution was added to each well and was further incubated for 2 h at 37 • C. The solution was removed from the wells and the formed formazan crystals were dissolved in 200 µL of DMSO. Finally, absorbance was measured at 570 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific Co., Waltham, MA, USA).

Measurement of NO Production
The Griess test was used to determine NO production. RAW 264.7 cells were plated into a 24-well plate at a density of 6 × 10 4 cells/well. After 24 h, RAW 264.7 cells were pre-treated with 50, 100, and 150 µM of wistin for 30 min, followed by treatment with LPS (0.1 µg/mL) and further incubation for 24 h. The supernatant from each well was transferred to a 96-well plate, followed by the addition of Griess reagent in a 1:1 ratio. After 15 min, absorbance was measured at 540 nm using a microplate reader (Varioskan LUX Multimode Microplate Reader, Thermo Fisher Scientific Co.).

Measurement of ROS Production
ROS production was determined using the 2',7'-dichlorofluorescin-diacetate (DCF-DA) assay. RAW 264.7 cells were plated into a 24-well plate at a density of 6 × 10 4 cells/well. After 24 h, RAW264.7 cells were pre-treated with 50, 100, and 150 µM wistin for 30 min, followed by LPS (0.1 µg/mL) treatment and further incubation for 24 h. The solution was completely removed from all the wells before incubation with a DCF-DA probe for 30 min at 37 • C. The cells were washed three times with HBSS before measuring fluorescence. The fluorescence intensity was measured at excitation and emission wavelengths of 485 nm and 530 nm, respectively, using a microplate reader (Varioskan LUX Multimode Microplate Reader, Thermo Fisher Scientific Co.). ROS in intact cells were detected with fluorescence microscopy using a Zeiss microscope (Zeiss, Jena, Germany). ROS production was further analyzed using flow cytometry (BD FACSVerse™, BD Bioscience).

QRT-PCR
RAW264.7 cells plated into a 6-well plate at a density of 3 × 10 5 cells/well were pre-treated with 150 µM of wistin for 30 min, followed by LPS (0.1 µg/mL) treatment. After 0, 2, 8, 12, and 24 h, cells were washed with DPBS and harvested. Total RNA was isolated using the TRIzol reagent (Invitrogen, Waltham, MA, USA). cDNA was synthesized from total RNA using a SimpliAmp Thermal Cycler (Applied Biosystems Co., Waltham, MA, USA), and then qRT-PCR was performed on a Step One Plus Real-time PCR System Cycler (Applied Biosystems Co.) using a Power SYBR Green PCR mix Cycler (Applied Biosystems Co.). The PCR primer sequences (forward and reverse) used are listed in Table 1, and β-actin was used as the reference gene. Table 1. Primers used in the quantitative reverse transcription polymerase chain reaction.

Western Blot Analysis
Protein isolation was performed using RAW264.7 cells plated in a 60 mm culture dish at a density of 7 × 10 5 cells/well. After 24 h, RAW264.7 cells were pre-treated with 150 µM wistin for 30 min, followed by LPS (0.1 µg/mL) treatment. The protein was isolated at two time points, 2 and 24 h. Cells were harvested and lysed with lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA pH 8, 1% Triton X-100, 1% sodium deoxycholate, 30 mM NaF, 1.5 mM NaVO4, 1 mM PMSF, and 1 mg/mL each of aprotinin, leupeptin, and pepstatin A) and sonicated for 10 s. The cell lysates were centrifuged at 13,000 rpm for 10 min at 4 • C (Centrifuge 5424 R (rotor type: FA-45-30-11), Eppendorf, Hamburg, Germany) and quantified with the help of the Bradford (Abcam, Cambridge, UK) assay. The proteins (15 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBS-T) at room temperature (RT) for 30 min. Membranes were then incubated with suitable primary antibodies overnight at RT with gentle shaking. The following day, the membranes were washed with TBS-T for 30 min. Membranes were then incubated with secondary horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse IgG antibodies for 1 h at RT. Finally, the membranes were detected using an ECL reagent (Cytiva, Malborough, MS, USA), and the bands were visualized using Chemidoc (iBright™ CL1500, Invitrogen). Band intensities were analyzed using ImageJ software. β-actin was used as the loading control.

Statistical Analysis
Results are presented as mean ± standard deviation (SD). Two statistical analysis methods were used. ANOVA and Dunnett's post hoc tests were conducted to compare three or more groups. The Student's t-test was conducted to compare two groups. Statistical significance was set at p < 0.05(*).