Calcification enhancer for osteoblast MC3t3-e1 cell

Abstract

[Problem] To provide: an inhibitor which is capable of providing a novel strategy for protecting and treating bone formation disorders and calcification disorders in osteoblasts associated with inflammatory conditions in bone marrow microenvironments, the inhibitor containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient; and others. [Solution] The present invention is characterized by containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and having an effect of enhancing the calcification of MC3T3-E1 cells that are osteoblasts.

Description

Calcification enhancer for osteoblastic MC3T3-E1 cells

[0001] The present invention relates to a mineralization promoter for osteoblastic MC3T3-E1 cells, for example.

[0002] The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), found in Pacific oysters (Crassostrea gigas) and other organisms, functions as a better peroxyl radical scavenger than other strong antioxidants such as Trolox, and therefore may play an important role in preventing health problems.

[0003] In the present invention, mouse macrophage RAW264.7 cells were used in vitro to clarify whether DHMBA prevents impaired mineralization of mouse osteoblastic MC3T3-E1 cells induced under inflammatory conditions.

[0004] In an osteoblast culture system, the addition of DHMBA (1-100 μM) did not affect the proliferation or death of MC3T3-E1 cells. Under these conditions, DHMBA was found to enhance the mineralization of osteoblasts. DHMBA inhibited the decrease in mineralization of MC3T3-E1 cells induced by the addition of the inflammatory cytokine TNF-α.

[0005] DHMBA inhibited lipopolysaccharide (LPS)-stimulated TNF-α production in cultured macrophage RAW264.7 cells. Under LPS stimulation, MC3T3-E1 cell proliferation was suppressed through crosstalk with RAW264.7 cells in coculture.

[0006] Interestingly, we found that incubation of macrophages with the culture medium obtained by LPS treatment suppressed the proliferation of MC3T3-E1 cells. This effect was suppressed by the presence of DHMBA or Bay 11-7082, an inhibitor of the TNF-α signaling pathway. This inhibitory effect of DHMBA was not further enhanced in the presence of Bay 11-7082. The mechanism underlying this was found to be that DHMBA reduced the level of NF-κB p65 and the activity of the NF-κB reporter gene in MC3T3-E1 cells. DHMBA was found to prevent the impairment of osteoblast mineralization by the inflammatory cytokine TNF-α through the suppression of TNF-α signaling, which is involved in macrophage activation in the bone marrow microenvironment. This discovery also provides a new strategy for the treatment of inflammation-induced osteoblast disorders.

[0007] (Introduction) Bone homeostasis is regulated by the functions of osteoblasts, osteocytes, and osteoclasts, which are the main cells in bone tissue. In bone homeostasis, bone resorption is first induced by osteoclasts. Osteoclasts differentiate from hematopoietic precursor cells and develop at the bone surface. Next, the cavity created by osteoclast formation is refilled by osteoblasts, which arise from local mesenchymal stem cells. Osteoblasts gather at the bottom of the cavity and initiate bone formation. This cellular mechanism allows bone tissue to maintain fresh, flexible tissue. Bone homeostasis, which involves this mechanism of bone remodeling, plays a physiological role in maintaining new bone mass.

[0008] The bone remodeling process is intricately regulated by hormones, cytokines, stresses acting on the skeletal system due to physical activity and weight bearing, various growth factors such as transforming growth factor-β1, platelet-derived growth factor, and insulin-like growth factor, as well as systemic influences and their interactions. Furthermore, immune cells such as lymphocytes and macrophages present in the bone marrow microenvironment contribute to the regulation of bone remodeling.

[0009] Postmenopausal osteoporosis, which physiologically causes bone loss in women with aging, is caused by age-related estrogen deficiency. Furthermore, notably, bone metastatic cancer cells can induce severe bone loss, leading to fractures and death in patients. In particular, inflammatory macrophages contribute to the development of bone damage through bone metastasis by cancer cells. Inflammatory cytokines, primarily tumor necrosis factor (TNF)-α, cause osteoarthritis, osteoporosis, and bone metastasis of cancer. Interestingly, TNF-α produced by inflammatory macrophages has been shown to suppress osteoblast mineralization and stimulate osteoclast bone resorption through activation of NF-κB signaling. Macrophage RAW264.7 cells are monocyte / macrophage-like cells that have been well characterized in terms of macrophage-mediated immune, metabolic, and phagocytic functions. RAW264.7 cells have been used in research as a cellular model of osteoclastogenesis, in which osteoclasts differentiate from the monocyte-macrophage lineage. The host immune system is stimulated by lipopolysaccharide (LPS), a major antigen of Gram-negative bacteria. In the bone marrow microenvironment, inflammatory macrophages activated by LPS produce TNF-α, which plays a major role in bone loss. Clinically, it is important to develop therapeutic factors to prevent bone loss and fractures caused by various diseases, such as inflammation and cancer bone metastasis.

[0010] The marine phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was first discovered in the Pacific oyster Crassostrea gigas. DHMBA has been shown to prevent oxidative stress by scavenging radicals in several cell types. Interestingly, DHMBA is an excellent peroxyl radical scavenger, approximately 15-fold and four orders of magnitude more potent than the known strong radical scavenger Trolox in lipid and aqueous media, respectively. DHMBA has also been shown to react with HOO(·) more rapidly than other known antioxidants, such as resveratrol and ascorbic acid.

[0011] Japanese Patent Application Laid-Open No. 2023-080697

[0012] The above-mentioned properties of DHMBA suggest that it may play an important role in regulating cellular function and maintaining health. In particular, we have demonstrated that DHMBA exhibits inhibitory effects on the proliferation and metastatic activity of bone-metastatic prostate cancer cells. Bone morphogenetic proteins are clinically important for repairing bone loss and preventing osteopenia and osteoporosis associated with aging and various diseases. The development of such substances is therefore crucial.

[0013] In this study, we investigated whether DHMBA stimulates osteoblast mineralization and prevents the impairment of osteoblast mineralization associated with TNF-α produced by inflammatory macrophages RAW264.7 cells stimulated with LPS. We found that DHMBA stimulates mineralization in mouse osteoblastic MC3T3-E1 cells and further inhibits the impairment of cell proliferation and mineralization of osteoblastic MC3T3-E1 cells via signal transduction associated with TNF-α produced by inflammatory macrophages RAW264.7 cells activated with LPS.

[0014] The purpose of this invention is to provide an inhibitor containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient, which can provide a new strategy to prevent and treat disorders of osteoblast bone formation and mineralization associated with inflammatory conditions in the bone marrow microenvironment.

[0015] The present invention is characterized in that it is an agent for promoting calcification of osteoblastic MC3T3-E1 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and has the effect of promoting calcification of osteoblastic MC3T3-E1 cells; or it is characterized in that it is an agent for controlling the effect of TNF-α on the proliferation of osteoblastic MC3T3-E1 cells or the cell death of osteoblastic MC3T3-E1 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and has the effect of controlling the effect of TNF-α on the proliferation or cell death of osteoblastic MC3T3-E1 cells; or it is characterized in that it is an agent for controlling the effect of TNF-α on the proliferation or cell death of osteoblastic MC3T3-E1 cells, which contains 3,5-dihydroxy-4-methoxybenzyl an agent for preventing TNF-α-induced calcification disorders in MC3T3-E1 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and has the effect of suppressing the effect of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cells, thereby preventing TNF-α-induced calcification disorders in MC3T3-E1 cells; or an inhibitor / control agent for controlling the suppression of proliferation and the promotion of cell death of osteoblast MC3T3-E1 cells co-cultured with inflammatory macrophage RAW264.7 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and has the effect of suppressing proliferation and the promotion of cell death of osteoblast MC3T3-E1 cells co-cultured with inflammatory macrophage RAW264.7 cells; or This product contains DHMBA as its active ingredient and has the effect of protecting against the effects of conditioned medium obtained from the culture of inflammatory macrophages RAW264.7 cells on the proliferation or cell death of osteoblastic MC3T3-E1 cells.7 cells, or an inhibitor of gene promoter activity associated with the transcription factor NF-κB in osteoblasts, containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and having the effect of suppressing gene promoter activity associated with the transcription factor NF-κB in osteoblasts.

[0016] The inhibitors according to the present invention, which contain 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient, can provide a new strategy for treating osteoblast disorders caused by inflammation, for example, and have the excellent effect of providing a new strategy for preventing and treating disorders of bone formation and calcification in osteoblasts associated with inflammatory conditions in the bone marrow microenvironment.

[0017] Effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on proliferation or cell death of mouse osteoblastic MC3T3-E1 cells in vitro. (A) Cells (1 x 10 per well of a 24-well plate) 5 (1x10 cells / ml culture medium) were cultured in α-MEM containing 10% FBS and 1% P / S in the presence of DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. (B) To measure the effect of increasing concentrations of DHMBA, cells (1x10 cells / ml culture medium) were cultured in α-MEM containing 10% FBS and 1% P / S in the presence of DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. 5 (1x10 cells / ml) were cultured in the presence of DHMBA (0.1, 1, 10, 100, or 1000 μM) for 3 days. (C) To examine the effect of DHMBA on cell death, cells (1x10 cells / ml) were cultured in the presence of DHMBA (0.1, 1, 10, 100, or 1000 μM) for 3 days. 5Cells (1x10 cells / ml culture medium) were cultured for 3 days until subconfluence was reached, after which DHMBA (0.1, 1, 10, 100, or 1000 μM) was added and cultured for an additional 48 hours. After culture, the number of cells attached to the dish was counted. Data are shown as the mean ± SD of values ​​obtained from 8 wells of a total of two replicate plates using different cell preparations. *: Significant difference compared to the DHMBA-free control group (gray bar) at p<0.001. One-way ANOVA with Tukey-Kramer post-hoc test was used. The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) enhances mineralization of mouse osteoblastic MC3T3-E1 cells. Cells (1x10 cells / well of a 24-well plate) were cultured for 3 days until subconfluence was reached, after which DHMBA (0.1, 1, 10, 100, or 1000 μM) was added and cultured for an additional 48 hours. After culture, the number of cells attached to the dish was counted. Data are shown as the mean ± SD of values ​​obtained from 8 wells of a total of two replicate plates using different cell preparations. *: Significant difference compared to the DHMBA-free control group (gray bar) at p<0.001. One-way ANOVA with Tukey-Kramer post-hoc test was used. 5 Cells (100 cells / ml) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days. After reaching subconfluence, the medium was replaced with α-MEM containing 10% FBS, 1% P / S, 4 mM β-glycerophosphate, and L-ascorbic acid (100 μg / ml) (mineralization-enhancing medium). The medium containing DHMBA (1 or 10 μM) was replaced every 3 days, and the cells were cultured in the presence of DHMBA for 18 days. (A) Representative photograph (40x magnification). (B) Absorbance at 570 nm after solubilization of stained cells. Results are shown as the mean ± SD of values ​​obtained from 8 wells of a total of two replicate plates using different cell preparations. *: Significant difference compared to the DHMBA-free control group (gray bar) at p<0.001. One-way ANOVA with Tukey-Kramer post-hoc test was used. The effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) prevents the effects of TNF-α on the proliferation or cell death of mouse osteoblastic cells MC3T3-E1. (A) To examine the effect of TNF-α on cell proliferation, cells (1 x 10 per well of a 24-well plate) were cultured. 5 (1x10 cells / ml culture medium) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days in the presence of DHMBA (0.1, 1, or 10 μM) or TNF-α (1 or 10 ng / ml culture medium). (B) To examine the effect of TNF-α on cell death, cells (1x10 cells / ml culture medium) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days in the presence of DHMBA (0.1, 1, or 10 μM) or TNF-α (1 or 10 ng / ml culture medium). (C) To examine the effect of TNF-α on cell death, cells (1x10 cells / ml culture medium) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days in the presence of DHMBA (0.1, 1, or 10 μM) or TNF-α 5Cells (0.1, 1, or 10 μM) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days to reach subconfluence, and then cultured for an additional 48 hours in the presence of DHMBA (0.1, 1, or 10 μM) or TNF-α (10 ng / ml medium). After incubation, the number of cells attached to the dish was counted. Data are presented as the mean ± SD of values ​​obtained from eight wells of two replicate plates using different cell preparations. *: Significant difference at p<0.001 compared to the control group (light gray bar) that did not contain either TNF-α or DHMBA. # : Significant difference at p<0.001 compared to the TNF-α (10 ng / ml) group without DHMBA (dark gray bar). One-way ANOVA with Tukey-Kramer post-hoc test was used. Marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) prevents the inhibition of mineralization in mouse osteoblastic MC3T3-E1 cells cultured with TNF-α. Cells (1x10 per well of a 24-well plate) 5 Cells (100 cells / ml) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days. After reaching subconfluence, the cells were replaced with α-MEM containing 10% FBS, 1% P / S, 4 mM β-glycerophosphate, and L-ascorbic acid (100 μg / ml) (mineralization-enhancing medium) and cultured for 18 days in the presence of TNF-α (1 ng / ml medium) and DHMBA (1 or 10 μM). The medium was replaced every 3 days. After culture, cells were stained with alizarin red solution. (A) Representative photograph (40x magnification). (B) Absorbance at 570 nm after solubilization of stained cells. Data are presented as the mean ± SD of values ​​obtained from 8 wells of a total of two replicate plates using different cell preparations. *: Significant difference compared to the DHMBA-free group (white bars) or the control group (gray bars) at p<0.001. #: Significant difference (p<0.001) compared with the group treated with TNF-α without DHMBA. One-way ANOVA with Tukey-Kramer post-hoc test was used. Marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits the inhibition of proliferation and the promotion of cell death in mouse osteoblastic MC3T3-E1 cells co-cultured with inflammatory macrophage RAW264.7 cells. (A) MC3T3-E1 cells (1x10 per well of a 24-well plate) 5 (10 cells / ml) were cultured for 3 days in the presence of lipopolysaccharide (LPS, 100 ng / ml medium). (B) MC3T3-E1 cells that had reached subconfluence after 3 days of culture were cultured for an additional 48 hours in the presence of LPS (100 ng / ml). (C) To measure the effect of co-culture on cell proliferation, MC3T3-E1 cells (10 per well) were cultured in the presence of LPS (10 5 Macrophage RAW264.7 cells (5x10 cells / 1 ml culture medium) were added to the lower compartment of the transwell chamber in the presence of DHMBA (1 or 10 μM). The upper chamber of each transwell contained macrophage RAW264.7 cells (5x10 cells / 1 ml culture medium). 4 The upper chamber contained LPS (100 ng / ml culture medium). The cells were cultured for 3 days. After culture, the MC3T3-E1 cells attached to the lower compartment were counted. (D) To examine the effect of co-culture on cell death, MC3T3-E1 cells (10 per well) were cultured in 0.5 ml DMEM (10% FBS). The upper chamber contained LPS (100 ng / ml culture medium). The cells were cultured for 3 days. After culture, the MC3T3-E1 cells attached to the lower compartment were counted. (E) To examine the effect of co-culture on cell death, MC3T3-E1 cells (10 per well) were cultured in 0.5 ml DMEM (10% FBS). 5 RAW264.7 cells (5x10 cells / 1 ml) were added to the lower compartment of the transwell chamber and cultured for 3 days until subconfluence was reached. After culture, DHMBA (1 or 10 μM) was added to the lower compartment, and RAW264.7 cells (5x10 cells / 1 ml) were added to the upper chamber of each transwell. 4The upper chamber was filled with 0.5 ml of DMEM (10% FBS) containing 0.5 ml of cells / 0.5 ml of culture medium. The cells were cultured in the presence of LPS (100 ng / ml culture medium) for an additional 48 hours in the upper chamber. After incubation, the MC3T3-E1 cells attached to the lower compartment were counted. Results are shown as the mean ± SD of values ​​obtained from a total of eight wells using two plates containing different cell preparations. *: Significant difference at p<0.001 compared to the control group cultured without LPS (gray bar). # : Significant difference (p<0.001) compared with the group without DHMBA in the presence of LPS (black bar). One-way ANOVA with Tukey-Kramer post-hoc test was used. The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) prevents the inhibition of proliferation and enhanced cell death of mouse osteoblastic MC3T3-E1 cells caused by conditioned medium obtained from the culture of inflammatory macrophage RAW264.7 cells. The conditioned medium was obtained by culturing macrophage RAW264.7 cells in the presence or absence of LPS (100 ng / ml medium). (A) MC3T3-E1 cells (1x10 per well of a 24-well plate) 5(cells / ml) were cultured for 3 days in α-MEM containing 10% FBS and 1% P / S. The conditioned medium obtained with LPS stimulation (0.25 or 0.5 ml, made up to 1 ml with normal medium) or the conditioned medium obtained with LPS stimulation (0.5 ml, made up to 1 ml with normal medium) was cultured in the presence of DHMBA (1 or 10 μM) for 3 days. (B) To measure the effect on cell death, subconfluent MC3T3-E1 cells were cultured for 48 hours in the conditioned medium obtained with LPS stimulation (0.25 or 0.5 ml, made up to 1 ml with normal medium) or the conditioned medium obtained with LPS stimulation (0.5 ml, made up to 1 ml with normal medium). Attached MC3T3-E1 cells were counted. Results are shown as the mean ± SD of values ​​obtained from a total of eight wells using two plates containing different cell preparations. *: Significant difference (p < 0.001) compared to the control group using RAW264.7 cells and culture medium without LPS (light or dark gray bars). # : Significant difference (p<0.001) compared to the group cultured in conditioned medium (0.5 ml) with LPS stimulation (dark gray bar). One-way ANOVA with Tukey-Kramer post-hoc test was used. NF-κB signaling inhibitors suppress the effect of conditioned medium obtained by culturing macrophage RAW264.7 cells with LPS stimulation on the proliferation and cell death of mouse osteoblastic MC3T3-E1 cells. Conditioned medium was obtained by culturing macrophage RAW264.7 cells in the presence or absence of LPS (100 ng / ml culture medium). (A) TNF-α concentration in conditioned medium. RAW264.7 cells (1x10 per well in a 24-well plate) 5 (1x10 cells / ml culture medium) were cultured for 3 days, after which the conditioned medium was collected. (B) Effect of Bay11-7082, an inhibitor of the NF-κB signaling pathway, on the proliferation of MC3T3-E1 cells. MC3T3-E1 cells (1x10 cells / ml culture medium) were cultured for 3 days, after which the conditioned medium was collected. (C) Effect of Bay11-7082, an inhibitor of the NF-κB signaling pathway, on the proliferation of MC3T3-E1 cells. 5(C) To measure the effect on cell proliferation, MC3T3-E1 cells were cultured for 3 days in α-MEM containing Bay11-7082 (1, 10, 50, 100, or 1000 nM). (C) To measure the effect on cell proliferation, MC3T3-E1 cells were cultured for 3 days in conditioned medium (0.5 ml / well) obtained by stimulation of RAW264.7 cells with LPS (100 ng / ml) in the presence of Bay11-7082 (1 or 10 nM) or Bay11-7082 (10 nM) and DHMBA (10 μM). (D) To examine the effect on cell death, MC3T3-E1 cells were cultured for 3 days until they reached subconfluence, then cultured for 48 hours in conditioned medium (0.5 ml / well) from RAW264.7 cells stimulated with LPS (100 ng / ml medium) in the presence of Bay11-7082 (1 or 10 nM) or Bay11-7082 (10 nM) and DHMBA (10 μM). After incubation, adherent MC3T3-E1 cells were counted. Results are shown as the mean ± SD of values ​​obtained from a total of eight wells using two plates containing different cell preparations. *: Significant difference (p<0.001) compared to the control group cultured without RAW264.7 cells (A and B, white bars; C and D, gray bars). # : Significant difference at p<0.001 compared to the group without Bay11-7082 (black bar). One-way ANOVA with Tukey-Kramer post-hoc test was used. Marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) suppresses TNF-α-induced NF-κB activation in mouse osteoblastic MC3T3-E1 cells. Cells (10 per dish) 6 Cells (10 ml) were cultured for 3 days in the presence or absence of DHMBA (10 μM) and with the addition of TNF-α (10 ng / ml). After culture, cell lysates were prepared. 40 micrograms of supernatant protein per lane was used for Western blot analysis. (A) Representative data are shown. (B) Bands are expressed as fold increase compared to the control (no DHMBA). Results are shown as the mean ± SD of values ​​obtained from a total of four dishes using different cell preparations. *: Significant difference compared to the control group at p<0.01. #: Significant difference at p<0.01 compared to the group without TNF-α (gray bar). One-way ANOVA with Tukey-Kramer post-hoc test was used. (C) To measure NF-κB promoter activity, MC3T3-E1 cells (2x10 per 100 μl in a 96-well plate) were cultured. 4 Cells (100 cells / well) were cultured in α-MEM containing 10% FBS and 1% P / S for 24 hours. After incubation, the α-MEM medium was replaced with α-MEM medium without 10% FBS and 1% P / S, and the cells were transfected with the reporter plasmid or empty vector control. After 5 hours, the medium was replaced with α-MEM containing 10% FBS and 1% P / S, and NF-κB was stimulated by adding TNF-α (1 or 10 ng / ml) for 24 hours. Control groups included either ethanol (final concentration 1%) or DHMBA (1 or 10 μM). After incubation, cells were extracted with passive lysis buffer, and luciferase activity was measured. Results are shown as the mean ± SD of values ​​obtained from a total of eight wells using different cell preparations. *: Significant difference compared to the control group (white bar) at p<0.001. # Significant difference (p<0.001) compared with the DHMBA-free group (B, dark gray bars; C, black bars). One-way ANOVA with Tukey-Kramer post-hoc test was used. The mechanism by which 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) protects against TNF-α-induced mineralization impairment in osteoblastic MC3T3-E1 cells. DHMBA inhibits TNF-α production in inflammatory macrophages. Furthermore, DHMBA reduces the level of NF-κB p65, inhibiting the NF-κB signaling process. Nuclear translocation of DHMBA acts on the NF-κB response element in the promoter region of DNA, inhibiting transcriptional activity enhanced by TNF-α stimulation. DHMBA protects against mineralization impairment caused by activation of the NF-κB signaling process associated with TNF-α production by inflammatory macrophages. DHMBA is thought to have the effect of regulating and repairing osteoblast mineralization that is hindered by inflammatory conditions.

[0018] Detailed experiments were conducted using mouse osteoblastic MC3T3-E1 cells to verify the effects of various agents according to the present invention, namely, agents containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient. The details of these experiments are described below.

[0019] Materials and Methods: Reagents. Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / liter (l) glucose, L-glutamine, 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). α-Minimum Essential Medium (α-MEM) was purchased from Invitrogen Corp. (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Lipopolysaccharide (LPS), Bay11-7089, and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Lipopolysaccharide (LPS) and Bay11-7089 were dissolved in 100% ethanol and stored at -20°C until use.

[0020] 3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA), a novel amphiphilic phenolic compound, was first isolated from the Pacific oyster (Crassostrea gigas) as an antioxidant. Synthetic DHMBA was used in this study. The structure of DHMBA has been previously shown. The purity of the synthesized DHMBA was 100%. DHMBA was dissolved in 100% ethanol and stored at -20°C until use.

[0021] Mouse osteoblastic MC3T3-E1 cells were obtained from the American Type Culture Collection (HTB-77™, ATCC; Rockville, MD, USA). MC3T3-E1 cells were cultured in α-MEM containing 10% FBS and 1% P / S at 37°C in a CO2 incubator in plastic dishes, as previously used.

[0022] Macrophage RAW264.7 Cells Mouse RAW264.7 cells were purchased from the American Type Culture Collection (Rockville, MD, USA) as previously described. RAW264.7 cells are a monocyte / macrophage-like cell line derived from an Abelson leukemia virus-transformed cell line derived from BALB / c mice.

[0023] (Cell proliferation assay) To confirm the progress of cell proliferation, MC3T3-E cells (1x10 per well) were cultured. 5 MC3T3-E1 cells (1x10 per well) were cultured in α-MEM supplemented with 10% FBS and 1% P / S in a water-saturated atmosphere containing 5% CO2 and 95% air at 37°C in 24-well plates. Cells were cultured in the presence of vehicle (1% ethanol as a final concentration) or DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. In a separate experiment, to measure the effect of increasing concentrations of DHMBA on MC3T3-E1 cells, cells (1x10 per well) were cultured in α-MEM supplemented with 10% FBS and 1% P / S in 24-well plates. 5 The cells were cultured in the presence of either reagent vehicle (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 μM) for 3 days.

[0024] To evaluate the effect of DHMBA on cell death of osteoblastic MC3T3-E1 cells (1 x 10 per well of a 24-well plate), 5Cells (1 / ml culture medium) were cultured in α-MEM containing 10% FBS and 1% P / S. They were cultured for 3 days to reach subconfluence, after which either a reagent medium (1% ethanol as a final concentration) or DHMBA (0.1, 1, 10, 100, or 1000 μM) was added and cultured for an additional 48 hours. After culture, Ca was used to measure the number of cells attached to each well. 2+ / Mg 2+ After adding 0.1 ml of a solution containing 0.05% trypsin and EDTA in PBS (per well), the culture dishes were incubated at 37°C for 2 minutes. After adding 0.9 ml of α-MEM containing 10% FBS and 1% P / S, the cells were detached and mixed by pipetting. The suspended cell solution (0.1 ml) was mixed with 0.1 ml of 0.5% trypan blue staining solution. This stain stains live cells but not dead cells. Viable cells were counted under a microscope (Olympus MTV-3) using a hemocytometer (Sigma-Aldrich, St. Louis, MO) and a cell counter (Line Seiki H-102P, Tokyo, Japan). The average of duplicate counts was used. Cell numbers were expressed as the number per well.

[0025] (Osteoblast differentiation assay and Alizarin Red S staining) MC3T3-E1 cells (10 per well in a 12-well plate) 5Cells (1.0 ml / ml culture medium) were cultured in α-MEM containing 10% FBS and 1% P / S (1.0 ml / well) for 3 days. The medium was then changed to mineralization-enhancing α-MEM supplemented with 10% FBS, 1% P / S, L-ascorbic acid (100 μg / ml), and 4 mM β-glycerophosphate, as previously described. DHMBA was added at 1 or 10 μM, and the cells were cultured for 18 days with fresh medium every 3 days. On day 18, the cells were rinsed with PBS and fixed with ice-cold 75% ethanol at 4°C for 30 minutes. Calcium deposition was visualized by staining with Alizarin Red S (40 mM, pH 6.2). After 30 minutes at room temperature, the stain was removed by thorough washing with distilled water. The stained cells on the plates were photographed under a microscope (40x magnification) (Olympus IX71, Olympus Corporation, Tokyo, Japan) and imaged using Image J2. For quantification, each plate containing stained cells was solubilized by the addition of 10% cetylpyridinium chloride solution, and the absorbance was measured at 570 nm.

[0026] Crosstalk between MC3T3-E1 and RAW264.7 cells. To investigate the crosstalk between osteoblastic MC3T3-E1 and macrophage RAW264.7 cells, a Transwell chamber (Corning, Life Sciences, Cat. No. CLS3464-48EA, USA) equipped with a polycarbonate filter (pore size 8 μm, diameter 6.5 mm) was used. MC3T3-E1 cells (1 x 10 per ml of culture medium) were placed in the lower part of the chamber. 5 RAW264.7 cells were suspended in DMEM containing 10% FBS and 1% P / S, and 0.5 ml (5 × 10 cells) was added to the upper chamber of each Transwell. 4The plates were filled with 1000 cells / ml of culture medium. RAW264.7 cells were incubated at 37°C under 5% CO2 and 95% air for 1 hour to allow them to adhere to the upper chamber. The plate with transwells was placed in an incubator at 37°C under 5% CO2 and 95% air. To induce an inflammatory state in RAW264.7 cells, LPS (100 ng / ml culture medium) was added to the upper chamber containing RAW264.7 cells. Culture plates containing MC3T3-E1 (lower compartment) or RAW264.7 cells (upper compartment) were cultured for 3 days until they reached subconfluence. After incubation, the culture medium in the upper and lower compartments was aspirated, and the filters were removed. MC3T3-E1 cells attached to the dishes (wells) were counted as described in the "Cell Proliferation Assay" section.

[0027] Furthermore, to examine the effect of co-culture with RAW264.7 cells on the death of MC3T3-E1 cells, osteoblasts (1 x 10 per well) were added. 5 The cells / ml were cultured for 3 days until they reached subconfluence, after which the culture medium was replaced with fresh medium containing either the reagent vehicle (1% ethanol as a final concentration) or DHMBA (1 or 10 μM). Transwell chambers were then placed in each culture well, and RAW264.7 cells were added to the upper chamber in 0.5 ml (5 x 10 cells / ml) with or without LPS (100 ng / ml culture medium). 4 After culturing for an additional 48 hours, the medium in the upper and lower compartments was aspirated and the filter was removed. The cells attached to the wells were counted as described in the "Cell proliferation assay" section.

[0028] (Effect of Conditioned Medium on MC3T3-E1 Cells) Conditioned medium was obtained by culturing RAW264.7 cells. RAW264.7 cells (1 × 10 per well of a 24-well plate) were cultured in a conditioned medium. 5MC3T3-E1 cells (100 cells / ml) were cultured for 3 days in DMEM containing 10% FBS and 1% P / S with or without LPS (100 ng / ml in culture medium) at subconfluence. After cell culture, the culture medium (conditioned medium) was collected. To measure the effect of conditioned medium on the proliferation of MC3T3-E1 cells, cells were cultured for 3 days in α-MEM containing 10% FBS and 1% P / S. Conditioned medium cultured without LPS (1 ml, control) or with LPS in the presence or absence of DHMBA (1 or 10 μM) (1 ml total in normal medium, 0.25 or 0.50 ml per well) was used for 3 days.

[0029] Furthermore, to examine the effect on the death of MC3T3-E1 cells, cells (1x10 per well) were 5 Cells (cells / ml) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days. After reaching subconfluence, the medium was replaced. Cells were further cultured for 48 hours in normal medium, conditioned medium (1 ml) cultured without LPS, or conditioned medium (0.25 or 0.50 ml / well added to bring the normal medium to 1 ml) containing either reagent vehicle (1% ethanol to a final concentration) or DHMBA (1 or 10 μM). After incubation, cells were detached from each culture well and counted as described above.

[0030] (TNF-α production assay) RAW264.7 cells (1x10 per well) 5RAW264.7 cells (100 ng / ml) were cultured in DMEM containing 10% FBS and 1% P / S in 24-well plates for 3 days until subconfluence. After 5 hours of incubation, the culture medium was collected for cytokine assays, and cells were detached from each culture dish and counted as described in the "Cell Proliferation Assay" section. Culture with LPS did not significantly alter the number of RAW264.7 cells. TNF-α concentrations in the culture medium were analyzed using a mouse TNF-α ELISA kit (catalog number KHC301) purchased from Cayman Chemical (Ann Arbor, MI, USA) according to the manufacturer's instructions. TNF-α production was expressed as picograms (pg) of TNF-α secreted into the culture medium (pg / ml).

[0031] (Effect of NF-κB signaling pathway inhibitors) Furthermore, the effect of Bay11-7082, an NF-κB signaling inhibitor, on the proliferation or cell death of MC3T3-E1 cells was investigated. First, to examine the effect of Bay11-7082 on the proliferation of MC3T3-E1 cells, cells (1x10 per well of a 24-well plate) were cultured. 5 MC3T3-E1 cells (1 ml each) were cultured for 3 days in the presence of 10% FBS, 1% P / S, and reagent medium (final concentration: 1% ethanol) or Bay11-7082 (1, 10, 50, 100, or 1000 nM) until subconfluent. MC3T3-E1 cells were then cultured for 3 days in the presence or absence of Bay11-7082 (1 or 10 nM) and DHMBA (10 μM) in the presence or absence of conditioned medium (0.5 ml per well, plus regular medium for a total of 1 ml).

[0032] In other experiments, to evaluate the effect of Bay11-7082 on MC3T3-E1 cell death, cells (1 x 10 per well of a 24-well plate) were cultured in 100 wells of MC3T3-E1 cells. 5Cells (1 ml each) were cultured in α-MEM containing 10% FBS and 1% P / S. After 3 days of culture to reach subconfluence, the medium was replaced with α-MEM (1 ml) containing either normal medium or conditioned medium (0.5 ml per well, plus normal medium for a total of 1 ml). Cells were further cultured for 48 hours in the presence of Bay11-7082 (1 or 10 nM), or Bay11-7082 (10 nM) plus DHMBA (10 μM). After incubation, cells were detached from each well and counted as previously described.

[0033] (Western blotting) MC3T3-E1 cells (1x10 6Cells (in 100 mm dishes containing 10 ml of culture medium) were cultured for 3 days in α-MEM containing 10% FBS and 1% P / S in the presence of reagent vehicle (final concentration: 1% ethanol), DHMBA (10 μM), TNF-α (10 ng / ml culture medium), or DHMBA (10 μM) plus TNF-α (10 ng / ml). After incubation, dishes were washed three times with 10 ml of cold PBS, and attached cells were removed from the dishes by scraping in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). A 40-microgram sample of protein from the cell lysate was loaded per lane and separated by SDS-polyacrylamide gel electrophoresis (12% SDS-PAGE) as previously described. After electrophoresis, the membranes were incubated overnight at 4°C with primary antibodies (1:1,000 dilution) for the target proteins NF-κB p65 (Cat. No. 3034, rabbit) and β-actin (Cat. No. 3700, mouse). After incubation, the membranes were further incubated in horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., mouse sc-2005 or rabbit sc-2305, 1:1,000 dilution) at room temperature for 60 minutes. Protein bands were then detected using a chemiluminescent substrate (Cat. No. 34577, Thermo Scientific, Rockford, IL, USA) on X-ray film. A total of four films from four independent experiments on separate membranes were scanned with an Epson Perfection 1660 Photo scanner, and bands were quantified using Image J2 software (National Institutes of Health, Bethesda, MD, USA).

[0034] NF-κB reporter assay: NF-κB-responsive reporter luciferase assay (pNF-κB-Luc, BD Biosciences) was performed as previously described. MC3T3-E1 cells (2 × 10 cells per 100 μl in a 96-well plate) were cultured. 4Cells (100 μl / well) were cultured in α-MEM containing 10% FBS and 1% P / S for 24 hours. After incubation, the medium was removed and washed twice with α-MEM without FBS or antibiotics. 100 μl of α-MEM without FBS or antibiotics was added to the wells containing the cells. MC3T3-E1 cells were transfected with reporter plasmids or an empty vector control (pGL3-Basic) using Lipofectamine 2000 reagent (Invitrogen) in α-MEM without FBS or antibiotics. After 5 hours, the medium was replaced with α-MEM containing 10% FBS and 1% P / S, and cells were stimulated with TNF-α (1 or 10 ng / ml) for 24 hours. In parallel, cells were cultured in either the reagent vehicle (final concentration of 1% ethanol) or DHMBA (1 or 10 μM). After incubation, cells were extracted with passive lysis buffer (Promega Corporation, Madison, WI), and luciferase activity was measured using a luciferase assay system (Promega) with a microplate reader (Turner Designs, Sunnyvale, CA, USA). Equivalent transfection efficiency was verified for all plasmids by using the luciferase reporter plasmid pRL-SV40 (Promega, Madison, WI).

[0035] Statistical Analysis: Statistical significance was determined using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc., La Jolla, CA). Data are presented as mean ± standard deviation (SD). Multiple comparisons were performed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons after testing of parametric data. A p value of <.05 was considered statistically significant.

[0036] (Results) Effects of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on proliferation and cell death of MC3T3-E1 osteoblastic cells. To investigate the effect of DHMBA on proliferation and cell death of mouse osteoblastic MC3T3-E1 cells, cells (1x10 per well of a 24-well plate) were cultured. 5 MC3T3-E1 cells (1x10 cells / ml culture medium) were cultured in α-MEM medium containing 10% FBS and 1% P / S in the presence of a reagent vehicle (1% ethanol as a final concentration) or DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. Five days of culture did not significantly alter the proliferation of MC3T3-E1 cells in vitro (Figure 1A). To confirm the effect of increasing the concentration of DHMBA, ... for 1, 2, 3, 4, or 5 days. 5 MC3T3-E1 cells (1000 cells / ml) were cultured for 3 days in the presence of reagent medium (final concentration 1% ethanol) or DHMBA (0.1, 1, 10, 100, or 1000 μM). MC3T3-E1 cell proliferation was unchanged when cultured with DHMBA (0.1, 1, 10, or 100 μM) (Figure 1B). At the highest dose (1000 μM), DHMBA caused a significant decrease in cell proliferation.

[0037] Furthermore, to evaluate the effect of DHMBA on cell death, cells (1x10 per well) 5 MC3T3-E1 cells (1000 μM / ml) were cultured for 3 days until they reached subconfluence, after which DHMBA (0.1, 1, 10, 100, or 1000 μM) was added and cultured for an additional 48 hours. The number of cells attached to the dish did not change with the addition of DHMBA (0.1, 1, 10, or 100 μM), but the highest dose of DHMBA (1000 μM) significantly reduced the cell number (Figure 1C). Therefore, it was found that culture with relatively low concentrations of DHMBA did not cause any change in MC3T3-E1 cell number.

[0038] (DHMBA stimulates the mineralization of osteoblastic MC3T3-E1 cells.) MC3T3-E1 cells (1x10 per well) 5MC3T3-E1 cells (100 cells / ml) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days. After reaching subconfluence, the cells were replaced with 10% FBS, 1% P / S, 4 mM β-glycerophosphate, and L-ascorbic acid (100 μg / ml) (mineralization-enhancing medium). This medium was replaced with fresh medium every 3 days. The medium contained either reagent vehicle (final concentration: 1% ethanol) or DHMBA (1 or 10 μM) for 18 days. After incubation, the cells were stained with alizarin red solution. Incubation with DHMBA (1 or 10 μM) enhanced mineralization of MC3T3-E1 cells (Figure 2A and B). These results suggest that incubation with DHMBA enhances mineralization of osteoblastic MC3T3-E1 cells in vitro.

[0039] DHMBA regulates the effects of TNF-α on the proliferation or cell death of osteoblastic MC3T3-E1 cells. TNF-α has been shown to inhibit bone formation and mineralization in osteoblasts. Therefore, we investigated whether DHMBA regulates the effects of TNF-α on osteoblasts in vitro. To measure the effect of TNF-α on cell proliferation, cells (1x10 per well) were cultured. 5 Cells (1x10 cells / ml) were cultured in α-MEM containing 10% FBS in the presence of TNF-α (1 or 10 ng / ml per ml of culture medium) for 3 days. When cells were cultured with TNF-α (1 or 10 ng / ml), cell proliferation was inhibited (Figure 3A). This inhibition was suppressed by the presence of DHMBA (1 or 10 μM) in the culture medium (Figure 3A). Furthermore, to examine the effect on cell death, cells (1x10 cells / ml) were cultured in α-MEM containing 10% FBS in the presence of TNF-α (1 or 10 ng / ml per ml of culture medium). 5MC3T3-E1 cells (1000 cells / ml) were cultured for 3 days until they reached subconfluence, and then cultured for 48 hours in a culture medium containing TNF-α (1 or 10 ng / ml culture medium) and DHMBA (0.1, 1, or 10 μM). Culture in the presence of TNF-α (1 or 10 ng / ml) promoted cell death (Figure 3B). This effect was suppressed by the presence of DHMBA (1 or 10 μM) (Figure 3B). Thus, DHMBA was found to suppress the inhibition of proliferation and stimulation of cell death of MC3T3-E1 cells induced by TNF-α.

[0040] Next, we investigated whether DHMBA prevents impaired mineralization in osteoblastic MC3T3-E1 cells cultured with TNF-α. 5 Osteoblasts (100 cells / ml) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days. After reaching subconfluence, the cells were cultured in α-MEM containing 10% FBS, 1% P / S, 4 mM β-glycerophosphate, and L-ascorbic acid (100 μg / ml) (mineralization-promoting medium) for 18 days. This medium was replaced with fresh medium every 3 days. After culture, the cells were stained with alizarin red solution. Osteoblast mineralization was inhibited when cultured in the presence of TNF-α (1 ng / ml) (Figure 4). This inhibition was also suppressed by the presence of DHMBA (1 or 10 μM) (Figure 4). DHMBA was found to prevent TNF-α-induced mineralization impairment in MC3T3-E1 cells in vitro.

[0041] DHMBA suppresses proliferation and promotes cell death of osteoblastic MC3T3-E1 cells cocultured with inflammatory macrophage RAW264.7 cells. Inflammatory macrophages, which produce the inflammatory cytokine TNF-α, may play a pathophysiological role in the bone marrow environment. Therefore, we investigated whether inflammatory macrophages affect osteoblast proliferation and death by examining crosstalk between osteoblastic MC3T3-E1 cells and mouse macrophage RAW264.7 cells (Figure 5). First, we examined the effect of lipopolysaccharide (LPS) on the number of MC3T3-E1 cells (Figures 5A and 5B). MC3T3-E1 cells (1 × 10 per well) were cultured in a 200-well culture. 5MC3T3-E1 cells (1000 cells / ml) were cultured in α-MEM containing 10% FBS for 3 days in the presence or absence of LPS (1-1000 ng / ml in culture medium). LPS did not affect proliferation of MC3T3-E1 cells (Figure 5A). Next, to evaluate the effect of LPS on cell death, subconfluent MC3T3-E1 cells were cultured for an additional 48 hours in the presence of LPS (1-1000 ng / ml). LPS did not induce cell death (Figure 5B).

[0042] Next, we investigated whether crosstalk between mouse macrophage RAW264.7 cells and MC3T3-E1 cells altered proliferation and cell death of MC3T3-E1 cells (Figure 5C and D). MC3T3-E1 cells were added to the lower compartment of a Transwell chamber. The upper chamber of each Transwell was filled with RAW264.7 cells containing LPS (100 ng / ml) and cultured for 3 days. After culture, the MC3T3-E1 cells attached to the lower compartment were counted. We found that coculture with RAW264.7 cells suppressed proliferation of MC3T3-E1 cells (Figure 5C). This decrease was suppressed by the presence of DHMBA (1 or 10 μM).

[0043] To further investigate the effect of coculture on cell death, MC3T3-E1 cells were added to the lower compartment of the Transwell chamber and cultured for 3 days until they reached subconfluence. After culture, DHMBA (1 or 10 μM) was added to the lower compartment, and the upper chamber of each Transwell was filled with RAW264.7 cells, which were then cultured for 48 hours in the presence of LPS (100 ng / ml culture medium) (Figure 5D). Coculture with RAW264.7 cells enhanced MC3T3-E1 cell death (Figure 5D). This enhancement was prevented by the presence of DHMBA (1 or 10 μM) (Figure 5D). These results suggest that inflammatory macrophages suppress osteoblast proliferation and stimulate cell death, resulting in a decrease in cell number. Thus, DHMBA was found to inhibit the effects of macrophages on osteoblasts in vitro.

[0044] (DHMBA protects against the effects of conditioned medium obtained from the culture of inflammatory macrophage RAW264.7 cells.) Conditioned medium was obtained by culturing macrophage RAW264.7 cells in the presence or absence of LPS (100 ng / ml culture medium). MC3T3-E1 cells (1x10 per well) 5 MC3T3-E1 cells (200 cells / ml culture medium) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days. After reaching subconfluence, the cells were replaced with conditioned medium (0.25 or 0.5 ml of conditioned medium, brought to 1 ml with normal medium) (Figure 6A). Growth of MC3T3-E1 cells was inhibited by the presence of conditioned medium. Notably, this inhibition was prevented in the presence of DHMBA (1 or 10 μM) (Figure 6A).

[0045] To further measure the effect on cell death, subconfluent MC3T3-E1 cells were cultured for an additional 48 hours in conditioned medium (0.25 or 0.5 ml of conditioned medium, brought to 1 ml with normal medium) or in conditioned medium containing DHMBA (1 or 10 μM) (0.5 ml) (Fig. 6B). MC3T3-E1 cell death was enhanced by incubation in conditioned medium (Fig. 6B). This enhancement was suppressed by incubation in conditioned medium containing DHMBA (1 or 10 μM) (Fig. 6B). Thus, LPS-conditioned medium inhibited proliferation, enhanced cell death, and caused a decrease in cell number in MC3T3-E1 cells.

[0046] Involvement of NF-κB signaling in osteoblastic MC3T3-E1 cells cultured in conditioned medium. It has been demonstrated that osteoblast mineralization is suppressed by activation of TNF-α-induced NF-κB signaling. Therefore, we investigated whether TNF-α and its signaling processes are involved in the expression of the conditioned medium's effects in inhibiting proliferation and promoting cell death of MC3T3-E1 cells by inhibiting NF-κB signaling. Incubation with LPS (10 or 100 ng / ml) increased TNF-α production in RAW264.7 cells, but this increase was suppressed by the presence of DHMBA (1 or 10 μM) (Figure 7A). Incubation with Bay11-7082 (1, 10, or 50 nM), an inhibitor of NF-κB signaling, did not significantly decrease the number of MC3T3-E1 cells (Figure 7B). Incubation with Bay11-7082 (1 or 10 nM) suppressed the effects of the conditioned medium on inhibiting proliferation (Figure 7C) and promoting cell death (Figure 7D) of MC3T3-E1 cells. These results suggest that TNF-α and its NF-κB signaling pathway are involved in the expression of the effects of the conditioned medium on inhibiting proliferation and promoting cell death of osteoblastic MC3T3-E1 cells.

[0047] We further investigated whether DHMBA regulates the NF-κB signaling process in MC3T3-E1 cells. Culture with DHMBA (10 μM) reduced the expression level of NF-κB p65 in MC3T3-E1 cells (Figures 8A and B). NF-κB p65 levels were elevated by culturing in the presence of TNF-α (10 ng / ml) (Figures 8A and B). This increase was suppressed by culturing with DHMBA (10 μM) (Figures 8A and B). Furthermore, NF-κB promoter activity in MC3T3-E1 cells was increased by treatment with TNF-α (1 or 10 ng / ml) (Figure 8C). This increase was suppressed by the addition of DHMBA (1 or 10 μM) (Figure 8C). Thus, we found that the increased NF-κB promoter activity induced by TNF-α stimulation in MC3T3-E1 cells was suppressed in the presence of DHMBA. DHMBA also reduced NF-κB promoter activity in MC3T3-E1 cells in the absence of TNF-α (Fig. 8C). These results support the notion that DHMBA suppresses gene promoter activity related to the transcription factor NF-κB in osteoblasts.

[0048] Discussion: The marine phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has been discovered as an excellent peroxyl radical scavenger. Its potency has been shown to be approximately 15 times, or four orders of magnitude, superior to that of Trolox in both lipid and aqueous media, suggesting its important role in regulating cellular function and maintaining health. Recently, DHMBA has been demonstrated to inhibit the proliferation and metastatic activity of bone-metastatic prostate cancer cells, suggesting that DHMBA may play a pharmacological role in preventing bone loss. Furthermore, this study demonstrated that DHMBA stimulates osteoblast mineralization and prevents the impairment of osteoblast mineralization associated with TNF-α signaling produced by inflammatory macrophage RAW264.7 cells upon LPS stimulation. DHMBA is believed to play an important pharmacological role in restoring impaired mineralization.

[0049] Factors that promote bone formation are clinically important for preventing and repairing bone loss in osteoporosis caused by aging and various diseases. The development of such factors is urgently needed. In vitro incubation of osteoblastic MC3T3-E1 cells with DHMBA was found to promote osteoblast mineralization. DHMBA exerts a promoting effect on osteoblast mineralization and differentiation, suggesting that this may lead to increased bone formation. Therefore, DHMBA is considered to be an important factor promoting bone formation. Bone loss is well known to be caused by postmenopausal estrogen deficiency associated with an inflammatory state. The cytokine TNF-α, produced by inflammation, plays an important role in skeletal pathology, causing bone loss. This cytokine inhibits osteoblast mineralization by activating NF-κB signaling in osteoblastic MC3T3-E1 cells.

[0050] In this study, we investigated whether incubation with DHMBA prevented the impaired osteoblast mineralization associated with NF-κB signaling in MC3T3-E1 cells. Mineralization of MC3T3-E1 cells was impaired by incubation with TNF-α, which activates NF-κB signaling. This impairment was found to be suppressed by the presence of DHMBA. DHMBA was found to exert an inhibitory effect on the NF-κB signaling process. Inflammatory macrophages may play a pathophysiological role in the bone marrow microenvironment by increasing cytokine production. Furthermore, we investigated the crosstalk between osteoblasts and macrophages (RAW264.7 cells) in vitro.

[0051] Co-culture of MC3T3-E1 and RAW264.7 cells with LPS stimulation inhibited the proliferation of osteoblastic MC3T3-E1 cells. This inhibition was induced by culturing osteoblasts in conditioned medium obtained from LPS-stimulated RAW264.7 cell cultures. Notably, the inhibitory effect of conditioned medium on MC3T3-E1 cell proliferation was suppressed by the presence of an inhibitor of NF-κB signaling, suggesting the involvement of NF-κB signaling. Furthermore, the TNF-α concentration in the conditioned medium was significantly increased by culturing RAW264.7 cells with LPS stimulation. These results support the idea that osteoblast mineralization is impaired by the activation of the TNF-α signaling process produced by inflammatory macrophages in the bone marrow microenvironment. Notably, incubation with DHMBA suppressed TNF-α production in inflammatory RAW264.7 cells. Furthermore, the inhibitory effect of conditioned medium on cell proliferation was not expressed in the presence of DHMBA in the culture medium, suggesting that this factor suppresses the process of TNF-α signaling in MC3T3-E1 cells. Therefore, DHMBA may be useful in preventing impaired osteoblast mineralization associated with inflammatory conditions.

[0052] We further investigated whether DHMBA affects the NF-κB signaling process in MC3T3-E1 cells. Culture in the presence of DHMBA reduced the level of NF-κB p65 in osteoblasts. Furthermore, DHMBA was found to suppress the enhanced reporter activity of NF-κB response elements in the promoter regions of nuclear genes in MC3T3-E1 cells induced by TNF-α stimulation. This suggests that DHMBA may be an important underlying mechanism for suppressing TNF-α-induced impairment of osteoblast mineralization. As summarized in Figure 9, inflammatory macrophages in the bone marrow microenvironment produce TNF-α, which activates the NF-κB signaling process in osteoblasts. DHMBA suppresses TNF-α production in inflammatory macrophages and regulates the activation of the associated signaling process. Furthermore, DHMBA can translocate to the nucleus of osteoblasts. Once translocated into the nucleus, DHMBA may inhibit NF-κB binding to response elements in the promoter regions of genes. Furthermore, DHMBA may exert a direct regulatory effect on the transcriptional activity of promoter regions in the nucleus.

[0053] In conclusion, we have confirmed that DHMBA stimulates mineralization in mouse osteoblastic MC3T3-E1 cells in vitro. Furthermore, we found that DHMBA inhibits the development of impaired osteoblast mineralization, which is manifested in vitro by activating the TNF-α signaling pathway produced by inflammatory macrophages. Thus, DHMBA exerts its osteogenic effects by preventing impaired osteoblast mineralization. The marine factor DHMBA may play a role in preventing and repairing inflammation-related bone loss. This study provides a new strategy for treating impaired osteoblast bone formation associated with inflammatory conditions in the bone marrow microenvironment.

Claims

1. A calcification enhancer for osteoblast MC3T3-E1 cells, characterized in that the active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) and the agent has the effect of enhancing the calcification of osteoblast MC3T3-E1 cells.

2. An agent for controlling the effect of TNF-α on the proliferation or cell death of osteoblastic MC3T3-E1 cells, characterized in that the active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) and the agent has the effect of controlling the effect of TNF-α on the proliferation or cell death of osteoblastic MC3T3-E1 cells.

3. An agent for preventing TNF-α-induced calcification disorder in MC3T3-E1 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient, controls the effect of TNF-α on the proliferation or cell death of osteoblastic MC3T3-E1 cells, and has the effect of preventing TNF-α-induced calcification disorder in MC3T3-E1 cells.

4. An inhibitory regulator for suppressing the proliferation and promoting cell death of osteoblasts MC3T3-E1 co-cultured with inflammatory macrophages RAW264.7 cells, characterized in that the inhibitory regulator contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and has the effect of suppressing the proliferation and promoting cell death of osteoblasts MC3T3-E1 co-cultured with inflammatory macrophages RAW264.7 cells.

5. A protective agent for protecting against the effects of a conditioned medium obtained from the culture of inflammatory macrophage RAW264.7 cells on the proliferation or cell death of osteoblastic MC3T3-E1 cells, characterized in that the active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) and the protective agent has an effect of protecting against the effects of a conditioned medium obtained from the culture of inflammatory macrophage RAW264.7 cells on the proliferation or cell death of osteoblastic MC3T3-E1 cells.

6. An inhibitor of gene promoter activity associated with the osteoblast transcription factor NF-κB, characterized in that the active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) and the inhibitor has the effect of suppressing gene promoter activity associated with the osteoblast transcription factor NF-κB.

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