The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol expresses the anti-inflammatory effects in mouse macrophages RAW264.7 cells in vitro

The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol expresses the anti-inflammatory effects in mouse macrophages RAW264.7 cells in vitro | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol expresses the anti-inflammatory effects in mouse macrophages RAW264.7 cells in vitro Masayoshi Yamaguchi, Kenji Yoshiike, Hideaki Watanabe, Mitsugu Watanabe This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2019515/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Inflammation is implicated in the pathogenesis of many diseases. Inflammatory cytokines are produced in macrophages with stimulation of lipopolysaccharide (LPS) and are used as biomarkers participating in diverse disease conditions. The novel marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was initially identified in the Pacific oyster Crassostrea Gigas. DHMBA has properties to reduce oxidative stress as radical scavenging and increase the production of antioxidant proteins. The pharmacologic role of DHMBA, however, has been poorly understood. Methods and Results: This study has been undertaken to investigate whether DHMBA attenuates growth, cytokine production, and osteoclastogenesis in inflammatory mouse macrophage RAW264.7 cells. Culturing with DHMBA (1-1000 µM) suppressed the growth and stimulated the death of RAW264.7 cells in vitro, leading to decrease in cell number. Mechanistically, DHMBA treatment decreased the levels of Ras, PI3K, Akt, MAPK, phospho-MAPK, and mTOR of signaling factors to promote the proliferation, and it raised the levels of p53, p21, Rb, and regucalcin, which are cell growth suppressors. The levels of caspase-3 and cleaved caspase-3 were increased by DHMBA treatment. Culturing with DHMBA suppressed productions of inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, interleukin-1β, or prostaglandin E2, were enhanced by LPS stimulation. Notably, the levels of NF-κB p65 were increased by LPS treatment, and this increase was repressed by DHMBA treatment. LPS treatment stimulated osteoclastogenesis of RAW264.7 cells. This stimulation was blocked by DHMBA treatment. Conclusion: DHMBA was found to potentially suppress the activity of inflammatory macrophages in vitro , suggesting therapeutic usefulness in inflammatory conditions. 3 5-dihydroxy-4-methoxybenzyl alcohol DHMBA an inflammatory cytokine osteoclastogenesis macrophage RAW264.7 cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Inflammation is a complicated biological response of body tissues to damaging motivation [ 1 ], and it also is a protective response connecting immune cells, blood vessels, and molecular mediators [ 1 ]. Inflammatory cytokines, including interleukins (ILs) and tumor necrosis factor (TNF)-α, are known as the biomarkers in chronic and experimental human muscle pain [ 2 ] and osteoarthritis [ 3 ]. These cytokines are produced by macrophages under inflammatory conditions [ 4 , 5 ]. Of note, inflammatory macrophages potentially contribute to the enhancement of progression, metastasis, and angiogenesis of cancer cells [ 6 , 7 ]. RAW264.7 cells are monocyte/macrophage-like cell lineage [ 4 ]. This RAW264.7 cell lineage is characterized by the macrophage-mediatedMetabnMetabtab,olic and phagocytic functions [ 4 ]. RAW264.7 cells are progressively used as modeled macrophages in inflammatory conditions in vitro , This cell line is also accepted as a modeled cell of osteoclastogenesis study [ 4 , 8 ]. Osteoclasts are differentiated from the monocyte-macrophage lineage [ 9 ]. Lipopolysaccharide (LPS) is a core antigen of gram-negative bacteria, which activates the innate immune system of the host [ 10 ]. LPS is an endotoxin that provides a persistent inflammatory stimulus to the tissue [ 11 ]. LPS induces osteoclastogenesis of RAW264.7 cells by regulating NF-κB-related signaling pathways and transcriptional activity [ 9 ]. The novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was initially found in the Pacific oyster Crassostrea Gigas [ 12 , 13 ]. DHMBA has dual properties to prevent oxidative stress as radical scavenging in cells [ 12 – 17 ]. DHMBA has been also reported to reveal a preventive effect on excess glutamatergic neuron activity in rats and mice in vivo [ 15 ]. Thus, DHMBA may play a role in the regulation of cell function as an antioxidant [ 16 , 17 ]. Furthermore, our recent study has demonstrated that DHMBA suppresses the growth of metastatic prostate cancer cells via targeting diverse signaling pathways, providing a new strategy for prostate cancer therapy with DHMBA [ 18 ]. Elucidating the pharmacologic effects of DHMBA may be significant in the therapy of various diseases. This study has been undertaken to elucidate whether DHMBA impacts the activity of inflammatory macrophages in vitro. Here, we demonstrate that culturing with DHMBA blocks the proliferation and stimulates the death of inflammatory mouse macrophages RAW264.7 cells in vitro , leading to a diminishing of cell number. Moreover, DHMBA was found to repress the enhancement of inflammatory cytokine production in RAW264.7 cells cultured with LPS in vitro. Interestingly, we found that osteoclastogenesis of RAW264.7 cells with LPS stimulation was suppressed by DHMBA treatment. Thus, the novel marine factor DHMBA may have a pharmacologic effect on inflammation implicated in macrophages. Our study may offer a useful therapeutic tool for the inflammatory condition with DHMBA. 2. Materials And Methods 2.1. Reagents Dulbecco’s Modification of Eagle’s Medium (DMEM) with 4.5 g/L glucose, L-glutamine and sodium pyruvate and antibiotics (100 units/mL penicillin and 100 µg/mL streptomycin; 1% P/S) was obtained from Corning (Mediatech, Inc. Manassas, VA, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Amphotericin B (fungizone), caspase-3 inhibitor (CAS 169332-60-9-Calbiochem), lipopolysaccharide (LPS), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The caspase-3 inhibitor was diluted in sterile phosphate-bufferedfered saline (PBS). Other reagents were dissolved in 100% ethanol, and these reagents were stored at -20℃ until use. 2.2. 3, 5-dihydroxy-4-methoxybenzyl alcohol 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a novel amphipathic phenolic compound, was initially isolated from the pacific oyster ( Crassostrea Gigas ) with a characterization of antioxidant [ 12 ]. We used the synthesized DHMBA in the present study [ 12 , 18 ]. The purity of synthesized DHMBA was 100% [ 12 , 18 ]. DHMBA was dissolved in 100% ethanol and stored at -20℃ until use. 2.3. RAW264.7 cells RAW264.7 cells are monocyte/macrophage-like cell lineage, originating from Abelson leukemia virus-transformed cell lineages derived from BALB/c mice [ 8 ]. Mouse macrophage RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) [ 19 ]. RAW264.7 cells were cultured in DMEM containing 10% FBS, 1% P/S, and 1% fungizone. 2.4. Assay of cell growth To determine development of cell proliferation, RAW264.7 cells (1x10 5 /ml per well) were cultured using 24-well plates in DMEM containing 10% FBS, 1% P/S and 1% fungizone in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 µM) for 1, 2, 3, or 4 days in a water-saturated atmosphere containing 5% CO 2 and 95% air at 37 o C [ 20 , 21 ]. In other experiments, to investigate the effects of DHMBA on the growth of RAW264.7 cells in the presence of LPS, the cells (1x10 5 /ml per well) were cultured using 24-well plates in DMEM (containing 10% FBS, 1% P/S, and 1% fungizone) in the presence of either vehicle (1% ethanol as a final concentration) or LPS (1, 10, 50, 100, or 500 ng/ml of medium) with or without DHMBA (1 or 10 µM) for 3 days. After culture, the cells were detached from each well by adding a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca 2+ /Mg 2+ -free PBS (Thermo Fisher Scientific, Waltham, MA, USA) with incubation for 2 min at 37℃. Each well was then added 0.9 ml of DMEM containing 10% FBS, and 1% P/S. The number of cells in the cell suspension was counted as described below in the section “Cell counting”. 2.5. Assay of cell death RAW264.7 cells (1x10 5 /ml per well in 24-well plates) were cultured using 24-well plates in DMEM containing 10% FBS, 1% P/S, and 1% fungizone for 3 days. Cells on reaching subconfluence were cultured for an additional 24 or 48 hours in the presence of either vehicle (PBS or 1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 µM) [ 22 , 23 ]. In other experiments, the cells (1x10 5 /ml per well) on reaching subconfluence with culturing for 3 days were additionally cultured for 48 hours in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (1 or 10 µM) with or without caspase-3 inhibitor (10 µM) [ 22 ]. In additional experiments, to establish the effects of DHMBA on the death of RAW264.7 cells cultured in the presence of LPS, the cells (1x10 5 /ml per well) were cultured using 24-well plates in DMEM (containing 10% FBS, 1% P/S, and 1% fungizone) for 3 days on reaching subconfluence, and then the cells were furthermore cultured for 48 hours in the presence of either vehicle (1% ethanol as a final concentration) or LPS (1, 10, 50, 100, or 500 ng/ml of medium) with or without DHMBA (1 or 10 µM). After culture, the cells were detached by adding a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca 2+ /Mg 2+ -free PBS per well as explained in the section “Cell growth assay”, and the cell number was counted as described below in the section “Cell counting”. 2.6. Cell counting After culture, to detach cells on each well, the culture dishes were incubated for 2 min at 37℃ after adding a solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca 2+ /Mg 2+ -free PBS, and then the cells were detached and mixed through pipetting after addition of DMEM (0.9 ml) containing 10% FBS and 1% P/S as explained in the previous study [ 20 – 23 ]. The number of viable cells was counted under a microscope (Olympus MTV-3) with a Hemocytometer (Sigma-Aldrich) by using a cell counter (Line Seiki H-102P, Tokyo, Japan). For each dish, we took the average of two counts. Cell numbers were shown as numbers per well. 2.7. Assay of cytokine production RAW264.7 cells (1x10 5 /ml per well) were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S for 3 days in reaching upon subconfluence [ 5 ], and then the cells were further cultured for 5 hours after the treatment with either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 µM) with or without LPS (100 ng/ ml). After incubation, the medium was collected to assay cytokines, and then the cells were detached from each culture dish to determine the number of cells as described in “cell counting”. The concentrations of TNF-α, IL-1β, IL-6, or PGE2 in the medium were analyzed using ELISA Kits for mouse TNF-α [catalog number (cat. no. BM), KHC301)] and IL-1β (cat. no., BMS6002) obtained from (ThermoFisher Scientific, Waltham, MA, USA) or IL-6 (cat. no. 583371) and PGE2 (cat. no. 514010) purchased from Cayman Chemical (Ann Arbor, MI, USA), according to the manufacturer’s instructions. The production of each cytokine was presented as a pictogram (pg) secreted into a culture medium (ml). In separate experiments, to determine the levels of Cox-1, Cox-2, NF-κB p65, and STAT3, which are implicated in cytokine signaling, RAW264.7 cells (1x10 6 cells/10 ml of 100 mm dishes) were cultured for 3 days on reaching subconfluence in DMEM containing 10% FBS, 1% P/S and 1% fungizone, and then the cells were additionally cultured for 5 hours in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (10 µM) with or without LPS (100 ng/ml of medium). After culture, the attached cells were removed from the dish by scraping in cell lysis buffer as described in the section “Western blot assay”. 2.8. Western blotting RAW264.7 cells (1x10 6 cells/10 ml of 100 mm dishes) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (10 µM), and then the dishes were washed three times with cold PBS (10 ml) to exclude floating and dead cells and the attached cells removed from the dish by scraping in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease and protein phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA) as explained in the previous study [ 23 ]. The lysates were then centrifuged at 17,000x g , at 4℃ for 10 min. Protein concentration in the supernatant was determined using the Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin as a standard. The cell lysate was stored at -80℃ until use. Samples of forty micrograms of supernatant protein per lane were separated by SDS polyacrylamide gel electrophoresis (12% SDS-PAGE) and then transferred to PVDF membranes. Transferred membranes were immunoblotted using specific antibodies against various proteins obtained from Cell Signaling Technology (Danvers, MA, USA), including Ras (cat. no. 3339, rabbit), Akt (cat. no. 9272, rabbit), mitogen-activated protein kinase (MAPK; cat. no. 4695, rabbit), phosphorylated-MAPK (cat. no. 4370, rabbit), mechanistic target of rapamycin (mTOR, cat. no. 4517, mouse), Rb (cat. no. 9309, mouse), p21 (cat. no. 2947, rabbit), STAT3 (cat. no. 12640, rabbit), COX-1 (cat. no. 48415), COX-2 (cat. no. 4842), and β-actin (cat. no. 3700, mouse), and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), including p53 (cat. no. sc-126, mouse), and NF-κB p65 (cat. no. sc-109, rabbit). Rabbit anti-regucalcin antibody was obtained from Sigma-Aldrich (cat. no. HPA029103, rabbit). Target proteins were incubated with one of the primary antibodies (1:1,000) as described above overnight at 4℃. After incubation, the membranes were additionally incubated for 60 min at room temperature in horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., mouse sc-2005 or rabbit sc-2305; diluted 1:2,000) at room temperature, and protein bands were detected using a Chemiluminescence substrate (cat. no. 34577, Thermo Scientific, Rockford, IL, USA) on X-ray film. A total of 3 or 4 films from 4 independent experiments on separate membranes were scanned on an Epson Perfection 1660 Photo scanner, and the bands were quantified using Image J2 software (National Institutes of Health, Bethesda, MD, USA). For immunoblotting with additional antibodies, we used the restored Western blot stripping buffer (cat. no. 21059; Thermo Scientific, Rockford, IL, USA) to remove the attached Chemiluminescence substrate (Thermo Scientific) by incubation at room temperature for 30 min. 2.9. Assay of osteoclastogenesis RAW264.7 cells (1 x 10 5 cells/1 ml per well in 24-well plates) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml of medium) with or without DHMBA (0.1, 1, 10, or 100 µM), and then 0.5 ml of old medium was replaced with fresh medium (0.5 ml) including above LPS or DHMBA. The cells were additionally cultured for 3 days [ 19 ]. In a separate experiment, RAW264.7 cells (1 x 10 5 cells/1 ml per well in 24-well plates) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone in either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml of medium) without DHMBA, and then 0.5 ml of old medium was replaced with fresh medium (0.5 ml) including either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml of medium) with DHMBA (as a final concentration of 0.1, 1, 10, or 100 µM).. The cells were additionally cultured for 3 days [ 19 ]. After culture, RAW264.7 cells adherent to the plates were fixed and stained for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts [ 24 ]. Cells were washed with PBS solution and fixed with 10% neutralized formalin-phosphate (pH7.2) for 1 minute. The fixed cells were incubated for 10 hours at room temperature in acetate buffer (pH 5.0) containing naphthol AS-MX phosphate (Sigma-Aldrich) as a stain for the reaction product in the presence of 10 mM sodium tartrate. TRACP-positive multinucleated cells (MNCs) containing three or more nuclei were counted as osteoclast-like cells under a microscope (40x) (Olympus MTV-3; Olympus Corporation, Tokyo, Japan). To assess the number of osteoclast-like TRACP-positive MNCs, one field per well was photographed and measured using ImageJ2 software. The number of osteoclast-like TRACP-positive MNCs was counted in five random fields under a light microscope (40x) (Olympus MTV-3; Olympus Corporation, Tokyo, Japan) and averages were calculated. 2.10. Statistical analysis Statistical significance was estimated using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc. La Jolla, CA). Data are presented as the mean ± standard deviation (SD). We used Student- t -test to calculate statistical significance between the 2 groups. As indicated, Multiple comparisons were performed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons post-test for the parametric data. A p-value of < .05 was considered statistically significant. 3. Results 3.1. DHMBA represses the growth of RAW264.7 cells. First, we investigated whether DHMBA influences the growth of mouse macrophage RAW264.7 cells (Fig. 2 ). RAW264.7 cells were cultured for 1, 2, 3, and 4 days in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 µM). The growth of RAW264.7 cells was blocked by culturing with DHMBA (1, 10, 100, or 1000 µM) for 1–4 days. Thus, DHMBA was found to repress the growth of RAW264.7 cells in vitro . To better understand the underlying mechanism by which DHMBA suppresses the proliferation of RAW264.7 cells, we determined whether DHMBA regulates the expression of key proteins linked to the proliferation of RAW264.7 cells (Fig. 3 ). Culturing with DHMBA (10 µM) diminished the levels of Ras, PI3 kinase, Akt, MAPK, phospho-MAPK, and mTOR, which are implicated in the promotion of the proliferation of RAW264.7 cells [ 25 ], while it increased the levels of p53, Rb, p21, and regucalcin that lead to repression of cell proliferation [ 23 , 26 ]. These results suggest that the alterations of signaling proteins and cell growth suppressors are involved in the mechanism by which DHMBA blocks the growth of RAW264.7 cells. 3.2. DHMBA promotes the death of RAW264.7 cells. Furthermore, it was elucidated whether DHMBA impacts the death of mouse macrophage RAW264.7 cells. Cells were cultured for 3 days on reaching subconfluence, and then they were further cultured for 24 or 48 hours in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 µM). The death of RAW264.7 cells was promoted by culturing with DHMBA (1, 10, 100, or 1000 µM) for 24 (Fig. 4 A) or 48 hours (Fig. 4 B). The stimulatory effects of DHMBA (1 or 10 µM) on the death of RAW264.7 cells were blocked by the presence of caspase-3 inhibitor (10 µM) (Fig. 4 C). The results of Western blotting indicated that the levels of caspase-3 and cleaved caspase-3 in the cells were increased by culturing with DHMBA (10 µM) (Fig. 4 D). These results suggest that DHMBA stimulates apoptotic cell death of mouse macrophage RAW264.7 cells. 3.3. Effects of DHMBA on RAW264.7 cells cultured with LPS Next, we investigated the suppressive effects of DHMBA on inflammatory macrophage RAW264.7 cells with LPS treatment. LPS is well known to enhance the inflammatory activity of mouse macrophage RAW264.7 cells [ 5 , 9 ]. It was determined whether DHMBA attenuates the repressive effects on the proliferation or the stimulatory effects on the death of RAW264.7 cells cultured in the presence of LPS in vitro (Fig. 5 ). RAW264.7 cells were cultured in the presence of LPS (1, 10, 50, 100, or 500 ng/ml of medium) for 3 days. The growth of RAW264.7 cells was not altered by culturing with LPS (1, 10, 50, and 100 ng/ml), while it was suppressed by culturing with a higher concentration of LPS (500 ng/ml) (Fig. 5 A). In the presence of LPS (100 ng/ml), DHMBA (1 or 10 µM) also suppressed the growth of RAW264.7 cells (Fig. 5 B). To determine the effects of DHMBA on cell death cultured in the presence of LPS, moreover, RAW264.7 cells on reaching subconfluence by culturing for 3 days were additionally cultured in the presence of LPS (1, 10, 50, 100, or 500 ng/ml of medium) for 48 hours (Fig. 5 C). Culturing with LPS (1, 10, 50, and 100 ng/ml) did not cause a significant effect on cell number, while a higher level of LPS (500 ng/ml) stimulated the death of RAW264.7 cells. In the presence of LPS (100 ng/ml), DHMBA (1 or 10 µM) stimulated the death of RAW264.7 cells (Fig. 5 B). These results indicate that DHMBA keeps activity that decreases the number of RAW264.7 cells in the presence of LPS (100 ng/ml). 3.4. DHMBA suppresses inflammatory cytokine production in RAW264.7 cells Furthermore, we elucidated whether DHMBA impacts the production of inflammatory cytokines in mouse macrophage RAW264.7 cells cultured in the presence of LPS (100 ng/ml) in vitro. RAW264.7 cells were cultured for 3 days on reaching subconfluence, and then the cells were moreover incubated for 5 hours after the addition of LPS (100 ng/ml) with or without DHMBA (0.1, 1, 10, 100, or 1000 µM). The number of RAW264.7 cells was not changed by adding DHMBA without (Fig. 6 A) and with LPS (100 ng/ml of medium) (Fig. 6 B). Under the same condition of culture which did not cause an alteration of cell number, we determined the production of inflammatory cytokines, including TN F-α (Fig. 7 A), IL-6 (Fig. 7 B), IL-1β (Fig. 7 C), or PGE2 (Fig. 6 D), in the medium obtained by culturing with RAW264.7 cells. In the absence of LPS, the productions of IL-6, or IL-1β in RAW264.7 cells were repressed by the addition of DHMBA (100 or 1000 µM) (Figs. 7 B and F). Notably, the treatment with LPS caused a remarkable increase in the production of TNF-α (Fig. 7 A), IL-6 (Fig. 7 B), IL-1β (Fig. 7 C), or PGE 2 (Fig. 7 D). These increases were suppressed by the treatment of DHMBA (1, 10, 100, or 1000 µM). Thus, the production of inflammatory cytokines in RAW264.7 cells was found to be suppressed by DHMBA treatment. It was elucidated whether DHMBA regulates the levels of proteins implicated in cytokine production and is linked to intracellular signaling processes of cytokines (Fig. 8 ). RAW264.7 cells were incubated for 5 hours in the presence of LPS (100 ng/ml) with or without DHMBA (10 µM) in vitro . In a culture of RAW264.7 cells without LPS, the levels of COX-1, COX-2, MAPK, phosphor-MAPK, NF-κB p65, and STAT3 were not altered by the treatment with DHMBA as compared with those of control (Figs. 8 A and B). The levels of MAPK, phospho-MAPK, NF-κB p65, and STAT3 in RAW264.7 cells were increased by culturing with LPS (100 ng/ml) (Figs. 8 A and C). These increases were repressed by the treatment with DHMBA (10 µM) (Figs. 8 A and D). These results suggest that DHMBA treatment suppresses the levels of signaling factors implicated in the production of cytokines in inflammatory RAW264.7 cells. 3.5. DHMBA inhibits osteoclastogenesis of RAW264.7 cells RAW264.7 cells are well used as modeled cells in osteoclastogenesis studies [ 4 , 8 ]. Osteoclasts are differentiated from the monocyte-macrophage lineage [ 9 ]. LPS has been shown to stimulate osteoclastogenesis of RAW264.7 cells by activating the NF-κB pathway [ 9 ]. We investigated whether DHMBA influences osteoclastogenesis of RAW264.7 cells cultured with LPS. RAW264.7 cells (1 x 10 5 cells/1 ml per well in 24-well plates) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S and 1% fungizone either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml of medium) with or without DHMBA (0.1, 1, 10, or 100 µM). After culture for 3 days, 0.5 ml of old medium was replaced with fresh medium (0.5 ml) including LPS (100 ng/ml) or DHMBA (0.1, 1, 10, or 100 µM), and then the cells were furthermore cultured for 3 days [ 19 ]. In culturing with both LPS and DHMBA for 6 days (Figs. 9 A and C), LPS enhanced osteoclastogenesis of RAW264.7 cells. This enhancement was suppressed by culturing with DHMBA (0.1, 1, 10, or 100 µM) (Figs. 9 A and C). In separate experiments, RAW267.4 cells were cultured in the presence of LPS (100 ng/ml) for 3 days without DHMBA, and then the cells were additionally cultured for 3 days with the replacement of fresh medium including LPS (100 ng/ml) with or without DHMBA (0.1, 1, 10, or 100 µM). Osteoclastogenesis at a later stage enhanced by LPS was also suppressed by DHMBA (Figs. 8Band D). These results support the view that DHMBA suppresses osteoclastogenesis enhanced with LPS stimulation in RAW264.7 cells. 4. Discussion The novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has dual characteristics to weaken oxidative stress as radical scavenging in cells [ 12 – 17 ]. DHMBA may play a role in the regulation of cell function as an antioxidant [ 16 , 17 ]. Our previous study demonstrated that DHMBA suppressed the growth and activity of metastatic prostate cancer cells via targeting diverse signaling pathways, providing a new strategy for prostate cancer therapy [ 18 ], Furthermore, we elucidated whether DHMBA reveals anti-inflammatory effects by using mouse macrophage RAW264.7 cells in vitro . We found that DHMBA revealed anti-inflammatory effects, leading to a reduction of the number of inflammatory macrophages, repression of inflammatory cytokine production, and inhibition of osteoclastogenesis development in RAW264.7 cells in vitro . Culturing with DHMBA suppressed the growth and stimulated the death of RAW264.7 cells in vitro , causing to decline in the number of macrophages. DHMBA treatment diminished the levels of Ras, PI3 kinase, Akt, MAPK, phospho-MAPK, and mTOR, which lead to the promotion of the proliferation of RAW264.7 cells [ 25 ], while it increased the levels of p53, Rb, p21, and regucalcin that induce repression of cell proliferation [ 23 , 26 ]. The diminish of these cell signaling-related protein levels and the enhancement of cell growth suppressor with DHMBA treatment may contribute to the underlying mechanism by which the compound blocks the growth of RAW264.7 cells. DHMBA may regulate the expression of various proteins linked to cell signaling and transcriptional activity. Furthermore, the levels of caspase-3 and cleaved caspase-3 implicated in apoptotic cell death were increased by DHMBA treatment. These augmentations may cause activation of nuclear DNA fragmentation that induces apoptotic cell death [ 25 , 26 ]. Thus, DHMBA may impact the levels of diverse proteins linked to the control of cell numbers. DHMBA was also found to powerfully suppress the production of inflammatory cytokines, including TNF-α, IL-6, IL-1β, or PGE2, with the treatment of LPS that stimulates the inflammatory condition of macrophage RAW264.7 cells. These reductions were observed under culture conditions that did not cause a decrease in the number of RAW264.7 cells when cultivated with both LPS and DHMBA.DH revealed inhibition of cytokine production independent of alteration of the number of RAW264.7 cells. LPS treatment has been shown to enhance the production of TNF-α, IL-6, IL-1β, or PGE 2 in RAW264.7 cells [ 27 – 29 ]. We found that the production of these cytokines enhanced by LPS treatment was suppressed by culturing with DHMBA in RAW264.7 cells in vitro. DHMBA treatment may be a useful tool in the suppression of cytokines production under inflammatory conditions. Further study was undertaken to better understand the underlying mechanism by which DHMBA represses cytokine production in inflammatory RAW264.7 cells with LPS stimulation. LPS binds to Toll-like receptor 4 (TLR4) on the plasma membranes of macrophage RAW264.7 cells, and the signaling with LPS/TLR4 pathway is transmitted into the cells [ 30 – 32 ]. Notably, TLR4 signaling activates NF-κB and MAPK signaling in RAW264.7 cells [ 33 – 35 ]. Also, there is growing evidence that LPS internalizes and binds intracellular proteins and receptors [ 30 – 32 ]. The production of TNF-α, IL-6, IL-1β, or PGE 2 with LPS treatment may be implicated in intracellular signaling NF-κB p65, and/or MAPK in RAW264.7 cells [ 33 – 35 ]. COX-1 and COX-2 may be implicated in the production of PGE2 in RAW264.7 cells [ 27 – 29 ]. Our studies demonstrated that the levels of NF-κB p65, MAPK, and phosphorylated MAPK were increased by culturing with LPS of RAW264.7 cells. These augmentations were repressed by DHMBA treatment. This repression may lead to the suppression of the LPS-enhanced production of inflammatory cytokines in RAW264.7 cells. Moreover, it is possible that DHMBA disturbs the binding of LPS to TLR4 and/or intracellular receptor proteins to weaken the production of inflammatory cytokines in RAW264.7 cells. DHMBA is also speculated to regulate transcriptional activity linked to the production of inflammatory cytokines in RAW264.7 cells. In addition, DHMBA may play a role in the repression of cytokine production as an antioxidant [ 16 , 17 ]. Further study remains to be elucidated the molecular mechanism. Interestingly, the levels of STAT3 were increased by LPS treatment, and this enhancement was repressed by culturing with DHMBA in RAW264.7 cells. STAT3 is involved in the intracellular signaling of IL-6 [ 36 ]. LPS stimulation caused the production of IL-6 and IL-1β in RAW264.7 cells. These cytokines may impact the activity of RAW264.7 cells with their autocrine effects. DHMBA treatment may disturb the signaling process which is mediated via IL-6 and IL-1β produced in RAW264.7 cells. LPS stimulates osteoclastogenesis via activation of NF-κB signaling in RAW264.7 cells [37–40]. LPS treatment leads to activation of TLR4 signaling linked to NF-κB in macrophages [ 30 – 32 ]. Osteoclastogenesis enhanced by culturing with LPS was suppressed by the treatment of DHMBA in RAW264.7 cells. This suppression was also observed by DHMBA treatment at the earlier and later stages of osteoclastogenesis. Presumably, the suppressive effects of DHMBA on osteoclastogenesis may be related to the inhibition of NF-κB signaling implicated in the decreased NF-κB p65 levels by DHMBA treatment. In conclusion, this study demonstrates that DHMBA represses the proliferation and stimulates the death of RAW264.7 cells, leading to a reduction in the number of inflammatory macrophages. DHMBA was also demonstrated to repress the production of inflammatory cytokines. Furthermore, DHMBA treatment repressed osteoclastogenesis of RAW264.7 cells with LPS stimulation in vitro . DHMBA may reveal anti-inflammatory effects by using modeled mouse macrophage RAW264.7 cells in vitro , although it remains to be elucidated whether DHMBA has an anti-inflammatory effect in vivo. DHMBA is a functional-medical food factor with very low toxicity. DHMBA may be a pharmacologic significant in the treatment of inflammatory conditions, providing a new strategy for prevention and therapy of inflammation Declarations Authorship contribution statement Masayoshi Yamaguchi, PhD : Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Kenji Yosiike , MS : Investigation, Resources, Writing - review & editing. Hideaki Watanabe, MD : Investigation, Resources, Writing - review & editing. Mitsugu Watanabe, PhD : Funding acquisition, Investigation, Resources, Writing - review & editing. Funding This study was supported in part by funds provided by the University of Hawaii Cancer Center (M.Y.) and the Foundation of Watanabe Oyster Laboratory (M.W.). Author declaration of competing Interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Human and animal ethics This article does not contain any studies with human participants or animals performed by any of the authors. All experimental protocols used databases or cell culture in vitro . Availability of data and materials The datasets used during the present study are available from the corresponding author upon reasonable request. Consent for publication Not applicable. Acknowledgments Not applicable References Abdelhamid RE, Sluka KA (2015). ASICs mediate pain and inflammation in musculoskeletal diseases. Physiology 30:449–459. Berenbaum F (2013) Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!). Osteoarthritis Cartilage. 21: 16–21. Pomari E, Stefanon B, Colitti M (2014) Effect of plant extracts on H 2 O 2 -induced inflammatory gene expression in macrophage. J Inflamm Res. 27:103–112. Kong L, Smith W, Hao D (2019) Overview of RAW264.7 for osteoclastogenesis study: Phenotype and stimuli, J Cell Mol Med 23: 3077–3087 Yamaguchi M, Levy RM (2020) Metaxalone suppresses production of inflammatory cytokines associated with painful conditions in mouse macrophages RAW264.7 cells in vitro : Synergistic effect with β-caryophyllene. Curr Mol Med 20:643–652 Xu Y, Wang X, Liu L, Wang H, Wu H, Sun C (2022) Role of macrophages in tumor progression and therapy (Review). Int J Oncol 60:57–75 Geindreau M, Bruchard M, Vegran F (2022) Role of cytokines and chemokines in angiogenesis in a tumor context. Cancers 14:2446. Doi.org/10:3390/cancers14102446. Hartley JW, Evanse LH, Green KY, Naghashfar Z, Macias AR, Zerfas PM, Ward JM. (2008) Expression of infectious murine leukemia viruses by RAW264.7 cells, a potentiated complication for studies with a widely used mouse macrophage cell line. Retrovirology 5:1. Li Y, Zhang J, Yan C, Chen Q, Xiang C, Zhang Q, Wang X, Jiang K (2022) Martin prevented LPS-induced osteoclastogenesis by regulating the NF-κB pathway in vitro. J Mem Biotech 32:141–148. Maldonado RF, Sa-Correia I, Valvano MA480–493 (2016) Lipopolysaccharide modification in Gram-negative bacteria during chronic infection. FEMS Microbiol Rev 40: 480–493 Ohanian SH, Schwab JH (1967) Persistence of group a streptococcal cell walls related in chronic inflammation of rabbit dermal connective tissue. J Exp Med 125:1137–1148 Watanabe M, Fuda H, Jin S, Sakurai T, Ohkawa F, Hui S-P, Takeda S, Watanabe T, Koike T, Chiba H (2012) Isolation and characterization of a phenolic antioxidant from the pacific oyster ( Crassostrea Gigas ). J Agric Food Chem 60: 830–835. Fuda H, Watanabe M, Hui S-P, Joko S, Okabe H, Jin S, Takeda S, Miki E, Watanabe T, CGigasH (2015) Anti-apoptotic effects of novel phenolic antioxidant isolated from the Pacific oyster ( Crassostrea Gigas ) on cultured human hepatocytes under oxidative stress. Food Chem 176: 226–233. Joko S, Watanabe M, Fuda H, Takeda S, Furukawa T, Hui S-P, Shrestha R, Chiba H (2017) Comparison of chemical structures and cytoprotection abilities between direct and indirect antioxidants. J Funct Foods 35 :245–255. Tamano H, Shakushi Y, Watanabe M, Ohashi K, Uematsu C, Otsubo T, Ikeda K, Takeda A (2019) Preventive effect of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) and zinc, components of the pacific oyster Crassostrea gigas, on glutamatergic neuron activity in the hippocampus. J Agric Food Chem 67:12844–12853. Fukai M, Nakayabu T, Ohtani S, Shibata K, Shimada S, Sakamoto S, Fuda H, Furukawa T, Watanabe M, Hui S-P, Chiba H, Shimamura T, Taketomi A (2021) The Phenolic Antioxidant 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) prevents enterocyte cell death under oxygen-dissolving cold conditions through polyphyletic antioxidant actions. J Clin Med, 10: 1972. doi.org/10.3390/jcm10091972 . Okabe H, Hui S.-P, Fuda H, Furukawa T, Takeda S, Shrestha R, Miura, Watanabe M, Chiba H (2015) Mass Spectrometric quantification of amphipathic, polyphenolic antioxidant of the pacific oyster ( Crassostrea Gigas ). Anal Sci 31:1341–1344. Yamaguchi M, Yosiike K, Watanabe H, Watanabe M (2022) The marine factor 3,5- dihydroxy-4-methoxybenzyl alcohol suppresses growth, migration, and invasion and stimulates death of metastatic human prostate cancer cells: Targeting diverse signaling processes. Anticancer Drug 33:424–436 Yamaguchi M, Weitzmann MN (2011) Vitamin K2 stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF-κB activation. Int J Mol Med 27:3–14. Misawa H, Inagaki S, Yamaguchi M (2001) Suppression of cell proliferation and deoxyribonucleic acid synthesis in cloned rat hepatoma H4-II-E cells overexpressing regucalcin. J Cell Biochem 84: 143–149. Yamaguchi M, Daimon Y (2005) Overexpression of regucalcin suppresses cell proliferation in cloned rat hepatoma H4-II-E cells: Involvement of intracellular signaling factors and cell cycle-related genes. J Cell Biochem 95: 1169–1177. Izumi T, Yamaguchi M: Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-α or thapsigargin. J Cell Biochem 92: 296–306, 2004. Yamaguchi M, Osuka S, Weitzmann MN, El-Rayes BF, Shoji S, Murata T (2016) Prolonged survival in pancreatic cancer patients with increased regucalcin gene expression: Overexpression of regucalcin suppresses the proliferation in human pancreatic cancer MIA PaCa-2 cells in vitro. Int J Oncol 48:1955–1964. Minkin C (1982) Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker osteoclast function. Calcif Tissue Int 34:285–290. Peleck SL, Charest DL, Mordret GP, Siow YL, Palaty C, Campbell D, Chaslton L, Samiei M, Sanghera JS (1993) Networking with mitogen-activated protein kinases. Mol Cell Biochem 127: 157–169. Yamaguchi M (2013) Suppressive role of regucalcin in liver cell proliferation: Involvement in carcinogenesis. Cell Prolif 46: 243–253. Echizen K, Hirose O, Maeda Y, Oshima M. Inflammation in gastric cancer: The interplay of the COX-2/prostaglandin E2 and Toll-like receptor/MyD88 pathways. Cancer Sci. 2016; 107:391–397. Guan F, Wang H, Shan Y, Chen Y, Wang M, Wang Q, Yin M, Zhao Y, Feng Z, Zhang J (2014) Inhibition of COX-2 and PGE 2 in LPS-stimulated RAW264.7 cells by lonimacranthoide VI, a chlorogenic acid ester saponin. Biomed Rep 2:760–764. Lin CC, Chan CM, Huang YP, Hsu SH, Huang CL, Tsai SJ. Methylglyoxal activates NF-κB nuclear translocation and induces COX-2 expression via a p38-dependent pathway in synovial cells. Life Sci. 2016; 149:25–33. Fenton MJ, Golenbock DT ( 1998 ) LPS-binding proteins and receptors . J Leukoc Biol 64 :25–32 Nunes-Alves C ( 2014 ) New LPS receptors discovered . Nat Rev Immunol 14:583 Huang M-H, Lin Y-H, Lyu P-C, Liu Y-C, Chang Y-S, Chung J-G, Lin W-Y, Hsieh W-T (2021) Imperatorin interferes with LPS binding to the TLR4 co-receptor and activates the Nrf2 antioxidative pathway in RAW264.7 murine macrophage cells. Antioxidants 10:362.doi: 10.3390/antiox10030362 . Zhou C, Gao J, Ji H, Li W, Xing X, Liu D, Guo Q, Zhou L, Jing F ( 2021 ) Modulates LPS-induced responses through inhibition of Toll-like receptor-mediated NF-κB and MAPK signaling in RAW264.7 cells . Inflammation 44 :2018–2031 Li N, Liu BW, Ren WZ, Liu JX, Li SN, Fu SP, Zeng YL, Xu SY, Yan X, Gao YJ, Liu DF, Wang W ( 2016 ) GLP-2 attenuates LPS-induced inflammation in BV-2 cells by inhibiting ERK1/2, JNK1/2 and NF-κB signaling pathway . Int J Mol Sci. 17 : E190 Doi : 10.3390/ijms17020190 . Cha SM, Cha JD, Jang EJ, Kim GU, Lee KY (2016) Sophoraflavone G prevents streptococcus mutants surface antigen/II-induced production of NO and PGE 2 by inhibiting MAPK-mediated pathways in RAW 264.7 macrophages. Arch Oral Biol. 68:97–104 Huang X, Fang J, Lai W, Hu Y, Li L, Zhong Y, Yang S, He D, Liu R, Tang Q (2022) IL-6/STAT3 axis activates glut5 to regulate fructose metabolism and tumorigenesis. Int J Biol Sci 18:3668–3675. Yamaguchi M, Weitzmann MN (2011) Vitamin K2 stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF-κB activation. Int J Mol Med 27:3–14. Xiao Y, Cao Y, Song C, Ren X, Yan L, Hao D, Kong L (2020) Cellular study of the LPS-induced osteoclastic multinucleated cell formation from RAW264.7 cells. J Cell Physiol 235:421–428. Liu Y, Fang S, Li X, Feng J, Du J, Guo L, Su Y, Zhou J, Ding G, Bai Y, Wang S, Wang H, Liu Y (2017) Aspirin inhibits LPS-induced macrophage activation via the NF-κB pathway. Sci Rep 7:11549. Sapkota M, Li L, Kim S-W, Soh Y (2018) Thymol inhibits RANKL-induced osteoclastogenesis in RAW264.7 and BMM cells and LPS-induced bone loss in mice. Food Chem Toxicol 120:418–429. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2019515","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":133518332,"identity":"2dc6e18b-132b-43fe-82ac-1f5bb4594b9c","order_by":0,"name":"Masayoshi Yamaguchi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDADNmb+559/VDAwGEAFmAlq4WPnYWNmOEOKFjl+oBbGNoQWnMDg+OnETzdq6uTZmHmPPS6cd1jenL35AAPQhezmuLScyd0snXPssGEbM1+68cxthw139hxLYOw5w8Bs2YBDy4HcDdI5bAcY25gZDCR4tx1m3HAjxwDkQmaDAzi0nH+7+XfOvzp7iJY5h+0Ja7mRu006t405sY2Zx0yat+FwIkEtkjfebrPO7Tuc3MbMlmw441h68oYzxxIO9pyRwOkXvvO5m2/nfKuznd9/+OCDDzXWthuONx988KPCJhlXiCmg2d4MJoGCEsm4IkgezfY6OMuOYJyOglEwCkbBSAEAzDtfZFmC3lUAAAAASUVORK5CYII=","orcid":"","institution":"University of Hawaii Cancer Center, the University of Hawaii at Manoa","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Masayoshi","middleName":"","lastName":"Yamaguchi","suffix":""},{"id":133518333,"identity":"237c0aa4-a133-468a-818a-ef703c2aa1bf","order_by":1,"name":"Kenji Yoshiike","email":"","orcid":"","institution":"Watanabe Oyster Laboratory Co. Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Yoshiike","suffix":""},{"id":133518334,"identity":"4081a300-94e5-46fe-af9d-99a37da3de13","order_by":2,"name":"Hideaki Watanabe","email":"","orcid":"","institution":"Watanabe Oyster Laboratory Co. Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hideaki","middleName":"","lastName":"Watanabe","suffix":""},{"id":133518335,"identity":"e89059ce-daac-410f-871e-0c5a28c51d34","order_by":3,"name":"Mitsugu Watanabe","email":"","orcid":"","institution":"Watanabe Oyster Laboratory Co. Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mitsugu","middleName":"","lastName":"Watanabe","suffix":""}],"badges":[],"createdAt":"2022-08-31 22:59:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2019515/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2019515/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":26118067,"identity":"4598c3df-4116-4ff2-a391-dd4964cc17fa","added_by":"auto","created_at":"2022-09-06 14:37:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53092,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical structure of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA). The molecular formula of DHMBA is C8H10O4 and its molecular weight is 170.164. [12, 18].\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/f26b68d0ef8e67b945ed76fa.jpg"},{"id":26118412,"identity":"a2f2c204-7815-4ccc-a4ea-6c4a80791797","added_by":"auto","created_at":"2022-09-06 14:42:56","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62533,"visible":true,"origin":"","legend":"\u003cp\u003eThe marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) represses the growth of mouse macrophage RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e.\u0026nbsp;Cells (1x10\u003csup\u003e5\u003c/sup\u003e cells/ml per well in 24-well plates) were cultured for 1 (A), 2 (B), 3 (C), or 4 (D) days in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 µM). After the culture, the number of cells attached to the dish was counted. Data are presented as the mean ± SD of the value obtained from 8 wells in a total of 2 replicate plates by using different cell preparations. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 versus control group without DHMBA (gray bar). 1-way ANOVA, Tukey-Kramer post-test.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/07cddc3b106db649debbff01.jpg"},{"id":26118408,"identity":"1b21797f-93a4-4695-b0d5-3b73c99d89a4","added_by":"auto","created_at":"2022-09-06 14:42:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69901,"visible":true,"origin":"","legend":"\u003cp\u003eThe marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) regulates the levels of various proteins linked to the proliferation of mouse macrophage RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e. \u0026nbsp;Cells (1x10\u003csup\u003e6 \u003c/sup\u003ecells/10 ml of medium in 100 mm dishes) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone in the presence of either (1% ethanol as a final concentration) or DHMBA (10 µM). After culture, the cells were removed from the dish with a cell scraper in cell lysis buffer containing protease inhibitors. Forty micrograms of supernatant protein per lane were separated by SDS-PAGE (12%) and transferred to nylon membranes for Western blotting by using antibodies against various proteins. (A) Representative data are presented. (B) The band was presented as a fold of control. Data are presented as the mean ± SD of the value obtained from 4 dishes by using different cell preparations. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus control. 1-way ANOVA, Tukey-Kramer post-test.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/f950950fbe1e63ef7e6bd17c.jpg"},{"id":26118072,"identity":"996b28d5-4fe7-4a40-b49b-9dee6e803533","added_by":"auto","created_at":"2022-09-06 14:37:56","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":69945,"visible":true,"origin":"","legend":"\u003cp\u003eThe marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) stimulates the death of mouse macrophage RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e. Cells (1x10\u003csup\u003e5\u003c/sup\u003e cells/ml per well in 24-well plates) were cultured in DMEM containing 10% FBS and 1% P/S and 1% fungizone for 3 days on reaching subconfluence, and then the cells were additionally cultured for 24 (A) or 48 (B) hours in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 µM), respectively. (C) RAW264.7 cells on reaching subconfluence were additionally cultured for 48 hours in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (1 or 10 µM) with or without caspase-3 inhibitor (10 µM). Several cells attached to the dish were counted. Data are presented as the mean ± SD obtained from 8 wells in a total of 2 replicate plates using different cell preparations. (D) To determine the levels of caspase-3 or cleaved caspase-3, PC-3 (1x10\u003csup\u003e6 \u003c/sup\u003ecells/10 ml of medium in 100 mm dishes) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone in the presence of either (1% ethanol as a final concentration) or DHMBA (10 µM). After culture, the cell lysates were obtained for assay of Western blotting as described in the legends of Figure 2. (D) Representative data are presented. (E) The band was presented as a fold of control. Data are presented as the mean ± SD of the value obtained from 4 dishes by using different cell preparations. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus control without DHMBA (gray bar). 1-way ANOVA, Tukey-Kramer post-test.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/62bc131b7a347e5e57da7e2f.jpg"},{"id":26118066,"identity":"ac747cd0-dd24-45f0-87ee-886694d96b26","added_by":"auto","created_at":"2022-09-06 14:37:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":68412,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the proliferation and death of mouse macrophage RAW264.7 cells with or without stimulation of lipopolysaccharide (LPS) \u003cem\u003ein vitro\u003c/em\u003e. (A) To determine the effects of cell proliferation, cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) were cultured in DMEM containing 10% FBS and 1% P/S and 1% fungizone for 3 days in the presence of either vehicle (1% ethanol as a final concentration) or LPS (1, 10, 50, 100, or 500 ng/ml). (B) Cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) were cultured for 3 days in DMEM containing 10% FBS and 1% P/S and 1% fungizone in the presence of DHMBA (1 or 10 μM) with or without LPS (100 ng/ml). (C) To determine the effects on cell death, the cells on reaching subconfluency with culture for 3 days were furthermore cultured for 48 hours in the presence of either vehicle (1% ethanol) or lipopolysaccharide (LPS) (1, 10, 50, 100, or 500 ng/ml). (D) The cells on reaching subconfluency were additionally cultured for 48 hours in the presence of DHMBA (1 or 10 μM) with or without LPS (100 ng/ml). After the culture, the number of attached cells on the dish was counted. Data are presented as the mean ± SD of the value obtained from 8 wells in a total of 2 replicate plates by using different cell preparations. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus control without LPS (A and C) (gray bar) or DHMBA (B and D) (gray bar). 1-way ANOVA, Tukey-Kramer post-test.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/e25d91cfb0fa1a0e01594385.jpg"},{"id":26118409,"identity":"593f56f4-6b4e-4809-b0e2-1fab2d0c771a","added_by":"auto","created_at":"2022-09-06 14:42:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":42498,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the number of mouse macrophage RAW264.7 cells with or without stimulation of lipopolysaccharide (LPS) \u003cem\u003ein vitro\u003c/em\u003e. Cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) were cultured in DMEM containing 10% FBS and 1% P/S and 1% fungizone for 5 hours in the presence of either vehicle (1% ethanol as a final concentration) without (A) or with (A) LPS (1, 10, 50, 100, or 1000 ng/ml). (B) Cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) were cultured for 3 days in DMEM containing 10% FBS and 1% P/S and 1% fungizone in the presence of DHMBA (1 or 10 μM) with or without LPS (100 ng/ml). After the culture, the number of attached cells on the dish was counted. Data are presented as the mean ± SD of the value obtained from 8 wells in a total of 2 replicate plates by using different cell preparations. The number of cells was not significantly altered by DHMBA and/or LPS treatment as compared with that of control (gray bar). 1-way ANOVA, Tukey-Kramer post-test.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/c1b1dfd71df9f73639a3c7ec.jpg"},{"id":26118943,"identity":"88933a75-94e4-4f01-ac16-2e9f6ee73726","added_by":"auto","created_at":"2022-09-06 14:47:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":527059,"visible":true,"origin":"","legend":"\u003cp\u003eThe marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) overwhelms the production of TNF-α, IL-6, IL-1β, and PGE\u003csub\u003e2 \u003c/sub\u003eafter the stimulation of lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e. Cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well in 24-well plates) were cultured in DMEM in the absence of DHMBA for 3 days on reaching subconfluence Then, the cells were treated with the addition of either vehicle (1% ethanol), DHMBA (0.1, 1, 10, 100 or 1000 μM) without or with LPS (100 ng/ml), and they were additionally incubated for 5 hours. After culture, the medium was collected, and the concentration of cytokines, including TNF-α (A), IL-6 (B), IL-1β (C), and PGE\u003csub\u003e2 \u003c/sub\u003e(E)) in the medium was determined using ELISA Kit. Data are presented as the mean ± SD of the value obtained from 8 wells in a total of 2 replicate plates by using different cell preparations. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus control without DHMBA and LPS (white bar). \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus control with LPS and without DHMBA. 1-way ANOVA, Tukey-Kramer post-test.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/3d7a14ab1ed9b8e0a6ac8e7b.jpg"},{"id":26118074,"identity":"69f163df-db20-44de-afb7-de5778200d36","added_by":"auto","created_at":"2022-09-06 14:37:56","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":381197,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of\u003cstrong\u003e \u003c/strong\u003ethe marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the levels of COX-1, COX-2, MAPK, phosphorylated MAPK, NF-κB p65, and STAT3 with the stimulation of lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e. Cells (1x10\u003csup\u003e6\u003c/sup\u003e cells/10 ml of medium) were cultured in DMEM for 3 days on reaching subconfluence. Then, the cells were treated with either vehicle (1% ethanol) or DHMBA (10 μM) without or with LPS (100 ng/ml), and they were additionally incubated for 5 hours. After culture, the cell lysates were obtained for assay of Western blotting by using specific antibodies against COX-1, COX-2, MAPK, phosphor-MAPK, NF-κB p65, STAT3, and β-actin as described in the legends of Figure 2. (A) Representative data are presented. (B-D) The band was presented as a fold of control without LPS (B), with LPS (100 ng/ml) alone (C), and with LPS (100 ng/ml) and DHMBA (10 μM). Data are presented as the mean ± SD of the value obtained from 4 dishes by using different cell preparations. (C) *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus control without LPS. (D) *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 versus control with LPS without DHMBA. 1-way ANOVA, Tukey-Kramer post-test.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/1d7e3599e7df0d766d73f73a.jpg"},{"id":26118069,"identity":"13b9302f-4eb9-478a-b3b8-aa84b28cda0b","added_by":"auto","created_at":"2022-09-06 14:37:56","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":64591,"visible":true,"origin":"","legend":"\u003cp\u003eThe marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits osteoclastogenesis enhanced with the stimulation of lipopolysaccharide (LPS) in mouse macrophage RAW264.7 cells \u003cem\u003ein vitro. \u003c/em\u003e(A) Cells (1 x 10\u003csup\u003e5\u003c/sup\u003e cells/1 ml per well in 24-well plates) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone in the presence of either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml) with or without DHMBA (0.1, 1, 10, or 100 μM), and then 0.5 ml of old medium was replaced with fresh conditioned medium (0.5 ml) containing LPS and/or DJMBA and were additionally cultured for 3 days. (B) Cells (1 x 10\u003csup\u003e5\u003c/sup\u003e cells/1 ml per well in 24-well plates) were cultured for 3 days in the above DMEM containing LPS (100 ng/ml) without DHMBA, and then 0.5 ml of old medium was replaced with fresh medium (0.5 ml) containing LPS (100 ng/ml) and/or DHMBA (0.1, 1, 10, or 100 μM) and were additionally cultured for 3 days. After culture, cells adherent to the plates were fixed and stained for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. TRACP-positive multinucleated cells (MNCs) (3 or more nuclei) were counted as osteoclast-like cells under a microscope (40x). Data are presented as the mean ± SD of the value obtained from 8 wells in a total of 2 replicate plates per data set by using different cell preparations. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 versus control (white bar). \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 versus LPS (gray bar). 1-way ANOVA, Tukey-Kramer post-test.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/e43bb7c3d4be337575f7ede8.jpg"},{"id":26124038,"identity":"1c41ba80-96a8-48da-aae2-a69b8692b37a","added_by":"auto","created_at":"2022-09-06 15:59:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":915670,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/ec4cd64a-928f-4944-90a2-2d34fa3b3905.pdf"},{"id":26119099,"identity":"035a1dd0-f698-4f68-8c4c-6d2414565851","added_by":"auto","created_at":"2022-09-06 14:52:56","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":12196,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-2019515/v1/787ee110760ffc50a872a1cd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol expresses the anti-inflammatory effects in mouse macrophages RAW264.7 cells in vitro","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eInflammation is a complicated biological response of body tissues to damaging motivation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], and it also is a protective response connecting immune cells, blood vessels, and molecular mediators [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Inflammatory cytokines, including interleukins (ILs) and tumor necrosis factor (TNF)-α, are known as the biomarkers in chronic and experimental human muscle pain [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and osteoarthritis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These cytokines are produced by macrophages under inflammatory conditions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Of note, inflammatory macrophages potentially contribute to the enhancement of progression, metastasis, and angiogenesis of cancer cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRAW264.7 cells are monocyte/macrophage-like cell lineage [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This RAW264.7 cell lineage is characterized by the macrophage-mediatedMetabnMetabtab,olic and phagocytic functions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. RAW264.7 cells are progressively used as modeled macrophages in inflammatory conditions \u003cem\u003ein vitro\u003c/em\u003e, This cell line is also accepted as a modeled cell of osteoclastogenesis study [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Osteoclasts are differentiated from the monocyte-macrophage lineage [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Lipopolysaccharide (LPS) is a core antigen of gram-negative bacteria, which activates the innate immune system of the host [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. LPS is an endotoxin that provides a persistent inflammatory stimulus to the tissue [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. LPS induces osteoclastogenesis of RAW264.7 cells by regulating NF-κB-related signaling pathways and transcriptional activity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was initially found in the Pacific oyster \u003cem\u003eCrassostrea Gigas\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. DHMBA has dual properties to prevent oxidative stress as radical scavenging in cells [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. DHMBA has been also reported to reveal a preventive effect on excess glutamatergic neuron activity in rats and mice \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, DHMBA may play a role in the regulation of cell function as an antioxidant [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, our recent study has demonstrated that DHMBA suppresses the growth of metastatic prostate cancer cells via targeting diverse signaling pathways, providing a new strategy for prostate cancer therapy with DHMBA [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Elucidating the pharmacologic effects of DHMBA may be significant in the therapy of various diseases.\u003c/p\u003e \u003cp\u003eThis study has been undertaken to elucidate whether DHMBA impacts the activity of inflammatory macrophages \u003cem\u003ein vitro.\u003c/em\u003e Here, we demonstrate that culturing with DHMBA blocks the proliferation and stimulates the death of inflammatory mouse macrophages RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e, leading to a diminishing of cell number. Moreover, DHMBA was found to repress the enhancement of inflammatory cytokine production in RAW264.7 cells cultured with LPS \u003cem\u003ein vitro.\u003c/em\u003e Interestingly, we found that osteoclastogenesis of RAW264.7 cells with LPS stimulation was suppressed by DHMBA treatment. Thus, the novel marine factor DHMBA may have a pharmacologic effect on inflammation implicated in macrophages. Our study may offer a useful therapeutic tool for the inflammatory condition with DHMBA.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents\u003c/h2\u003e \u003cp\u003eDulbecco\u0026rsquo;s Modification of Eagle\u0026rsquo;s Medium (DMEM) with 4.5 g/L glucose, L-glutamine and sodium pyruvate and antibiotics (100 units/mL penicillin and 100 \u0026micro;g/mL streptomycin; 1% P/S) was obtained from Corning (Mediatech, Inc. Manassas, VA, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Amphotericin B (fungizone), caspase-3 inhibitor (CAS 169332-60-9-Calbiochem), lipopolysaccharide (LPS), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The caspase-3 inhibitor was diluted in sterile phosphate-bufferedfered saline (PBS). Other reagents were dissolved in 100% ethanol, and these reagents were stored at -20℃ until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. 3, 5-dihydroxy-4-methoxybenzyl alcohol\u003c/h2\u003e \u003cp\u003e3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a novel amphipathic phenolic compound, was initially isolated from the pacific oyster (\u003cem\u003eCrassostrea Gigas\u003c/em\u003e) with a characterization of antioxidant [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. We used the synthesized DHMBA in the present study [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The purity of synthesized DHMBA was 100% [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. DHMBA was dissolved in 100% ethanol and stored at -20℃ until use.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. RAW264.7 cells\u003c/h2\u003e \u003cp\u003eRAW264.7 cells are monocyte/macrophage-like cell lineage, originating from \u003cem\u003eAbelson leukemia\u003c/em\u003e virus-transformed cell lineages derived from BALB/c mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mouse macrophage RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. RAW264.7 cells were cultured in DMEM containing 10% FBS, 1% P/S, and 1% fungizone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Assay of cell growth\u003c/h2\u003e \u003cp\u003eTo determine development of cell proliferation, RAW264.7 cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) were cultured using 24-well plates in DMEM containing 10% FBS, 1% P/S and 1% fungizone in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 \u0026micro;M) for 1, 2, 3, or 4 days in a water-saturated atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air at 37\u003csup\u003eo\u003c/sup\u003eC [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In other experiments, to investigate the effects of DHMBA on the growth of RAW264.7 cells in the presence of LPS, the cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) were cultured using 24-well plates in DMEM (containing 10% FBS, 1% P/S, and 1% fungizone) in the presence of either vehicle (1% ethanol as a final concentration) or LPS (1, 10, 50, 100, or 500 ng/ml of medium) with or without DHMBA (1 or 10 \u0026micro;M) for 3 days. After culture, the cells were detached from each well by adding a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e-free PBS (Thermo Fisher Scientific, Waltham, MA, USA) with incubation for 2 min at 37℃. Each well was then added 0.9 ml of DMEM containing 10% FBS, and 1% P/S. The number of cells in the cell suspension was counted as described below in the section \u0026ldquo;Cell counting\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Assay of cell death\u003c/h2\u003e \u003cp\u003eRAW264.7 cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well in 24-well plates) were cultured using 24-well plates in DMEM containing 10% FBS, 1% P/S, and 1% fungizone for 3 days. Cells on reaching subconfluence were cultured for an additional 24 or 48 hours in the presence of either vehicle (PBS or 1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 \u0026micro;M) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In other experiments, the cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) on reaching subconfluence with culturing for 3 days were additionally cultured for 48 hours in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (1 or 10 \u0026micro;M) with or without caspase-3 inhibitor (10 \u0026micro;M) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In additional experiments, to establish the effects of DHMBA on the death of RAW264.7 cells cultured in the presence of LPS, the cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) were cultured using 24-well plates in DMEM (containing 10% FBS, 1% P/S, and 1% fungizone) for 3 days on reaching subconfluence, and then the cells were furthermore cultured for 48 hours in the presence of either vehicle (1% ethanol as a final concentration) or LPS (1, 10, 50, 100, or 500 ng/ml of medium) with or without DHMBA (1 or 10 \u0026micro;M). After culture, the cells were detached by adding a sterile solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e-free PBS per well as explained in the section \u0026ldquo;Cell growth assay\u0026rdquo;, and the cell number was counted as described below in the section \u0026ldquo;Cell counting\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Cell counting\u003c/h2\u003e \u003cp\u003eAfter culture, to detach cells on each well, the culture dishes were incubated for 2 min at 37℃ after adding a solution (0.1 ml per well) of 0.05% trypsin plus EDTA in Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e-free PBS, and then the cells were detached and mixed through pipetting after addition of DMEM (0.9 ml) containing 10% FBS and 1% P/S as explained in the previous study [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The number of viable cells was counted under a microscope (Olympus MTV-3) with a Hemocytometer (Sigma-Aldrich) by using a cell counter (Line Seiki H-102P, Tokyo, Japan). For each dish, we took the average of two counts. Cell numbers were shown as numbers per well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Assay of cytokine production\u003c/h2\u003e \u003cp\u003eRAW264.7 cells (1x10\u003csup\u003e5\u003c/sup\u003e/ml per well) were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S for 3 days in reaching upon subconfluence [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and then the cells were further cultured for 5 hours after the treatment with either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 \u0026micro;M) with or without LPS (100 ng/ ml). After incubation, the medium was collected to assay cytokines, and then the cells were detached from each culture dish to determine the number of cells as described in \u0026ldquo;cell counting\u0026rdquo;. The concentrations of TNF-α, IL-1β, IL-6, or PGE2 in the medium were analyzed using ELISA Kits for mouse TNF-α [catalog number (cat. no. BM), KHC301)] and IL-1β (cat. no., BMS6002) obtained from (ThermoFisher Scientific, Waltham, MA, USA) or IL-6 (cat. no. 583371) and PGE2 (cat. no. 514010) purchased from Cayman Chemical (Ann Arbor, MI, USA), according to the manufacturer\u0026rsquo;s instructions. The production of each cytokine was presented as a pictogram (pg) secreted into a culture medium (ml).\u003c/p\u003e \u003cp\u003eIn separate experiments, to determine the levels of Cox-1, Cox-2, NF-κB p65, and STAT3, which are implicated in cytokine signaling, RAW264.7 cells (1x10\u003csup\u003e6\u003c/sup\u003e cells/10 ml of 100 mm dishes) were cultured for 3 days on reaching subconfluence in DMEM containing 10% FBS, 1% P/S and 1% fungizone, and then the cells were additionally cultured for 5 hours in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (10 \u0026micro;M) with or without LPS (100 ng/ml of medium). After culture, the attached cells were removed from the dish by scraping in cell lysis buffer as described in the section \u0026ldquo;Western blot assay\u0026rdquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Western blotting\u003c/h2\u003e \u003cp\u003eRAW264.7 cells (1x10\u003csup\u003e6\u003c/sup\u003e cells/10 ml of 100 mm dishes) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (10 \u0026micro;M), and then the dishes were washed three times with cold PBS (10 ml) to exclude floating and dead cells and the attached cells removed from the dish by scraping in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease and protein phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA) as explained in the previous study [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The lysates were then centrifuged at 17,000x\u003cem\u003eg\u003c/em\u003e, at 4℃ for 10 min. Protein concentration in the supernatant was determined using the Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin as a standard. The cell lysate was stored at -80℃ until use. Samples of forty micrograms of supernatant protein per lane were separated by SDS polyacrylamide gel electrophoresis (12% SDS-PAGE) and then transferred to PVDF membranes. Transferred membranes were immunoblotted using specific antibodies against various proteins obtained from Cell Signaling Technology (Danvers, MA, USA), including Ras (cat. no. 3339, rabbit), Akt (cat. no. 9272, rabbit), mitogen-activated protein kinase (MAPK; cat. no. 4695, rabbit), phosphorylated-MAPK (cat. no. 4370, rabbit), mechanistic target of rapamycin (mTOR, cat. no. 4517, mouse), Rb (cat. no. 9309, mouse), p21 (cat. no. 2947, rabbit), STAT3 (cat. no. 12640, rabbit), COX-1 (cat. no. 48415), COX-2 (cat. no. 4842), and β-actin (cat. no. 3700, mouse), and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), including p53 (cat. no. sc-126, mouse), and NF-κB p65 (cat. no. sc-109, rabbit). Rabbit anti-regucalcin antibody was obtained from Sigma-Aldrich (cat. no. HPA029103, rabbit). Target proteins were incubated with one of the primary antibodies (1:1,000) as described above overnight at 4℃. After incubation, the membranes were additionally incubated for 60 min at room temperature in horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., mouse sc-2005 or rabbit sc-2305; diluted 1:2,000) at room temperature, and protein bands were detected using a Chemiluminescence substrate (cat. no. 34577, Thermo Scientific, Rockford, IL, USA) on X-ray film. A total of 3 or 4 films from 4 independent experiments on separate membranes were scanned on an Epson Perfection 1660 Photo scanner, and the bands were quantified using Image J2 software (National Institutes of Health, Bethesda, MD, USA). For immunoblotting with additional antibodies, we used the restored Western blot stripping buffer (cat. no. 21059; Thermo Scientific, Rockford, IL, USA) to remove the attached Chemiluminescence substrate (Thermo Scientific) by incubation at room temperature for 30 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Assay of osteoclastogenesis\u003c/h2\u003e \u003cp\u003eRAW264.7 cells (1 x 10\u003csup\u003e5\u003c/sup\u003e cells/1 ml per well in 24-well plates) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml of medium) with or without DHMBA (0.1, 1, 10, or 100 \u0026micro;M), and then 0.5 ml of old medium was replaced with fresh medium (0.5 ml) including above LPS or DHMBA. The cells were additionally cultured for 3 days [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In a separate experiment, RAW264.7 cells (1 x 10\u003csup\u003e5\u003c/sup\u003e cells/1 ml per well in 24-well plates) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S, and 1% fungizone in either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml of medium) without DHMBA, and then 0.5 ml of old medium was replaced with fresh medium (0.5 ml) including either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml of medium) with DHMBA (as a final concentration of 0.1, 1, 10, or 100 \u0026micro;M).. The cells were additionally cultured for 3 days [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. After culture, RAW264.7 cells adherent to the plates were fixed and stained for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Cells were washed with PBS solution and fixed with 10% neutralized formalin-phosphate (pH7.2) for 1 minute. The fixed cells were incubated for 10 hours at room temperature in acetate buffer (pH 5.0) containing naphthol AS-MX phosphate (Sigma-Aldrich) as a stain for the reaction product in the presence of 10 mM sodium tartrate. TRACP-positive multinucleated cells (MNCs) containing three or more nuclei were counted as osteoclast-like cells under a microscope (40x) (Olympus MTV-3; Olympus Corporation, Tokyo, Japan). To assess the number of osteoclast-like TRACP-positive MNCs, one field per well was photographed and measured using ImageJ2 software. The number of osteoclast-like TRACP-positive MNCs was counted in five random fields under a light microscope (40x) (Olympus MTV-3; Olympus Corporation, Tokyo, Japan) and averages were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance was estimated using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc. La Jolla, CA). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). We used Student-\u003cem\u003et\u003c/em\u003e-test to calculate statistical significance between the 2 groups. As indicated, Multiple comparisons were performed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons post-test for the parametric data. A p-value of \u0026lt;\u0026thinsp;.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.1. DHMBA represses the growth of RAW264.7 cells.\u003c/h2\u003e\n \u003cp\u003eFirst, we investigated whether DHMBA influences the growth of mouse macrophage RAW264.7 cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). RAW264.7 cells were cultured for 1, 2, 3, and 4 days in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 \u0026micro;M). The growth of RAW264.7 cells was blocked by culturing with DHMBA (1, 10, 100, or 1000 \u0026micro;M) for 1\u0026ndash;4 days. Thus, DHMBA was found to repress the growth of RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eTo better understand the underlying mechanism by which DHMBA suppresses the proliferation of RAW264.7 cells, we determined whether DHMBA regulates the expression of key proteins linked to the proliferation of RAW264.7 cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Culturing with DHMBA (10 \u0026micro;M) diminished the levels of Ras, PI3 kinase, Akt, MAPK, phospho-MAPK, and mTOR, which are implicated in the promotion of the proliferation of RAW264.7 cells [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], while it increased the levels of p53, Rb, p21, and regucalcin that lead to repression of cell proliferation [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. These results suggest that the alterations of signaling proteins and cell growth suppressors are involved in the mechanism by which DHMBA blocks the growth of RAW264.7 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.2. DHMBA promotes the death of RAW264.7 cells.\u003c/h2\u003e\n \u003cp\u003eFurthermore, it was elucidated whether DHMBA impacts the death of mouse macrophage RAW264.7 cells. Cells were cultured for 3 days on reaching subconfluence, and then they were further cultured for 24 or 48 hours in the presence of either vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 \u0026micro;M). The death of RAW264.7 cells was promoted by culturing with DHMBA (1, 10, 100, or 1000 \u0026micro;M) for 24 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) or 48 hours (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). The stimulatory effects of DHMBA (1 or 10 \u0026micro;M) on the death of RAW264.7 cells were blocked by the presence of caspase-3 inhibitor (10 \u0026micro;M) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results of Western blotting indicated that the levels of caspase-3 and cleaved caspase-3 in the cells were increased by culturing with DHMBA (10 \u0026micro;M) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results suggest that DHMBA stimulates apoptotic cell death of mouse macrophage RAW264.7 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.3. Effects of DHMBA on RAW264.7 cells cultured with LPS\u003c/h2\u003e\n \u003cp\u003eNext, we investigated the suppressive effects of DHMBA on inflammatory macrophage RAW264.7 cells with LPS treatment. LPS is well known to enhance the inflammatory activity of mouse macrophage RAW264.7 cells [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. It was determined whether DHMBA attenuates the repressive effects on the proliferation or the stimulatory effects on the death of RAW264.7 cells cultured in the presence of LPS \u003cem\u003ein vitro\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). RAW264.7 cells were cultured in the presence of LPS (1, 10, 50, 100, or 500 ng/ml of medium) for 3 days. The growth of RAW264.7 cells was not altered by culturing with LPS (1, 10, 50, and 100 ng/ml), while it was suppressed by culturing with a higher concentration of LPS (500 ng/ml) (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the presence of LPS (100 ng/ml), DHMBA (1 or 10 \u0026micro;M) also suppressed the growth of RAW264.7 cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eTo determine the effects of DHMBA on cell death cultured in the presence of LPS, moreover, RAW264.7 cells on reaching subconfluence by culturing for 3 days were additionally cultured in the presence of LPS (1, 10, 50, 100, or 500 ng/ml of medium) for 48 hours (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Culturing with LPS (1, 10, 50, and 100 ng/ml) did not cause a significant effect on cell number, while a higher level of LPS (500 ng/ml) stimulated the death of RAW264.7 cells. In the presence of LPS (100 ng/ml), DHMBA (1 or 10 \u0026micro;M) stimulated the death of RAW264.7 cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results indicate that DHMBA keeps activity that decreases the number of RAW264.7 cells in the presence of LPS (100 ng/ml).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003e3.4. DHMBA suppresses inflammatory cytokine production in RAW264.7 cells\u003c/h2\u003e\n \u003cp\u003eFurthermore, we elucidated whether DHMBA impacts the production of inflammatory cytokines in mouse macrophage RAW264.7 cells cultured in the presence of LPS (100 ng/ml) \u003cem\u003ein vitro.\u003c/em\u003e RAW264.7 cells were cultured for 3 days on reaching subconfluence, and then the cells were moreover incubated for 5 hours after the addition of LPS (100 ng/ml) with or without DHMBA (0.1, 1, 10, 100, or 1000 \u0026micro;M). The number of RAW264.7 cells was not changed by adding DHMBA without (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA) and with LPS (100 ng/ml of medium) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). Under the same condition of culture which did not cause an alteration of cell number, we determined the production of inflammatory cytokines, including TN F-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), IL-6 (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB), IL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC), or PGE2 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD), in the medium obtained by culturing with RAW264.7 cells. In the absence of LPS, the productions of IL-6, or IL-1\u0026beta; in RAW264.7 cells were repressed by the addition of DHMBA (100 or 1000 \u0026micro;M) (Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB and F). Notably, the treatment with LPS caused a remarkable increase in the production of TNF-\u0026alpha; (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA), IL-6 (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB), IL-1\u0026beta; (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC), or PGE\u003csub\u003e2\u003c/sub\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). These increases were suppressed by the treatment of DHMBA (1, 10, 100, or 1000 \u0026micro;M). Thus, the production of inflammatory cytokines in RAW264.7 cells was found to be suppressed by DHMBA treatment.\u003c/p\u003e\n \u003cp\u003eIt was elucidated whether DHMBA regulates the levels of proteins implicated in cytokine production and is linked to intracellular signaling processes of cytokines (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). RAW264.7 cells were incubated for 5 hours in the presence of LPS (100 ng/ml) with or without DHMBA (10 \u0026micro;M) \u003cem\u003ein vitro\u003c/em\u003e. In a culture of RAW264.7 cells without LPS, the levels of COX-1, COX-2, MAPK, phosphor-MAPK, NF-\u0026kappa;B p65, and STAT3 were not altered by the treatment with DHMBA as compared with those of control (Figs. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA and B). The levels of MAPK, phospho-MAPK, NF-\u0026kappa;B p65, and STAT3 in RAW264.7 cells were increased by culturing with LPS (100 ng/ml) (Figs. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA and C). These increases were repressed by the treatment with DHMBA (10 \u0026micro;M) (Figs. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA and D). These results suggest that DHMBA treatment suppresses the levels of signaling factors implicated in the production of cytokines in inflammatory RAW264.7 cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003e3.5. DHMBA inhibits osteoclastogenesis of RAW264.7 cells\u003c/h2\u003e\n \u003cp\u003eRAW264.7 cells are well used as modeled cells in osteoclastogenesis studies [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. Osteoclasts are differentiated from the monocyte-macrophage lineage [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. LPS has been shown to stimulate osteoclastogenesis of RAW264.7 cells by activating the NF-\u0026kappa;B pathway [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. We investigated whether DHMBA influences osteoclastogenesis of RAW264.7 cells cultured with LPS. RAW264.7 cells (1 x 10\u003csup\u003e5\u003c/sup\u003e cells/1 ml per well in 24-well plates) were cultured for 3 days in DMEM containing 10% FBS, 1% P/S and 1% fungizone either vehicle (1% ethanol as a final concentration) or LPS (100 ng/ml of medium) with or without DHMBA (0.1, 1, 10, or 100 \u0026micro;M). After culture for 3 days, 0.5 ml of old medium was replaced with fresh medium (0.5 ml) including LPS (100 ng/ml) or DHMBA (0.1, 1, 10, or 100 \u0026micro;M), and then the cells were furthermore cultured for 3 days [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. In culturing with both LPS and DHMBA for 6 days (Figs. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA and C), LPS enhanced osteoclastogenesis of RAW264.7 cells. This enhancement was suppressed by culturing with DHMBA (0.1, 1, 10, or 100 \u0026micro;M) (Figs. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA and C). In separate experiments, RAW267.4 cells were cultured in the presence of LPS (100 ng/ml) for 3 days without DHMBA, and then the cells were additionally cultured for 3 days with the replacement of fresh medium including LPS (100 ng/ml) with or without DHMBA (0.1, 1, 10, or 100 \u0026micro;M). Osteoclastogenesis at a later stage enhanced by LPS was also suppressed by DHMBA (Figs. 8Band D). These results support the view that DHMBA suppresses osteoclastogenesis enhanced with LPS stimulation in RAW264.7 cells.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe novel phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has dual characteristics to weaken oxidative stress as radical scavenging in cells [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. DHMBA may play a role in the regulation of cell function as an antioxidant [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our previous study demonstrated that DHMBA suppressed the growth and activity of metastatic prostate cancer cells via targeting diverse signaling pathways, providing a new strategy for prostate cancer therapy [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Furthermore, we elucidated whether DHMBA reveals anti-inflammatory effects by using mouse macrophage RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e. We found that DHMBA revealed anti-inflammatory effects, leading to a reduction of the number of inflammatory macrophages, repression of inflammatory cytokine production, and inhibition of osteoclastogenesis development in RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCulturing with DHMBA suppressed the growth and stimulated the death of RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e, causing to decline in the number of macrophages. DHMBA treatment diminished the levels of Ras, PI3 kinase, Akt, MAPK, phospho-MAPK, and mTOR, which lead to the promotion of the proliferation of RAW264.7 cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], while it increased the levels of p53, Rb, p21, and regucalcin that induce repression of cell proliferation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The diminish of these cell signaling-related protein levels and the enhancement of cell growth suppressor with DHMBA treatment may contribute to the underlying mechanism by which the compound blocks the growth of RAW264.7 cells. DHMBA may regulate the expression of various proteins linked to cell signaling and transcriptional activity. Furthermore, the levels of caspase-3 and cleaved caspase-3 implicated in apoptotic cell death were increased by DHMBA treatment. These augmentations may cause activation of nuclear DNA fragmentation that induces apoptotic cell death [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thus, DHMBA may impact the levels of diverse proteins linked to the control of cell numbers.\u003c/p\u003e \u003cp\u003eDHMBA was also found to powerfully suppress the production of inflammatory cytokines, including TNF-α, IL-6, IL-1β, or PGE2, with the treatment of LPS that stimulates the inflammatory condition of macrophage RAW264.7 cells. These reductions were observed under culture conditions that did not cause a decrease in the number of RAW264.7 cells when cultivated with both LPS and DHMBA.DH revealed inhibition of cytokine production independent of alteration of the number of RAW264.7 cells. LPS treatment has been shown to enhance the production of TNF-α, IL-6, IL-1β, or PGE\u003csub\u003e2\u003c/sub\u003e in RAW264.7 cells [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We found that the production of these cytokines enhanced by LPS treatment was suppressed by culturing with DHMBA in RAW264.7 cells \u003cem\u003ein vitro.\u003c/em\u003e DHMBA treatment may be a useful tool in the suppression of cytokines production under inflammatory conditions.\u003c/p\u003e \u003cp\u003eFurther study was undertaken to better understand the underlying mechanism by which DHMBA represses cytokine production in inflammatory RAW264.7 cells with LPS stimulation. LPS binds to Toll-like receptor 4 (TLR4) on the plasma membranes of macrophage RAW264.7 cells, and the signaling with LPS/TLR4 pathway is transmitted into the cells [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Notably, TLR4 signaling activates NF-κB and MAPK signaling in RAW264.7 cells [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Also, there is growing evidence that LPS internalizes and binds intracellular proteins and receptors [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The production of TNF-α, IL-6, IL-1β, or PGE\u003csub\u003e2\u003c/sub\u003e with LPS treatment may be implicated in intracellular signaling NF-κB p65, and/or MAPK in RAW264.7 cells [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. COX-1 and COX-2 may be implicated in the production of PGE2 in RAW264.7 cells [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our studies demonstrated that the levels of NF-κB p65, MAPK, and phosphorylated MAPK were increased by culturing with LPS of RAW264.7 cells. These augmentations were repressed by DHMBA treatment. This repression may lead to the suppression of the LPS-enhanced production of inflammatory cytokines in RAW264.7 cells. Moreover, it is possible that DHMBA disturbs the binding of LPS to TLR4 and/or intracellular receptor proteins to weaken the production of inflammatory cytokines in RAW264.7 cells. DHMBA is also speculated to regulate transcriptional activity linked to the production of inflammatory cytokines in RAW264.7 cells. In addition, DHMBA may play a role in the repression of cytokine production as an antioxidant [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Further study remains to be elucidated the molecular mechanism.\u003c/p\u003e \u003cp\u003eInterestingly, the levels of STAT3 were increased by LPS treatment, and this enhancement was repressed by culturing with DHMBA in RAW264.7 cells. STAT3 is involved in the intracellular signaling of IL-6 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. LPS stimulation caused the production of IL-6 and IL-1β in RAW264.7 cells. These cytokines may impact the activity of RAW264.7 cells with their autocrine effects. DHMBA treatment may disturb the signaling process which is mediated via IL-6 and IL-1β produced in RAW264.7 cells.\u003c/p\u003e \u003cp\u003eLPS stimulates osteoclastogenesis via activation of NF-κB signaling in RAW264.7 cells [37\u0026ndash;40]. LPS treatment leads to activation of TLR4 signaling linked to NF-κB in macrophages [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Osteoclastogenesis enhanced by culturing with LPS was suppressed by the treatment of DHMBA in RAW264.7 cells. This suppression was also observed by DHMBA treatment at the earlier and later stages of osteoclastogenesis. Presumably, the suppressive effects of DHMBA on osteoclastogenesis may be related to the inhibition of NF-κB signaling implicated in the decreased NF-κB p65 levels by DHMBA treatment.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that DHMBA represses the proliferation and stimulates the death of RAW264.7 cells, leading to a reduction in the number of inflammatory macrophages. DHMBA was also demonstrated to repress the production of inflammatory cytokines. Furthermore, DHMBA treatment repressed osteoclastogenesis of RAW264.7 cells with LPS stimulation \u003cem\u003ein vitro\u003c/em\u003e. DHMBA may reveal anti-inflammatory effects by using modeled mouse macrophage RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e, although it remains to be elucidated whether DHMBA has an anti-inflammatory effect \u003cem\u003ein vivo.\u003c/em\u003e DHMBA is a functional-medical food factor with very low toxicity. DHMBA may be a pharmacologic significant in the treatment of inflammatory conditions, providing a new strategy for prevention and therapy of inflammation\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMasayoshi Yamaguchi, PhD\u003c/strong\u003e:\u0026nbsp;Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKenji Yosiike\u003c/strong\u003e, \u003cstrong\u003eMS\u003c/strong\u003e: Investigation, Resources, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHideaki Watanabe, MD\u003c/strong\u003e: Investigation, Resources, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitsugu Watanabe, PhD\u003c/strong\u003e:\u0026nbsp;Funding acquisition, Investigation, Resources, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported in part by funds provided by the University of Hawaii Cancer Center (M.Y.) and\u0026nbsp;the Foundation of Watanabe Oyster Laboratory (M.W.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor declaration of competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman and animal ethics \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u0026nbsp;All experimental protocols used databases or cell culture \u003cem\u003ein vitro\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAbdelhamid RE, Sluka KA (2015). ASICs mediate pain and inflammation in musculoskeletal diseases. Physiology 30:449\u0026ndash;459.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBerenbaum F (2013) Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!). Osteoarthritis Cartilage. 21: 16\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePomari E, Stefanon B, Colitti M (2014) Effect of plant extracts on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced inflammatory gene expression in macrophage. J Inflamm Res. 27:103\u0026ndash;112.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKong L, Smith W, Hao D (2019) Overview of RAW264.7 for osteoclastogenesis study: Phenotype and stimuli, J Cell Mol Med 23: 3077\u0026ndash;3087\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYamaguchi M, Levy RM (2020) Metaxalone suppresses production of inflammatory cytokines associated with painful conditions in mouse macrophages RAW264.7 cells \u003cem\u003ein vitro\u003c/em\u003e: Synergistic effect with \u0026beta;-caryophyllene. Curr Mol Med 20:643\u0026ndash;652\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXu Y, Wang X, Liu L, Wang H, Wu H, Sun C (2022) Role of macrophages in tumor progression and therapy (Review). Int J Oncol 60:57\u0026ndash;75\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGeindreau M, Bruchard M, Vegran F (2022) Role of cytokines and chemokines in angiogenesis in a tumor context. Cancers 14:2446. Doi.org/10:3390/cancers14102446.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHartley JW, Evanse LH, Green KY, Naghashfar Z, Macias AR, Zerfas PM, Ward JM. (2008) Expression of infectious murine leukemia viruses by RAW264.7 cells, a potentiated complication for studies with a widely used mouse macrophage cell line. Retrovirology 5:1.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLi Y, Zhang J, Yan C, Chen Q, Xiang C, Zhang Q, Wang X, Jiang K (2022) Martin prevented LPS-induced osteoclastogenesis by regulating the NF-\u0026kappa;B pathway in vitro. J Mem Biotech 32:141\u0026ndash;148.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMaldonado RF, Sa-Correia I, Valvano MA480\u0026ndash;493 (2016) Lipopolysaccharide modification in Gram-negative bacteria during chronic infection. FEMS Microbiol Rev 40: 480\u0026ndash;493\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOhanian SH, Schwab JH (1967) Persistence of group a streptococcal cell walls related in chronic inflammation of rabbit dermal connective tissue. J Exp Med 125:1137\u0026ndash;1148\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWatanabe M, Fuda H, Jin S, Sakurai T, Ohkawa F, Hui S-P, Takeda S, Watanabe T, Koike T, Chiba H (2012) Isolation and characterization of a phenolic antioxidant from the pacific oyster (\u003cem\u003eCrassostrea Gigas\u003c/em\u003e). J Agric Food Chem 60: 830\u0026ndash;835.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFuda H, Watanabe M, Hui S-P, Joko S, Okabe H, Jin S, Takeda S, Miki E, Watanabe T, CGigasH (2015) Anti-apoptotic effects of novel phenolic antioxidant isolated from the Pacific oyster (\u003cem\u003eCrassostrea Gigas\u003c/em\u003e) on cultured human hepatocytes under oxidative stress. Food Chem 176: 226\u0026ndash;233.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJoko S, Watanabe M, Fuda H, Takeda S, Furukawa T, Hui S-P, Shrestha R, Chiba H (2017) Comparison of chemical structures and cytoprotection abilities between direct and indirect antioxidants. J Funct Foods \u003cem\u003e35\u003c/em\u003e:245\u0026ndash;255.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTamano H, Shakushi Y, Watanabe M, Ohashi K, Uematsu C, Otsubo T, Ikeda K, Takeda A (2019) Preventive effect of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) and zinc, components of the pacific oyster Crassostrea gigas, on glutamatergic neuron activity in the hippocampus. J Agric Food Chem 67:12844\u0026ndash;12853.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFukai M, Nakayabu T, Ohtani S, Shibata K, Shimada S, Sakamoto S, Fuda H, Furukawa T, Watanabe M, Hui S-P, Chiba H, Shimamura T, Taketomi A (2021) The Phenolic Antioxidant 3,5-dihydroxy-4-methoxybenzyl Alcohol (DHMBA) prevents enterocyte cell death under oxygen-dissolving cold conditions through polyphyletic antioxidant actions. J Clin Med, 10: 1972. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.3390/jcm10091972\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOkabe H, Hui S.-P, Fuda H, Furukawa T, Takeda S, Shrestha R, Miura, Watanabe M, Chiba H (2015) Mass Spectrometric quantification of amphipathic, polyphenolic antioxidant of the pacific oyster (\u003cem\u003eCrassostrea Gigas\u003c/em\u003e). Anal Sci 31:1341\u0026ndash;1344.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYamaguchi M, Yosiike K, Watanabe H, Watanabe M (2022) The marine factor 3,5- dihydroxy-4-methoxybenzyl alcohol suppresses growth, migration, and invasion and stimulates death of metastatic human prostate cancer cells: Targeting diverse signaling processes. Anticancer Drug 33:424\u0026ndash;436\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYamaguchi M, Weitzmann MN (2011) Vitamin K2 stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF-\u0026kappa;B activation. Int J Mol Med 27:3\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMisawa H, Inagaki S, Yamaguchi M (2001) Suppression of cell proliferation and deoxyribonucleic acid synthesis in cloned rat hepatoma H4-II-E cells overexpressing regucalcin. J Cell Biochem 84: 143\u0026ndash;149.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYamaguchi M, Daimon Y (2005) Overexpression of regucalcin suppresses cell proliferation in cloned rat hepatoma H4-II-E cells: Involvement of intracellular signaling factors and cell cycle-related genes. J Cell Biochem 95: 1169\u0026ndash;1177.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eIzumi T, Yamaguchi M: Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-\u0026alpha; or thapsigargin. J Cell Biochem 92: 296\u0026ndash;306, 2004.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYamaguchi M, Osuka S, Weitzmann MN, El-Rayes BF, Shoji S, Murata T (2016) Prolonged survival in pancreatic cancer patients with increased regucalcin gene expression: Overexpression of regucalcin suppresses the proliferation in human pancreatic cancer MIA PaCa-2 cells \u003cem\u003ein vitro.\u003c/em\u003e Int J Oncol 48:1955\u0026ndash;1964.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMinkin C (1982) Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker osteoclast function. Calcif Tissue Int 34:285\u0026ndash;290.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePeleck SL, Charest DL, Mordret GP, Siow YL, Palaty C, Campbell D, Chaslton L, Samiei M, Sanghera JS (1993) Networking with mitogen-activated protein kinases. Mol Cell Biochem 127: 157\u0026ndash;169.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYamaguchi M (2013) Suppressive role of regucalcin in liver cell proliferation: Involvement in carcinogenesis. Cell Prolif 46: 243\u0026ndash;253.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eEchizen K, Hirose O, Maeda Y, Oshima M. Inflammation in gastric cancer: The interplay of the COX-2/prostaglandin E2 and Toll-like receptor/MyD88 pathways. Cancer Sci. 2016; 107:391\u0026ndash;397.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGuan F, Wang H, Shan Y, Chen Y, Wang M, Wang Q, Yin M, Zhao Y, Feng Z, Zhang J (2014) Inhibition of COX-2 and PGE \u003csub\u003e2\u003c/sub\u003e in LPS-stimulated RAW264.7 cells by lonimacranthoide VI, a chlorogenic acid ester saponin. Biomed Rep 2:760\u0026ndash;764.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLin CC, Chan CM, Huang YP, Hsu SH, Huang CL, Tsai SJ. Methylglyoxal activates NF-\u0026kappa;B nuclear translocation and induces COX-2 expression via a p38-dependent pathway in synovial cells. Life Sci. 2016; 149:25\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFenton MJ, Golenbock DT \u003cstrong\u003e(\u003c/strong\u003e1998\u003cstrong\u003e) LPS-binding proteins and receptors\u003c/strong\u003e. J Leukoc Biol \u003cstrong\u003e64\u003c/strong\u003e:25\u0026ndash;32\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNunes-Alves C \u003cstrong\u003e(\u003c/strong\u003e2014\u003cstrong\u003e) New LPS receptors discovered\u003c/strong\u003e. Nat Rev Immunol 14:583\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHuang M-H, Lin Y-H, Lyu P-C, Liu Y-C, Chang Y-S, Chung J-G, Lin W-Y, Hsieh W-T (2021) Imperatorin interferes with LPS binding to the TLR4 co-receptor and activates the Nrf2 antioxidative pathway in RAW264.7 murine macrophage cells. Antioxidants 10:362.doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/antiox10030362\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhou C, Gao J, Ji H, Li W, Xing X, Liu D, Guo Q, Zhou L, Jing F \u003cstrong\u003e(\u003c/strong\u003e2021\u003cstrong\u003e) Modulates LPS-induced responses through inhibition of Toll-like receptor-mediated NF-\u0026kappa;B and MAPK signaling in RAW264.7 cells\u003c/strong\u003e. Inflammation \u003cstrong\u003e44\u003c/strong\u003e:2018\u0026ndash;2031\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLi N, Liu BW, Ren WZ, Liu JX, Li SN, Fu SP, Zeng YL, Xu SY, Yan X, Gao YJ, Liu DF, Wang W \u003cstrong\u003e(\u003c/strong\u003e2016\u003cstrong\u003e) GLP-2 attenuates LPS-induced inflammation in BV-2 cells by inhibiting ERK1/2, JNK1/2 and NF-\u0026kappa;B signaling pathway\u003c/strong\u003e. Int J Mol Sci. \u003cstrong\u003e17\u003c/strong\u003e: E190 \u003cstrong\u003eDoi\u003c/strong\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms17020190\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCha SM, Cha JD, Jang EJ, Kim GU, Lee KY (2016) Sophoraflavone G prevents streptococcus mutants surface antigen/II-induced production of NO and PGE\u003csub\u003e2\u003c/sub\u003e by inhibiting MAPK-mediated pathways in RAW 264.7 macrophages. Arch Oral Biol. 68:97\u0026ndash;104\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHuang X, Fang J, Lai W, Hu Y, Li L, Zhong Y, Yang S, He D, Liu R, Tang Q (2022) IL-6/STAT3 axis activates glut5 to regulate fructose metabolism and tumorigenesis. Int J Biol Sci 18:3668\u0026ndash;3675.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYamaguchi M, Weitzmann MN (2011) Vitamin K2 stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF-\u0026kappa;B activation. Int J Mol Med 27:3\u0026ndash;14.\u0026nbsp;\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXiao Y, Cao Y, Song C, Ren X, Yan L, Hao D, Kong L (2020) Cellular study of the LPS-induced osteoclastic multinucleated cell formation from RAW264.7 cells. J Cell Physiol 235:421\u0026ndash;428.\u0026nbsp;\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLiu Y, Fang S, Li X, Feng J, Du J, Guo L, Su Y, Zhou J, Ding G, Bai Y, Wang S, Wang H, Liu Y (2017) Aspirin inhibits LPS-induced macrophage activation via the NF-\u0026kappa;B pathway. Sci Rep 7:11549.\u0026nbsp;\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSapkota M, Li L, Kim S-W, Soh Y (2018) Thymol inhibits RANKL-induced osteoclastogenesis in RAW264.7 and BMM cells and LPS-induced bone loss in mice. Food Chem Toxicol 120:418\u0026ndash;429.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"3,5-dihydroxy-4-methoxybenzyl alcohol, DHMBA, an inflammatory cytokine, osteoclastogenesis, macrophage, RAW264.7 cells","lastPublishedDoi":"10.21203/rs.3.rs-2019515/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2019515/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Inflammation is implicated in the pathogenesis of many diseases. Inflammatory cytokines are produced in macrophages with stimulation of lipopolysaccharide (LPS) and are used as biomarkers participating in diverse disease conditions. The novel marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was initially identified in the\u003cstrong\u003e \u003c/strong\u003ePacific oyster \u003cem\u003eCrassostrea Gigas.\u003c/em\u003e DHMBA has properties to reduce oxidative stress as radical scavenging and increase the production of antioxidant proteins. The pharmacologic role of DHMBA, however, has been poorly understood.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods and Results:\u003c/strong\u003e This study has been undertaken to investigate whether DHMBA attenuates growth, cytokine production, and osteoclastogenesis in inflammatory mouse macrophage RAW264.7 cells. Culturing with DHMBA (1-1000 µM) suppressed the growth and stimulated the death of RAW264.7 cells \u003cem\u003ein vitro, \u003c/em\u003eleading to decrease in cell number. Mechanistically, DHMBA treatment decreased the levels of Ras, PI3K, Akt, MAPK, phospho-MAPK, and mTOR of signaling factors to promote the proliferation, and it raised the levels of p53, p21, Rb, and regucalcin, which are cell growth suppressors. The levels of caspase-3 and cleaved caspase-3 were increased by DHMBA treatment. Culturing with DHMBA suppressed productions of inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, interleukin-1β, or prostaglandin E2, were enhanced by LPS stimulation. Notably, the levels of NF-κB p65 were increased by LPS treatment, and this increase was repressed by DHMBA treatment. LPS treatment stimulated osteoclastogenesis of RAW264.7 cells. This stimulation was blocked by DHMBA treatment.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e DHMBA was found to potentially suppress the activity of inflammatory macrophages \u003cem\u003ein vitro\u003c/em\u003e, suggesting therapeutic usefulness in inflammatory conditions.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol expresses the anti-inflammatory effects in mouse macrophages RAW264.7 cells in vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-06 14:37:53","doi":"10.21203/rs.3.rs-2019515/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1274c8ee-1d18-4b69-9075-18c1f95d10d2","owner":[],"postedDate":"September 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-06T15:59:33+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-06 14:37:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2019515","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2019515","identity":"rs-2019515","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}