Methods to enhance mineralization in osteoblast MC3T3-E1 cell lines; methods to control the effects of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cell lines; methods to prevent TNF-α-induced mineralization disorders in MC3T3-E1 cell lines; and methods to inhibit the activity of gene promoters related to transcription factor NF-κB in osteoblast MC3T3-E1 cell lines.

Abstract

The subject of this invention is to provide an inhibitor with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as the active ingredient to defend against and treat the impairment of osteoblast bone formation and mineralization associated with inflammatory conditions in the bone marrow microenvironment, and to provide a new strategy. The solution of the present invention is characterized by using 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as the active ingredient, which has the effect of promoting the mineralization of osteoblast MC3T3-E1 cells.

Description

Mineralization enhancer for osteoblast MC3T3-E1 cells The present invention relates to, for example, a mineralization enhancer for osteoblast MC3T3-E1 cells. Yu Chang oyster ( The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), found in Crassostrea Gigas and other plants, acts as a superior peroxy radical scavenger compared to other strong antioxidants like Trolox. Therefore, DHMBA may play an important role in preventing health disorders. In the present invention, mouse macrophage RAW264.7 cells were used in vitro to investigate whether DHMBA prevents the mineralization disorder of mouse osteoblast MC3T3-E1 cells induced by inflammation. In an osteoblast culture system, the addition of DHMBA (1-100 μM) did not affect the proliferation or cell death of MC3T3-E1 cells. DHMBA was found to enhance osteoblast mineralization under these conditions. DHMBA also inhibited the reduction in MC3T3-E1 cell mineralization induced by the addition of the inflammatory cytokine TNF-α. DHMBA inhibits lipopolysaccharide (LPS)-induced TNF-α production in macrophage RAW264.7 cell cultures. The proliferation of MC3T3-E1 cells under LPS stimulation is inhibited through crosstalk caused by co-culture with RAW264.7 cells. Interestingly, it was discovered that culturing macrophages with LPS-derived culture medium inhibited the proliferation of MC3T3-E1 cells. This effect was inhibited by the presence of DHMBA or Bay 11-7082, an inhibitor of the TNF-α signaling pathway. The inhibitory effect of DHMBA was not further enhanced in the presence of Bay 11-7082. As its mechanism, DHMBA is known to reduce the level of NF-κB p65 and the activity of the NF-κB reporter gene in MC3T3-E1 cells. DHMBA was invented to prevent the mineralization impairment of osteoblasts caused by the inflammatory cytokine TNF-α by inhibiting TNF-α signaling associated with the activation of macrophages in the bone marrow microenvironment. This invention also provides a new strategy for the treatment of osteoblast disorders caused by inflammation. (Introduction) Bone homeostasis is regulated by the functions of osteoblasts, osteocytes and osteoclasts, the main cells of bone tissue. In bone homeostasis, bone resorption is first caused by osteoclasts. The osteoclasts differentiate from hematopoietic progenitor cells and mature at the surface of the bone. Then, the cavity created by bone erosion is refilled by osteoblasts produced by local mesenchymal stem cells. Osteoblasts gather at the bottom of the cavity and begin bone formation. Through such a cell mechanism, bone tissue is maintained as a fresh and flexible tissue. Bone homeostasis, which is accompanied by the mechanism of bone remodeling, plays a physiological role in maintaining new bone mass. The process of bone remodeling is intricately controlled by hormones and cytokines; stress on the skeletal system caused by physical activity or weight load; various growth factors, such as transforming growth factor-β1 produced by osteoblasts; platelet-derived growth factors and insulin-like growth factor; and systemic influences, all interacting with each other. Furthermore, immune cells such as lymphocytes and macrophages, residing in the bone marrow microenvironment, contribute to the control of bone remodeling. Postmenopausal osteoporosis, which physiologically causes bone loss in women with aging, is caused by the lack of estrogen that accompanies aging. Furthermore, it should be noted that severe bone loss induced by bone metastatic cancer cells causes the death of patients with fractures. Inflammatory macrophages, in particular, contribute to the manifestation of bone disorders mediated by bone metastasis of cancer cells. Inflammatory cytokines, mainly tumor necrosis factor (TNF)-α, can cause osteoarthritis, osteoporosis, and bone metastasis of cancer. Interestingly, TNF-α produced by inflammatory macrophages inhibits the mineralization of osteoblasts through the activation of NF-κB signaling, and has been shown to stimulate bone resorption by osteoclasts. Macrophage RAW264.7 cells are monocyte / macrophage-like cells with distinct characteristics regarding the immune, metabolic, and phagocytic functions mediated by macrophages. The RAW264.7 cell line is used as a model cell for osteoclast formation, which is differentiated from the monocyte-macrophage lineage. The host immune system is stimulated by lipopolysaccharide (LPS), a major antigen for 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, the development of therapeutic factors to prevent bone loss and fractures caused by various diseases, such as inflammation and cancer bone metastasis, has become important. The marine phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was originally derived from the long oyster Found in Crassostrea Gigas, DHMBA has been shown to scavenge free radicals and protect against oxidative stress in several cell types. Interestingly, DHMBA is an excellent peroxyl radical scavenger, approximately 15-fold and four-digitally more potent than the known strong free radical scavenger Trolox in lipid and aqueous media, respectively. Furthermore, DHMBA has been shown to react with HOO(･) more rapidly than other known antioxidants, such as resveratrol and ascorbic acid. [Prior Art] [Patent] [Patent Document 1] Japanese Patent Application Laid-Open No. 2023-080697 [Problem to be Solved by the Invention] DHMBA, possessing the aforementioned properties, suggests the potential for playing an important role in regulating cell function and maintaining a healthy state. In particular, we have demonstrated that DHMBA inhibits the proliferation and metastasis of bone-metastatic prostate cancer cells. Bone morphogenetic factors are clinically important for repairing bone loss and preventing osteopenia and osteoporosis associated with aging and various diseases. The development of such substances is crucial. This study examined whether DHMBA stimulates osteoblast mineralization and prevents osteoblast mineralization impairment associated with TNF-α production in inflammatory macrophages RAW264.7 cells stimulated by LPS. The findings demonstrate that DHMBA stimulates mineralization in mouse osteoblasts MC3T3-E1 cells and, furthermore, inhibits cell proliferation and mineralization impairment in MC3T3-E1 cells mediated by signaling associated with TNF-α production in LPS-activated inflammatory macrophages RAW264.7 cells. The present invention aims to provide a novel strategy for preventing and treating osteoblastic bone formation and mineralization disorders associated with inflammatory conditions in the bone marrow microenvironment, and to provide an inhibitor containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient. [Means for Solving the Problem] The present invention is characterized by a mineralization enhancer for osteoblast MC3T3-E1 cells, comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient, and having the effect of enhancing the mineralization of osteoblast MC3T3-E1 cells; or a characteristic of the present invention is a control agent for the effect of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cells, comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient, and having the effect of controlling the effect of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cells; or a characteristic of the present invention is a preventive agent for TNF-α-induced mineralization disorder in MC3T3-E1 cells, comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA). alcohol (DHMBA) as an active ingredient, and has the effect of controlling the effect of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cells, and preventing TNF-α-induced mineralization disorders in MC3T3-E1 cells; or it is characterized by being a controlling agent that inhibits the proliferation and inhibits the cell death of osteoblast MC3T3-E1 cells co-cultured with inflammatory macrophage RAW264.7 cells, and contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient, and has the effect of controlling the proliferation and inhibiting the cell death of osteoblast MC3T3-E1 cells co-cultured with inflammatory macrophage RAW264.7 cells; or it is characterized by being a protective agent that protects against the effect of conditioned medium obtained from the culture of inflammatory macrophage RAW264.7 cells on the proliferation or cell death of osteoblast MC3T3-E1 cells, and contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient. The invention comprises a novel inhibitor of the activity of a gene promoter associated with the transcription factor NF-κB in osteoblasts, containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient, and has the effect of protecting against the effects of conditioned medium obtained from culturing inflammatory macrophage RAW264.7 cells on the proliferation or cell death of osteoblasts MC3T3-E1 cells; or a novel inhibitor of the activity of a gene promoter associated with the transcription factor NF-κB in osteoblasts, containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient, and has the effect of inhibiting the activity of a gene promoter associated with the transcription factor NF-κB in osteoblasts. [Effects of the invention] The inhibitors of the present invention containing 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient may provide a novel strategy for treating inflammation-induced osteoblast disorders, and exhibit the excellent effect of providing a novel strategy for preventing and treating osteoblast bone formation and mineralization disorders associated with inflammatory conditions in the bone marrow microenvironment. Detailed experiments were conducted using mouse osteoblasts MC3T3-E1 cells to verify the effects of various agents of the present invention comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient. The details of the experiments are described below. (Materials and Methods) (Reagents) Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / 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, Virginia, USA). α-Minimum essential medium (α-MEM) was purchased from Invitrogen Corp. (Carlsbad, California, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, Utah, USA). Lipopolysaccharide (LPS), Bay11-7089, and all other reagents, unless otherwise specified, were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Lipopolysaccharide (LPS) and Bay11-7089 were dissolved in 100% ethanol and stored at −20°C until use. 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a novel amphiphilic phenolic compound, is an antioxidant and was first used in the study of long oysters ( This invention uses synthetic DHMBA. The structure of DHMBA was previously revealed. The purity of the synthesized DHMBA is 100%. DHMBA was dissolved in 100% ethanol and stored at -20°C until use. Mouse osteoblast 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 supplemented with 10% FBS and 1% P / S in a plastic dish in a CO2 incubator at 37°C, as previously used. (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 a BALB / c mouse-derived Abelson leukemia virus-transformed cell line. (Cell Proliferation Analysis) To confirm the progress of cell proliferation, MC3T3-E cells (1×10 5 100 μM DHMBA) in 24-well plates in DMEM (containing 10% FBS and 1% P / S) were cultured at 37°C in a water-saturated atmosphere containing 5% CO2 and 95% air. The cells were cultured in the presence of medium (final concentration of 1% ethanol) or DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. In other experiments, to determine the effect of increasing concentrations of DHMBA on MC3T3-E1 cells, cells (1×10 cells per well) were cultured in DMEM (containing 10% FBS and 1% P / S) in 24-well plates. 5 The cells were cultured for 3 days in the presence of either the test medium (final concentration: 1% ethanol) or DHMBA (0.1, 1, 10, 100, or 1000 μM). To evaluate the effect of DHMBA on cell death of osteoblast MC3T3-E1 cells (1×10 5 / ml culture medium) in α-MEM containing 10% FBS and 1% P / S. After culturing for 3 days to reach subconfluence, either the test medium (final concentration of 1% ethanol) or DHMBA (0.1, 1, 10, 100, or 1000 μM) was added and cultured for a further 48 hours. After culture, Ca was added to determine the number of cells attached to each well. 2+ / Mg 2+After adding a solution containing 0.05% trypsin and EDTA (0.1 ml per well), the culture dish was incubated at 37°C for 2 minutes. After adding α-MEM containing 10% FBS and 1% P / S (0.9 ml), 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 staining stains living cells, but not dead cells. Viable cells were counted using a hemacytometer (Sigma-Aldrich, St. Louis, MO) and a cell counter (Line Seiki H-102P, Tokyo, Japan) under a microscope (Olympus MTV-3), and the average of two counts was taken. The number of cells is expressed as the number per well. (Osteoblast differentiation analysis and Alizarin Red S staining) MC3T3-E1 cells (10 cells per well in a 12-well plate) were cultured in a 12-well plate. 5 Cells were cultured in α-MEM (1.0 ml / well) containing 10% FBS and 1% P / S for 3 days. The culture medium was then changed to α-MEM supplemented with 10% FBS, 1% P / S, L-ascorbic acid (100 μg / ml), and 4 mM β-glycerophosphate, as previously described. 1 or 10 μM DHMBA was added, and the cells were exchanged with fresh culture medium every 3 days for 18 days. On the 18th day of culture, the cells were rinsed with PBS, fixed with ice-cold 75% ethanol at 4°C for 30 minutes, and then stained with Alizarin Red S (40 mM, pH 6.2) to visualize calcium deposits. The cells were incubated at room temperature for 30 minutes, after which the staining solution was removed by thorough washing with distilled water. The stained cells on the plates were photographed using a microscope (40x magnification) (Olympus IX71, Olympus Corporation, Tokyo, Japan) and visualized using Image J2. For quantification, each plate containing stained cells was solubilized by adding a 10% cetylpyridinium chloride solution, and the absorbance was measured at 570 nm. (Crosstalk between MC3T3-E1 cells and RAW264.7 cells) To investigate the crosstalk between osteoblasts MC3T3-E1 cells and macrophages RAW264.7 cells, a perforated chamber (Corning, Life Sciences, catalog number CLS3464-48EA, USA) equipped with a polycarbonate filter (pore size 8 μm, diameter 6.5 mm) was used for analysis. MC3T3-E1 cells (1×10 cells per ml of culture medium) were placed in the lower chamber. 5Cells) were added to α-MEM culture medium (1 ml) containing 10% FBS and 1% P / S. In the upper part of the chamber, RAW264.7 cells were suspended in DMEM containing 10% FBS and 1% P / S, and 0.5 ml (5×10 4 The upper chamber was filled with 100 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 transparent holes was placed in a 37°C incubator under 5% CO2 and 95% air. To induce an inflammatory state in the RAW264.7 cells, LPS (100 ng / ml of culture medium) was added to the upper chamber containing the RAW264.7 cells. The culture plates containing MC3T3-E1 (lower chamber) or RAW264.7 cells (upper chamber) were cultured for 3 days to subconfluence. After incubation, the culture medium in the upper and lower chambers was aspirated and the filter membrane was removed. MC3T3-E1 cells attached to the culture dish (wells) were counted as described in the "Analysis of Cell Proliferation" section. Furthermore, in order to investigate the effect of co-culture with RAW264.7 cells on the cell death of MC3T3-E1 cells, osteoblasts (1×10 5 Cells were cultured for 3 days to subconfluence, after which the culture medium was exchanged with fresh medium. The culture medium contained either the test drug medium (final concentration: 1% ethanol) or DHMBA (1 or 10 μM). A permeable chamber was then placed in each well. RAW264.7 cells were plated in the upper chamber with 0.5 ml (5×10 cells / ml) of culture medium, regardless of the presence or absence of LPS. 4 After a further 48 hours of culture, the culture medium in the upper and lower chambers was aspirated and the filter membrane removed. Count the cells attached to the culture dish (wells) as described in the "Cell Proliferation Analysis" section. (Effect of Conditioned Medium on MC3T3-E1 Cells) Conditioned medium was obtained by culturing RAW264.7 cells. RAW264.7 cells (1×10 cells per well of a 24-well plate) were inoculated with LPS at subconfluence, with or without LPS (100 ng / ml in the culture medium). 5Cells / ml) were cultured in DMEM containing 10% FBS and 1% P / S for 3 days. After cell culture, the culture fluid (conditioned medium) was recovered. In order to determine the effect of conditioned medium on the proliferation of MC3T3-E1 cells, α-MEM containing 10% FBS and 1% P / S was cultured for 3 days. Conditioned medium (1 ml, control group) was used to culture without LPS, or conditioned medium (usually culture fluid, 1 ml in total, 0.25 or 0.50 ml of conditioned medium per well) was cultured for 3 days in the presence or absence of DHMBA (1 or 10 μM) or LPS. Furthermore, in order to investigate the effect of the cell death inhibitory effect on MC3T3-E1 cells, cells (1×10 5 Cells were cultured for 3 days in α-MEM supplemented with 10% FBS and 1% P / S (10 cells / ml). Upon reaching subconfluence, the medium was exchanged. Cells were then supplemented for 48 hours in either standard medium, conditioned medium without LPS (1 ml), or conditioned medium (0.25 or 0.50 ml / well of standard medium added to 1 ml) containing either the test drug medium (final concentration: 1% ethanol) or DHMBA (1 or 10 μM). Following incubation, cells were detached from each well and the cell count was determined as described above. (Analysis of TNF-α production) RAW264.7 cells (1×10 5 RAW264.7 cells were cultured in DMEM containing 10% FBS and 1% P / S for 3 days in 24-well plates until subconfluence. After treatment with or without LPS (100 ng / ml of culture medium), the culture medium was harvested for 5 hours. For cytokine analysis, the cells were detached from each dish and the cell count was determined as described in the "Cell Proliferation Analysis" section. The number of RAW264.7 cells did not change significantly when cultured with LPS. TNF-α concentrations in the culture medium were analyzed using a mouse TNF-α ELISA kit (Catalog No. 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). (Effect of NF-κB signaling pathway inhibitor) Further, the effect of NF-κB signaling pathway inhibitor Bay11-7082 on the proliferation or cell death of MC3T3-E1 cells was investigated. First, in order to investigate the effect of Bay11-7082 on the proliferation of MC3T3-E1 cells, cells (1×10 5Cells were cultured for 3 days in the presence of 10% FBS, 1% P / S, and test medium (final concentration 1% ethanol) or Bay11-7082 (1, 10, 50, 100, or 1000 nM) to subconfluence. 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 conditioned medium (0.5 ml per well, with standard medium added to a total of 1 ml). In other experiments, to evaluate the effect of Bay11-7082 on MC3T3-E1 cell death, cells (1×10 5 Cells were cultured at 10% FBS and 1% P / S in α-MEM (10% FBS / 1% P / S) for 3 days to reach subconfluence. Afterward, the culture medium was exchanged with 1 ml of α-MEM containing either standard culture medium or conditioned medium 1 (0.5 ml per well) plus standard culture medium to 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) and DHMBA (10 μM). Following incubation, cells were detached from each well, and cell count was determined as described previously. (Western blot) MC3T3-E1 cells (containing 1×10 6Cells were cultured in 100 mm dishes (10 ml of culture medium per 100 mm dish) in α-MEM supplemented with 10% FBS and 1% P / S for 3 days in the presence of test medium (final concentration: 1% ethanol), DHMBA (10 μM), TNF-α (10 ng / ml culture medium), or DHMBA (10 μM) plus TNF-α (10 ng / ml). Following incubation, the dishes were washed three times with 10 ml of cold PBS, and adherent cells were removed by scraping in lysis buffer (Cell Signaling Technology, Danvers, MA, USA). A 40 μg sample of protein obtained from the cell lysate was added to each 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 against the target proteins NF-κB p65 (Catalog No. 3034, rabbit) and β-actin (Catalog No. 3700, mouse) (diluted 1:1,000). Following incubation, the membranes were further incubated with a secondary antibody conjugated to wasabi peroxidase (Santa Cruz Biotechnology, Inc., mouse sc-2005 or rabbit sc-2305, diluted 1:1,000) for 60 minutes at room temperature. Protein bands were then detected using a chemiluminescent substrate (Catalog No. 34577, Thermo Scientific, Rockford, IL, USA) on an X-ray film. Four membranes from four independent experiments were scanned using an Epson Perfection 1660 Photo scanner, and the bands were quantified using Image J2 software (National Institutes of Health, Bethesda, MD, USA). (NF-κB reporter assay) We performed the NF-κB-responsive reporter luciferase assay (pNF-κB-Luc, BD Biosciences) using a previously described method. MC3T3-E1 cells (2 × 10 cells / 100 μl in a 96-well plate) were plated and cultured. 4Cells were cultured in α-MEM supplemented with 10% FBS and 1% P / S for 24 hours. Following incubation, the culture medium was removed and the cells were washed twice with α-MEM supplemented with FBS and antibiotics. 100 μl of α-MEM supplemented with FBS and antibiotics was added to the wells containing the cells. MC3T3-E1 cells were transfected with the reporter plasmid or an empty vector control (pGL3-Basic) in α-MEM supplemented with FBS and antibiotics using Lipofectamine 2000 reagent (Invitrogen). Five hours later, the culture medium was exchanged with α-MEM supplemented with 10% FBS and 1% P / S, and TNF-α (1 or 10 ng / ml) was added to the cells for 24 hours. In parallel, the cells were cultured with either the test medium (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) and a microplate reader (Turner Designs, Sunnyvale, CA, USA). Equivalent transfection efficiency was verified for all plasmids using the luciferase reporter plasmid pRL-SV40 (Promega, Madison, WI). 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 using one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons after parametric data testing. A p-value < .05 was considered statistically significant. (Results) Effects of marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the proliferation and cell death of MC3T3-E1 osteoblasts. To investigate the effect of DHMBA on the proliferation and cell death of mouse osteoblasts MC3T3-E1, cells (1×10 5 Cells were cultured in α-MEM medium containing 10% FBS and 1% P / S in the presence of test medium (final concentration of 1% ethanol) or DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. No significant changes were observed in the proliferation of MC3T3-E1 cells in vitro after 5 days of culture (Figure 1A). To confirm the effect of increasing DHMBA concentrations, cells (1×10 cells per well) were cultured in the presence of test medium (final concentration of 1% ethanol) or DHMBA (10 μM). 5 Cells were cultured for 3 days in the presence of test vehicle (final concentration 1% ethanol) or DHMBA (0.1, 1, 10, 100, or 1000 μM). MC3T3-E1 cell proliferation remained unchanged even when cultured with DHMBA (0.1, 1, 10, or 100 μM) (Figure 1B). At the highest dose (1000 μM), DHMBA significantly reduced cell proliferation. Furthermore, in order to evaluate the effect of DHMBA on cell death, cells (1×10 5 Cells were cultured for 3 days at 100 cells / ml until subconfluence, after which DHMBA (0.1, 1, 10, 100, or 1000 μM) was added and cultured for a further 48 hours. The number of cells attached to the culture 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 number of cells (Figure 1C). This indicates that the number of MC3T3-E1 cells did not change with lower DHMBA concentrations. (DHMBA stimulates the mineralization of osteoblast MC3T3-E1 cells) MC3T3-E1 cells (1×10 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 10% FBS, 1% P / S, 4 mM β-glycerophosphate, and L-ascorbic acid (100 μg / ml) (mineralization-enhancing culture medium). The culture medium was replaced with fresh one every 3 days. The culture medium contained the test medium (final concentration was 1% ethanol) or DHMBA (1 or 10 μM) and was cultured for 18 days. After culture, the cells were stained with Alizarin Red solution. If cultured with DHMBA (1 or 10 μM), the mineralization of MC3T3-E1 cells was enhanced (Figures 2A and B). These results suggest that DHMBA culture can enhance the mineralization of osteoblast MC3T3-E1 cells in vitro. (DHMBA inhibits the effects of TNF-α on the proliferation or cell death of osteoblasts MC3T3-E1 cells) TNF-α has been shown to inhibit bone formation and mineralization in osteoblasts. Therefore, we investigated whether DHMBA modulates the effects of TNF-α on osteoblasts in vitro. To determine the effect of TNF-α on cell proliferation, cells (1×10 cells per well) were plated. 5Cells (1×10 cells / ml) were cultured in α-MEM containing 10% FBS in the presence of TNF-α (1 or 10 ng / ml per 1 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 investigate the effect on cell death, cells (1×10 cells per well) were cultured in the presence of TNF-α (1 or 10 ng / ml). 5 MC3T3-E1 cells were cultured for 3 days at 400 μM (100 μM) to subconfluence. Afterwards, cells were cultured for 48 hours in a medium containing DHMBA (0.1, 1, or 10 μM) in the presence of TNF-α (1 or 10 ng / ml). Cell death was enhanced when MC3T3-E1 cells were cultured in the presence of TNF-α (1 or 10 ng / ml) ( FIG3B ). This effect was inhibited by the presence of DHMBA (1 or 10 μM) ( FIG3B ). Thus, DHMBA inhibits the inhibition of MC3T3-E1 cell proliferation and cell death induced by TNF-α. Next, we investigated whether DHMBA could prevent the mineralization disorder of osteoblast MC3T3-E1 cells cultured with TNF-α. 5 Cells 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) (a mineralization-enhancing medium) for 18 days. The culture medium was replaced with fresh one every 3 days. After culture, the cells were stained with Alizarin Red solution. If cultured in the presence of TNF-α (1 ng / ml), the mineralization of osteoblasts was inhibited (Figure 4). This inhibition was inhibited by the presence of DHMBA (1 or 10 μM) (Figure 4). DHMBA was found to prevent TNF-α-induced mineralization disorders in MC3T3-E1 cells in vitro. (DHMBA controls the inhibition of proliferation and the promotion of cell death of osteoblasts MC3T3-E1 co-cultured with inflammatory macrophages RAW264.7 cells. Inflammatory macrophages that produce the inflammatory cytokine TNF-α may play a pathological physiological role in the bone marrow environment. Therefore, we investigated whether inflammatory macrophages affect the proliferation and death of osteoblasts through the crosstalk between osteoblasts MC3T3-E1 cells and mouse macrophages RAW264.7 cells (Figure 5). First, we investigated the effect of lipopolysaccharide (LPS) on the number of MC3T3-E1 cells (Figures 5A and 5B). MC3T3-E1 cells (1×10 per well) were placed in the 1% MC3T3-E1 cell line. 5MC3T3-E1 cells were cultured in α-MEM supplemented with 10% FBS in the presence or absence of LPS (1-1000 ng / ml in the culture medium) for 3 days. LPS had no effect on the proliferation of MC3T3-E1 cells (Figure 5A). To evaluate the effect of LPS on cell death, subconfluent MC3T3-E1 cells were further cultured in the presence of LPS (1-1000 ng / ml) for 48 hours. LPS did not induce cell death (Figure 5B). Next, we investigated whether the proliferation and cell death of MC3T3-E1 cells changed by crosstalk between mouse macrophage RAW264.7 cells and MC3T3-E1 cells (Figures 5C and D). MC3T3-E1 cells were added to the lower compartment of the permeable well chamber. The upper chamber of each permeable well was filled with RAW264.7 cells containing LPS (100 ng / ml) and cultured for 3 days. After culture, the number of MC3T3-E1 cells attached to the lower compartment was counted. It was found that if co-cultured with RAW264.7 cells, the proliferation of MC3T3-E1 cells was inhibited (Figure 5C). This reduction was inhibited by the presence of DHMBA (1 or 10 μM). Furthermore, to investigate the effect of co-culture on cell death, MC3T3-E1 cells were added to the lower compartment of the permeable well chamber and cultured for 3 days to reach subconfluence. After culture, DHMBA (1 or 10 μM) was added to the lower compartment, and the upper chamber of each permeable well was filled with RAW264.7 cells and cultured for 48 hours in the presence of LPS (100 ng / ml culture medium) (Figure 5D). Co-culture with RAW264.7 cells increased MC3T3-E1 cell death (Figure 5D). This increase was prevented by the presence of DHMBA (1 or 10 μM) (Figure 5D). These results suggest that inflammatory macrophages inhibit the proliferation of osteoblasts, stimulate cell death, and cause a decrease in cell number. In this way, it was found that DHMBA inhibited the effect of macrophages on osteoblasts in vitro. (DHMBA protects against the effects of conditioned medium obtained by culturing inflammatory macrophages RAW264.7 cells) Conditioned medium was obtained by culturing macrophages RAW264.7 cells in the presence or absence of LPS (100 ng / ml culture medium). MC3T3-E1 cells (1×10 5Cells were cultured in α-MEM supplemented with 10% FBS and 1% P / S for 3 days (10 cells / ml culture medium). After reaching subconfluence, the cells were replaced with conditioned medium (containing 0.25 or 0.5 ml of conditioned medium, and the volume was increased to 1 ml with normal culture medium) (Figure 6A). MC3T3-E1 cell proliferation was inhibited by the presence of conditioned medium. Notably, this inhibition was prevented in the presence of DHMBA (1 or 10 μM) (Figure 6A). Further, in order to measure the effect on cell death, the MC3T3-E1 cells reaching sub-confluence were further cultured for 48 hours (Fig. 6B) with conditioned medium (containing 0.25 or 0.5 ml of conditioned medium, and added to 1 ml with normal culture medium) or conditioned medium (containing 0.5 ml) containing DHMBA (1 or 10 μM). MC3T3-E1 cell death was enhanced by culturing with conditioned medium (Fig. 6B). This enhancement was suppressed by conditioned medium containing DHMBA (1 or 10 μM) (Fig. 6B). Therefore, it was learned that the conditioned medium cultured with LPS could hinder the proliferation of MC3T3-E1 cells, enhance cell death, and cause a decrease in cell number. (Involvement of NF-κB Signaling in Conditioned Medium-Cultivated Osteoblasts MC3T3-E1) It has been demonstrated that osteoblast mineralization is inhibited by TNF-α-induced activation of NF-κB signaling. Therefore, by blocking NF-κB signaling, we investigated whether TNF-α and its signaling pathways are associated with the effects of conditioned medium on the inhibition of MC3T3-E1 cell proliferation and the enhancement of cell death. TNF-α production in RAW264.7 cells increased when cultured with LPS (10 or 100 ng / ml), but this increase was inhibited by the presence of DHMBA (1 or 10 μM) (Figure 7A). Culture with the NF-κB signaling inhibitor Bay11-7082 (1, 10, or 50 nM) did not significantly reduce the number of MC3T3-E1 cells (Figure 7B). When cultured with Bay11-7082 (1 or 10 nM), the effects of conditioned medium on the proliferation inhibition (Figure 7C) and cell death enhancement (Figure 7D) of MC3T3-E1 cells were suppressed. These results suggest that TNF-α and NF-κB signaling are involved in the effects of conditioned medium on the proliferation inhibition and cell death enhancement of MC3T3-E1 osteoblasts. Furthermore, we investigated whether DHMBA modulates NF-κB signaling in MC3T3-E1 cells. Incubation with DHMBA (10 μM) reduced the expression of NF-κB p65 in MC3T3-E1 cells (Figures 8A and B). Incubation in the presence of TNF-α (10 ng / ml) increased NF-κB p65 levels (Figures 8A and B). This increase was inhibited by incubation 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 inhibited by the addition of DHMBA (1 or 10 μM) (Figure 8C). Thus, it was found that the increase in NF-κB promoter activity induced by TNF-α stimulation in MC3T3-E1 cells was inhibited in the presence of DHMBA. Furthermore, DHMBA also reduced NF-κB promoter activity in MC3T3-E1 cells in the absence of TNF-α ( FIG8C ). These results support the view that DHMBA inhibits promoter activity of genes associated with the transcription factor NF-κB in osteoblasts. (Discussion) The marine phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) was discovered as an excellent peroxyl radical scavenger. Its activity in lipid and aqueous media is approximately 15-fold and four-digitally superior to Trolox, suggesting a key role in regulating cellular function and maintaining a healthy state. Recently, DHMBA has been demonstrated to inhibit the proliferation and metastatic activity of bone-metastatic prostate cancer cells, suggesting a pharmacological role in preventing osteopenia. Furthermore, the present invention demonstrates that DHMBA stimulates osteoblast mineralization and prevents osteoblast mineralization impairment associated with the signaling of TNF-α produced by inflammatory macrophages RAW264.7 cells following LPS stimulation. It is believed that DHMBA plays a key pharmacological role in repairing impaired mineralization. Factors that promote bone formation are clinically important for preventing and repairing osteoporosis caused by aging or various diseases. The development of such factors is essential. It has been discovered that culturing MC3T3-E1 osteoblasts with DHMBA in vitro enhances osteoblast mineralization. This suggests that DHMBA enhances the mineralization and differentiation of osteoblasts, potentially promoting bone formation, and is therefore considered an important factor in promoting bone formation. It is well known that osteopenia is caused by postmenopausal estrogen deficiency, which is associated with inflammatory conditions. The cytokine TNF-α, produced by inflammation, plays a key role in skeletal pathologies, causing bone loss. This cytokine inhibits osteoblast mineralization by activating NF-κB signaling in MC3T3-E1 osteoblasts. The present invention investigated whether culturing MC3T3-E1 cells with DHMBA could prevent mineralization impairment in osteoblasts associated with NF-κB signaling. Mineralization of MC3T3-E1 cells was impaired by culturing with TNF-α, which activates NF-κB signaling. It was found that this impairment was inhibited by the presence of DHMBA. DHMBA was shown to exert an inhibitory effect on the NF-κB signaling process. Inflammatory macrophages may play a pathological role in the bone marrow microenvironment by increasing cytokine production. Furthermore, crosstalk between osteoblasts and macrophage RAW264.7 cells was investigated in vitro. When MC3T3-E1 cells are co-cultured with RAW264.7 cells stimulated with LPS, the proliferation of the MC3T3-E1 osteoblasts is inhibited. This inhibition of proliferation is caused by culturing the osteoblasts in conditioned medium obtained from culturing RAW264.7 cells stimulated with LPS. It is noteworthy that the inhibitory effect of the conditioned medium on MC3T3-E1 cell proliferation is suppressed by the presence of an inhibitor of NF-κB signaling, suggesting a role for NF-κB signaling. Furthermore, the TNF-α concentration in the conditioned medium is significantly increased by culturing RAW264.7 cells with LPS. These results support the view that osteoblast mineralization is impaired by activation of the TNF-α signaling process produced by inflammatory macrophages in the bone marrow microenvironment. Notably, TNF-α production in inflammatory RAW264.7 cells is suppressed when they are cultured with DHMBA. Furthermore, the inhibitory effect of the conditioned medium on cell proliferation was not observed in the presence of DHMBA in the culture medium, suggesting that this factor inhibits TNF-α signaling in MC3T3-E1 cells. Therefore, DHMBA may be useful in preventing osteoblast mineralization disorders associated with inflammatory conditions. Regarding its mechanism, we further investigated whether DHMBA affects the NF-κB signaling process in MC3T3-E1 cells. When cultured in the presence of DHMBA, the levels of NF-κB p65 in osteoblasts decreased. Furthermore, it was found that DHMBA inhibited the enhanced reporter activity of the NF-κB response element in the promoter region of nuclear genes in MC3T3-E1 cells, which was caused by TNF-α stimulation. This is believed to be important as the fundamental mechanism by which DHMBA inhibits the mineralization impairment of osteoblasts caused by TNF-α. As summarized in Figure 9, inflammatory macrophages within the bone marrow microenvironment produce TNF-α, which activates the NF-κB signaling process in osteoblasts. DHMBA inhibits TNF-α production in inflammatory macrophages and controls the activation of the associated signaling process. Furthermore, DHMBA has the potential to translocate to the nucleus of osteoblasts. DHMBA, which migrates to the nucleus, may inhibit the binding of NF-κB to response elements in gene promoter regions. Furthermore, DHMBA may exert a direct regulatory effect on the transcriptional activity of promoter regions in the nucleus. In conclusion, the present invention confirmed that DHMBA stimulates the mineralization of mouse osteoblasts MC3T3-E1 cells in vitro. Furthermore, it was found that DHMBA controls the manifestation of osteoblast mineralization disorders caused by the activation of the signal transduction process of TNF-α produced by inflammatory macrophages in vitro. In this way, it can be considered that DHMBA exerts a bone formation effect by preventing the mineralization disorder of osteoblasts. It can be considered that the marine factor DHMBA plays a role in the prevention and repair of inflammation-related bone loss. This study provides a new strategy for treating osteoblast bone formation disorders related to inflammatory states in the bone marrow microenvironment. [Figure 1] Effects of marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on proliferation and cell death of mouse osteoblasts MC3T3-E1 in vitro. (A) Cells (1×10 per well of a 24-well plate) were placed in a 24-well plate. 5 cells / ml culture medium) 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 determine the effect of increasing concentrations of DHMBA, cells (1×10 cells / well in a 24-well plate) were cultured in the presence of DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. 5 cells / ml) were cultured in the presence of DHMBA (0.1, 1, 10, 100, or 1000 μM) for 3 days. (C) To investigate the effect of DHMBA on cell death, cells (1×10 cells / well in a 24-well plate) were cultured in the presence of DHMBA (0.1, 1, 10, 100, or 1000 μM). 5Cells / ml culture medium) were cultured for 3 days to reach subconfluent, after which DHMBA (0.1, 1, 10, 100 or 1000 μM) was added and cultured for a further 48 hours. After culture, the number of cells attached to the culture dish was measured. The data are expressed as the mean ± SD of the values ​​obtained from 8 wells of 2 replicate plates using different cell modulators. *: Compared with the control group without DHMBA (gray bar), p < 0.001 was considered significantly different. One-way analysis of variance and Tukey-Kramer post hoc test were used. [Figure 2] The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) promotes the mineralization of mouse osteoblasts MC3T3-E1. Cells (1×10 per well of a 24-well plate) were placed in the culture dish. 5 Cells (10 cells / ml) were cultured in α-MEM supplemented with 10% FBS and 1% P / S for 3 days. Upon reaching subconfluence, the medium was exchanged for α-MEM supplemented with 10% FBS, 1% P / S, 4 mM β-glycerophosphate, and L-ascorbic acid (100 μg / ml) (mineralization-enhancing medium). The medium was exchanged every 3 days with DHMBA (1 or 10 μM) for 18 days. (A) Representative photographs (40×). (B) Absorbance at 570 nm of stained cells after solubilization. Results are presented as mean ± SD of 8 wells from 2 replicate plates using different cell-based treatments. *: p < 0.001 indicates a significant difference compared to the control group without DHMBA (grey bars). One-way analysis of variance with Tukey-Kramer post hoc test was used. [Figure 3] The effect of marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the proliferation and cell death of mouse osteoblasts MC3T3-E1. (A) To investigate the effect of TNF-α on cell proliferation, cells (1×10 cells per well of a 24-well plate) were plated. 5 cells / ml culture medium) in α-MEM containing 10% FBS and 1% P / S, in the presence of DHMBA (0.1, 1, or 10 μM) or TNF-α (1 or 10 ng / ml culture medium). (B) To investigate the effect of TNF-α on cell death, cells (1×10 cells / well in a 24-well plate) were cultured for 3 days. 5Cells were cultured for 3 days at 400 μg / ml (100 cells / ml) to subconfluence. Afterwards, the cells were further cultured for 48 hours in α-MEM supplemented with 10% FBS and 1% P / S in the presence of DHMBA (0.1, 1, or 10 μM) or TNF-α (10 ng / ml of culture medium). Following culture, the number of cells attached to the culture dish was measured. Data are presented as the mean ± SD of 8 wells from two replicate plates using different cell-modulating agents. *: p < 0.001 indicates a significant difference compared to a control group lacking both TNF-α and DHMBA (light gray bars). # : Compared with the TNF-α (10 ng / ml) group without DHMBA (dark gray bar), p < 0.001 was considered significant. One-way analysis of variance with Tukey-Kramer post hoc test was used. [Figure 4] The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) protects against mineralization inhibition in mouse osteoblasts MC3T3-E1 cultured with TNF-α. Cells (1×10 cells per well of a 24-well plate) were placed in a 24-well plate. 5 Cells were cultured in α-MEM supplemented with 10% FBS and 1% P / S for 3 days. After reaching subconfluence, the cells were replaced with α-MEM supplemented with 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 exchanged every 3 days. After incubation, the cells were stained with Alizarin Red solution. (A) Representative photographs (40×). (B) Absorbance at 570 nm of stained cells after solubilization. Data are presented as the mean ± SD of 8 wells from two replicate plates using different cell-modulating agents. *: p < 0.001 indicates a significant difference compared to the group without DHMBA (white bars) or the control group (gray bars). # : Compared with the TNF-α-treated group without DHMBA, p < 0.001 was considered significant. One-way analysis of variance with Tukey-Kramer post hoc test was used. [Figure 5] The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits the proliferation and promotes cell death of mouse osteoblasts MC3T3-E1 co-cultured with inflammatory macrophages RAW264.7 cells. (A) MC3T3-E1 cells (1×10 cells per well of a 24-well plate) were placed in the 24-well plate. 5(C) To determine the effect of co-culture on cell proliferation, MC3T3-E1 cells (10 cells per well) were cultured in the presence of lipopolysaccharide (LPS, 100 ng / ml medium) for 3 days. (B) MC3T3-E1 cells that reached subconfluence after 3 days of culture were further cultured in the presence of LPS (100 ng / ml) for 48 hours. 5 cells / 1 ml of culture medium) were added to the lower compartment of the transwell chamber in the presence of DHMBA (1 or 10 μM). 4 The upper chamber of each permeable well was filled with 0.5 ml of DMEM (10% FBS) containing 10 cells (100 ng / ml of culture medium). The upper chamber contained LPS (100 ng / ml). The cells were cultured for 3 days. After culture, the number of MC3T3-E1 cells attached to the lower compartment was counted. (D) To investigate the effect of co-culture on cell death, MC3T3-E1 cells (10 cells per well) were cultured. 5 cells / 1 ml) were added to the lower compartment of the permeable well chamber and cultured for 3 days until subconfluence. After culture, DHMBA (1 or 10 μM) was added to the lower compartment containing RAW264.7 cells (5×10 4 The upper chamber of each permeable well was filled with 0.5 ml of DMEM (10% FBS) containing 0.5 ml of cells / culture medium. The cells were further cultured in the upper chamber in the presence of LPS (100 ng / ml culture medium) for 48 hours. After incubation, the number of MC3T3-E1 cells attached to the lower chamber was counted. Results are presented as mean ± SD of values ​​obtained from a total of 8 wells using two plates containing different cell preparations. *: p < 0.001 indicates a significant difference compared to the control group cultured without LPS (gray bar). #: Compared with the group in the presence of LPS and without the addition of DHMBA (black bars), p < 0.001 was considered significantly different. One-way analysis of variance and Tukey-Kramer post hoc test were used. [Figure 6] The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) protects against the proliferation inhibition and cell death enhancement of mouse osteoblasts MC3T3-E1 caused by the conditioned medium obtained by culturing inflammatory macrophages RAW264.7 cells. The conditioned medium was obtained by culturing macrophages RAW264.7 cells in the presence or absence of LPS (100 ng / ml culture medium). (A) MC3T3-E1 cells (1×10 per well of a 24-well plate) were placed in the conditioned medium. 5 Cells were cultured for 3 days in α-MEM supplemented with 10% FBS and 1% P / S (10 cells / ml). They were cultured for 3 days in conditioned medium obtained after stimulation with LPS (0.25 or 0.5 ml, added to normal culture medium to make a total of 1 ml) or in conditioned medium supplemented with LPS (0.5 ml, added to normal culture medium to make a total of 1 ml), in the presence of DHMBA (1 or 10 μM). (B) To determine the effect on cell death, subconfluent MC3T3-E1 cells were cultured for 48 hours in conditioned medium obtained after stimulation with LPS (0.25 or 0.5 ml, added to normal culture medium to make a total of 1 ml) or in conditioned medium supplemented with LPS (0.5 ml, added to normal culture medium to make a total of 1 ml), in the presence of DHMBA (1 or 10 μM). After incubation, the number of MC3T3-E1 cells attached to the culture dish was counted. Results are presented as the mean ± SD of values ​​obtained from a total of 8 wells using two plates containing different cell preparations. *: p < 0.001 was considered significant compared to the control group cultured in medium without RAW264.7 cells and LPS (light gray or dark gray bars). # : Compared with the group cultured with conditioned medium (0.5 ml) stimulated with LPS (dark gray bar), p < 0.001 was considered significantly different. One-way analysis of variance and Tukey-Kramer post hoc test were used. [Figure 7] NF-κB signaling inhibitors inhibit the effects of conditioned medium obtained by culturing macrophage RAW264.7 cells with LPS stimulation on the proliferation and cell death of mouse osteoblasts MC3T3-E1. 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 of conditioned medium. RAW264.7 cells (1×10 per well in a 24-well plate) were cultured. 5 Cells were cultured for 3 days at 1 x 10 cells / ml of culture medium, 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 (1 x 10 cells / ml per well in a 24-well plate) were cultured in α-MEM containing Bay11-7082 (1, 10, 50, 100, or 1000 nM) for 3 days. (C) To determine the effect on cell proliferation, MC3T3-E1 cells were cultured for 3 days in conditioned medium (0.5 ml / well) obtained by stimulating 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 investigate the effects on cell death, MC3T3-E1 cells were cultured for 3 days to subconfluence and then incubated for 48 hours in conditioned medium (0.5 ml / well) derived 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). Following incubation, MC3T3-E1 cells attached to the culture dish were counted. Results are presented as mean ± SD of 8 wells total, using two plates containing different cell-modulating agents. *: p < 0.001 indicates a significant difference compared to controls cultured without RAW264.7 cells (A and B, white bars; C and D, gray bars). # : Compared with the group without Bay11-7082 (black bars), p < 0.001 was considered significant. One-way analysis of variance with Tukey-Kramer post hoc test was used. [Figure 8] The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits TNF-α-induced NF-κB activation in mouse osteoblasts MC3T3-E1. Cells (106 cells / 10 ml per dish) were cultured for 3 days in the presence or absence of DHMBA (10 μM) and TNF-α (10 ng / ml). After incubation, cell lysates were prepared. 40 μg of supernatant protein per lane was used for Western blot analysis. (A) Representative data are shown. (B) Bands are expressed as multiples of the control group (no DHMBA). Results are presented as the mean ± SD of values ​​obtained from a total of four dishes using different cell culture media. *: Compared with the control group, p < 0.01 was considered a significant difference. #: Compared with the group without TNF-α (gray bar), p < 0.01 was considered significant. One-way analysis of variance and Tukey-Kramer post hoc test were used. (C) To measure NF-κB promoter activity, MC3T3-E1 cells (2 × 10 cells per 100 μl in a 96-well plate) were cultured. 4 Cells (10 cells / well) were cultured in α-MEM supplemented with 10% FBS and 1% P / S for 24 hours. Following incubation, the medium was replaced with α-MEM supplemented with 10% FBS and 1% P / S, and the cells were transfected with the reporter plasmid or an empty vector control. Five hours later, the medium was exchanged with α-MEM supplemented with 10% FBS and 1% P / S, and TNF-α (1 or 10 ng / ml) was added to the cells to stimulate NF-κB for 24 hours. Either (final concentration of 1% ethanol) or DHMBA (1 or 10 μM) was added to the control group. After incubation, the cells were extracted with passive lysis buffer, and luciferase activity was measured. Results are presented as the mean ± SD of values ​​obtained from a total of 8 wells using different cell modulators. *: p < 0.001 indicates a significant difference compared to the control group (white bar). # : Compared with the group without DHMBA (B, dark gray bars; C, black bars), p < 0.001 was considered significant. One-way analysis of variance with Tukey-Kramer post hoc test was used. [Figure 9] Mechanism by which 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) protects against TNF-α-induced mineralization impairment in MC3T3-E1 osteoblasts. DHMBA inhibits TNF-α production in inflammatory macrophages. Furthermore, DHMBA reduces NF-κB p65 levels, inhibiting NF-κB signaling. DHMBA, which translocates to the nucleus, 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 NF-κB signaling associated with TNF-α production by inflammatory macrophages. DHMBA is believed to have the effect of controlling and repairing osteoblast mineralization that is impaired by inflammation.

Claims

1. A method for enhancing the mineralization of osteoblast MC3T3-E1 cell line, characterized by using an agent with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as the active ingredient to enhance the mineralization of osteoblast MC3T3-E1 cell line in vitro.

2. A method for controlling the effect of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cell line, characterized by using an agent with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as the active ingredient to control the effect of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cell line in vitro.

3. A method for preventing TNF-α-induced mineralization disorders in the MC3T3-E1 cell line, characterized by using an agent with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as the active ingredient to control the effect of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cell line in vitro, and to prevent TNF-α-induced mineralization disorders in the MC3T3-E1 cell line.

4. A method for inhibiting the activity of gene promoters related to transcription factor NF-κB in osteoblast MC3T3-E1 cell line, characterized by using an agent with 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as the active ingredient to inhibit the activity of gene promoters related to transcription factor NF-κB in osteoblast MC3T3-E1 cell line in vitro.

Images