Enhancer for calcification of osteoblast MC3t3-e1 cells

Abstract

To provide an inhibitor comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient to provide novel strategy for preventing and treating disorders in osteogenesis and calcification of osteoblasts associated with inflammatory conditions in the bone marrow microenvironment.SOLUTION: The invention relates to use of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient capable of enhancing the calcification of osteoblast MC3T3-E1 cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

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

[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 may therefore 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 calcification in mouse osteoblast MC3T3-E1 cells induced under inflammatory conditions.

[0004] In osteoblast cultures, 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 caused 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, the proliferation of MC3T3-E1 cells was suppressed by crosstalk with RAW264.7 cells in coculture.

[0006] Interestingly, it was found that the proliferation of MC3T3-E1 cells was suppressed by culturing macrophages in the culture medium obtained by culturing them with LPS. 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. As a mechanism, DHMBA was found to reduce the level of NF-κB p65 and the activity of gene NF-κB reporter expression in MC3T3-E1 cells. It was invented that DHMBA prevents the impairment of osteoblast mineralization by the inflammatory cytokine TNF-α through the suppression of TNF-α signaling, which is involved in 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.

[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. The cavity created by osteoclasts is then refilled by osteoblasts that 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 the mechanism of bone remodeling, plays a physiological role in maintaining new bone mass.

[0008] The process of bone remodeling is intricately regulated by hormones, cytokines, skeletal stresses associated with 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 interactions among them. 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 age, is caused by age-related estrogen deficiency. Moreover, notably, severe bone loss can be induced by bone metastatic cancer cells, leading to the death of patients with fractures. In particular, inflammatory macrophages contribute to the development of bone disorders through bone metastasis of cancer cells. Inflammatory cytokines, mainly 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 studies as a cellular model of osteoclastogenesis, in which osteoclasts differentiate from the monocyte-macrophage lineage. The host immune system is stimulated by lipopolysaccharide (LPS), the main antigen of Gram-negative bacteria. In the bone marrow microenvironment, inflammatory macrophages activated by LPS stimulation 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 bone metastasis of cancer.

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

[0011] [Patent Document 1] JP 2023-080697 A Summary of the Invention [Problem to be solved by the invention]

[0012] The above-mentioned properties suggest that DHMBA may play an important role in regulating cell function and maintaining health. In particular, we demonstrated that DHMBA exhibits inhibitory effects on the proliferation and metastatic activity 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 will be important.

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

[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 bone formation and calcification of osteoblasts associated with inflammatory conditions in the bone marrow microenvironment. [Means for solving the problem]

[0015] The present invention relates to This is a calcification enhancer for osteoblast MC3T3-E1 cells, which contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and has the effect of promoting calcification of osteoblast MC3T3-E1 cells. It is characterized by: or The agent 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 osteoblast MC3T3-E1 cells. The agent is an agent for controlling the effect of TNF-α on the proliferation or cell death of osteoblast MC3T3-E1 cells. It is characterized by: or This product 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 and preventing TNF-α-induced calcification disorders in MC3T3-E1 cells. It is characterized by: or This product 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 osteoblastic cells MC3T3-E1 co-cultured with inflammatory macrophage cells RAW264.7. It is an inhibitory regulator that suppresses the proliferation and promotes cell death of osteoblastic cells MC3T3-E1 co-cultured with inflammatory macrophage cells RAW264.7. It is characterized by: or The present invention is a protective agent that contains 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and 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, and protects 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. The feature is, or The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), and it is an inhibitor of gene promoter activity related to the transcription factor NF-κB in osteoblasts. It is characterized by the above. Effect of the Invention

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

[0017] [Figure 1]Effects 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 (1x105 cells / ml culture medium per well of a 24-well plate) 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 (1x105 cells / ml culture medium per well of a 24-well plate) 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 (1x105 cells / ml culture medium per well of a 24-well plate) were cultured for 3 days to reach subconfluence, after which DHMBA (0.1, 1, 10, 100, or 1000 μM) was added and cultured for another 48 h. After incubation, the number of cells attached to the dish was counted. Data are presented as the mean ± SD of values ​​obtained from 8 wells of a total of 2 replicate plates using different cell preparations. *: Significant difference at p<0.001 compared to the control group without DHMBA (gray bar). One-way ANOVA with Tukey-Kramer post-hoc test was used. [Diagram 2]The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) enhances mineralization of mouse osteoblastic MC3T3-E1 cells. Cells (1x105 cells / ml per well of a 24-well plate) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days and, after reaching subconfluence, 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). The medium supplemented with 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 photographs (40x magnification). (B) Absorbance at 570 nm after solubilization of stained cells. Results 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 at p<0.001 compared to the control group without DHMBA (gray bar), using one-way ANOVA with Tukey-Kramer post-hoc test. [Diagram 3]The effect of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) prevents the effect of TNF-α on proliferation or cell death of mouse osteoblastic MC3T3-E1 cells. (A) To examine the effect of TNF-α on cell proliferation, cells (1x105 cells / ml culture medium per well of a 24-well plate) were cultured for 3 days in the presence of DHMBA (0.1, 1 or 10 μM) or TNF-α (1 or 10 ng / ml culture medium) in α-MEM containing 10% FBS and 1% P / S. (B) To examine the effect of TNF-α on cell death, cells (1x105 cells / ml per well of a 24-well plate) were cultured for 3 days to reach subconfluence, after which the cells were cultured for an additional 48 hours in the presence of DHMBA (0.1, 1 or 10 μM) or TNF-α (10 ng / ml culture medium) in α-MEM containing 10% FBS and 1% P / S. After incubation, the number of cells attached to the dishes was counted. Data are presented as the mean ± SD of values ​​obtained from 8 wells of a total of 2 replicate plates using different cell preparations. *: Significant difference at p<0.001 compared to the control group without both TNF-α and DHMBA (light gray bar). #: 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. [Figure 4]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 (1x105 cells / ml per well of a 24-well plate) 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 in the presence of TNF-α (1 ng / ml medium) and DHMBA (1 or 10 μM) for 18 days. 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 mean ± SD of values ​​obtained from 8 wells of a total of 2 replicate plates using different cell preparations. *: Significant difference at p < 0.001 compared to the group without DHMBA (white bars) or the control group (gray bars). #: Significant difference at p < 0.001 compared to the group treated with TNF-α without DHMBA. One-way ANOVA with Tukey-Kramer post-hoc test was used. [Diagram 5]Marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits the inhibition of proliferation and enhances cell death of mouse osteoblastic MC3T3-E1 cells co-cultured with inflammatory macrophage RAW264.7 cells. (A) MC3T3-E1 cells (1x105 cells / ml per well of a 24-well plate) were cultured for 3 days in the presence of lipopolysaccharide (LPS, 100ng / ml medium). (B) MC3T3-E1 cells that reached subconfluence after 3 days of culture were cultured for an additional 48 hours in the presence of LPS (100ng / ml). (C) To measure the effect of co-culture on cell proliferation, MC3T3-E1 cells (105 cells / 1ml medium per well) were added to the lower compartment of a transwell chamber in the presence of DHMBA (1 or 10μM). The upper chamber of each Transwell was filled with 0.5 ml DMEM (10% FBS) containing macrophage RAW264.7 cells (5x104 cells per ml of medium). The upper chamber contained LPS (100 ng / ml of medium). The cells were cultured for 3 days. After culture, the attached MC3T3-E1 cells in the lower compartment were counted. (D) To examine the effect of co-culture on cell death, MC3T3-E1 cells (105 cells / ml per well) were added to the lower compartment of the Transwell 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 Transwell was filled with 0.5 ml DMEM (10% FBS) containing RAW264.7 cells (5x104 cells / 0.5 ml of medium). The upper chamber was incubated with cells in the presence of LPS (100 ng / ml culture medium) for an additional 48 h. After incubation, the attached MC3T3-E1 cells in the lower compartment were counted. Results are presented 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 incubated without LPS (gray bars). #: Significant difference at p<0.001 compared to the group without DHMBA in the presence of LPS (black bars). One-way ANOVA with Tukey-Kramer post-hoc test was used. [Figure 6]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. 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 (1x105 cells / ml per well of a 24-well plate) were cultured for 3 days in α-MEM containing 10% FBS and 1% P / S. Conditioned medium obtained with LPS stimulation (containing 0.25 or 0.5 ml, with normal medium added to a total of 1 ml) or conditioned medium with LPS stimulation (containing 0.5 ml, with normal medium added to a total of 1 ml) were 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 h in the presence of DHMBA (1 or 10 μM) in conditioned medium obtained with LPS stimulation (0.25 or 0.5 ml, total of 1 ml was added to normal medium) or in conditioned medium with LPS stimulation (0.5 ml, total of 1 ml was added to normal medium). After incubation, the MC3T3-E1 cells attached to the dishes were counted. Results are presented 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 with RAW264.7 cells and medium cultured without LPS (light gray bar or dark gray bar). #: Significant difference at p<0.001 compared to the group cultured in conditioned medium with LPS stimulation (0.5 ml) (dark gray bar). One-way analysis of variance with Tukey-Kramer post-hoc test was used. [Figure 7]NF-κB signaling inhibitors suppress the effects of conditioned medium obtained by culturing macrophage RAW264.7 cells stimulated with LPS on proliferation and cell death of mouse osteoblast 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 (1x105 cells / ml culture medium per well in 24-well plates) 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 proliferation of MC3T3-E1 cells. MC3T3-E1 cells (1x105 cells / ml culture medium per well in 24-well plates) were cultured in α-MEM containing Bay11-7082 (1, 10, 50, 100, or 1000 nM) for 3 days. (C) To measure the effect on cell proliferation, MC3T3-E1 cells were cultured for 3 days in the presence of Bay11-7082 (1 or 10 nM) or Bay11-7082 (10 nM) and DHMBA (10 μM) in conditioned medium (0.5 ml / well) obtained by stimulation of RAW264.7 cells with LPS (100 ng / ml). (D) To examine the effect on cell death, MC3T3-E1 cells were cultured for 3 days until they reached subconfluence, and then cultured for 48 hours in the presence of Bay11-7082 (1 or 10 nM) or Bay11-7082 (10 nM) and DHMBA (10 μM) in conditioned medium (0.5 ml / well) obtained by stimulation of RAW264.7 cells with LPS (100 ng / ml). After incubation, the number of attached MC3T3-E1 cells on the dish was counted. Results are presented as 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 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 bars). One-way ANOVA with Tukey-Kramer post-hoc test was used. [Figure 8]Marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits TNF-α-induced NF-κB activation in mouse osteoblastic MC3T3-E1 cells. Cells (106 cells / 10 ml per dish) were cultured for 3 days with or without DHMBA (10 μM) and with the addition of TNF-α (10 ng / ml). After culture, cell lysates were prepared. Samples of 40 micrograms of supernatant protein per lane were used for Western blot analysis. (A) Representative data are shown. (B) Bands are shown as fold of control (no DHMBA). Results are shown as mean ± SD of values ​​obtained from a total of four dishes using different cell preparations. *: significant difference at p<0.01 compared to the control group. #: 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 (2x104 cells / well per 100 μl in a 96-well plate) were cultured for 24 h in α-MEM containing 10% FBS and 1% P / S. 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 reporter plasmids or empty vector control. After 5 h, the medium was replaced with α-MEM containing 10% FBS and 1% P / S, and cells were stimulated with NF-κB by adding TNF-α (1 or 10 ng / ml) for 24 h. In the control group, either TNF-α (1% ethanol at a final concentration) or DHMBA (1 or 10 μM) was added. After incubation, 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 eight wells using different cell preparations. *: Significant difference at p<0.001 compared to the control group (white bars). #: Significant difference at p<0.001 compared to the group without DHMBA (B, dark gray bars; C, black bars). One-way ANOVA 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 disorders in osteoblastic MC3T3-E1 cells. DHMBA causes inhibition of TNF-α production in inflammatory macrophages. In addition, DHMBA reduces the level of NF-κB p65 and inhibits the NF-κB signaling process. DHMBA translocated to the nucleus acts on the NF-κB response element in the promoter region of DNA and inhibits the transcriptional activity enhanced by TNF-α stimulation. DHMBA protects against mineralization disorders caused by activation of the NF-κB signaling process associated with TNF-α produced by inflammatory macrophages. DHMBA is considered to have the effect of controlling and repairing osteoblastic mineralization hindered by inflammatory conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0019] (material and method) (reagent) 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) were 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) 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 the present invention. The structure of DHMBA has been shown in a previous study. The purity of the synthesized DHMBA was 100%. DHMBA was dissolved in 100% ethanol and stored at -20°C until use.

[0021] (Osteoblast MC3T3-E1 cells) Mouse osteoblastic MC3T3-E1 cells were obtained from the American Type Culture Collection (HTB-77TM, 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) Murine 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 progression of cell proliferation, MC3T3-E cells (1x10 per well) were 5 / ml culture medium) were cultured in α-MEM supplemented with 10% FBS and 1% P / S in 24-well plates in a water-saturated atmosphere containing 5% CO2 and 95% air at 37 °C. Cells were cultured in the presence of vehicle (1% ethanol as final concentration) or DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. In separate experiments, to measure the effect of increasing concentrations of DHMBA on MC3T3-E1 cells, cells (1x10 per well) were cultured in DMEM (containing 10% FBS and 1% P / S) in 24-well plates. 5 The cells were cultured in a 1% ethanol final concentration in the presence of either reagent vehicle (1% ethanol as 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 in osteoblastic MC3T3-E1 cells (1x10 per well of a 24-well plate), 5 Cells (100 / 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 reagent vehicle (1% ethanol as 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 added to measure the number of cells attached to each well. 2+ / Mg 2+After adding a solution containing 0.05% trypsin and EDTA in 0.1 ml per well, the culture dishes were incubated at 37°C for 2 min. 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, which stains live cells but not dead cells. Live 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), and the average of two counts was taken. The cell number was expressed as number per well.

[0025] (Osteoblast differentiation assay and Alizarin Red S staining) MC3T3-E1 cells (10 per well in a 12-well plate) 5 Cells / ml culture medium) were cultured for 3 days in α-MEM (1.0 ml / well) containing 10% FBS and 1% P / S. The medium was then changed to mineralization-enhancing medium 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 the 18th day of culture, the cells were rinsed with PBS and fixed for 30 min at 4°C with ice-cold 75% ethanol, after which calcium deposition was visualized by staining with Alizarin Red S (40 mM, pH 6.2). After 30 min at room temperature, the staining solution was removed by extensive washing with distilled water. The stained cells on the plates were photographed under a microscope (40x) (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 cells and RAW264.7 cells) To investigate the crosstalk between osteoblastic MC3T3-E1 cells and macrophage RAW264.7 cells, a Transwell chamber (Corning, Life Sciences, Cat. No. CLS3464-48EA, USA) with a polycarbonate filter (pore size 8 μm, diameter 6.5 mm) was used for the assay. The lower part of the chamber contained MC3T3-E1 cells (1x10 per ml of culture medium). 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. 4 The plates were filled with 100 μl of 1000 μl of MC3T3-E1 (lower compartment) or RAW264.7 cells (upper compartment) at 37 °C (cells / ml culture medium). RAW264.7 cells were incubated for 1 h at 37 °C under 5% CO2 and 95% air to allow them to adhere to the upper chamber. The plates with transwells were 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 to reach 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 section "Assay for cell proliferation."

[0027] Furthermore, to examine the effect of co-culture with RAW264.7 cells on the death of MC3T3-E1 cells, osteoblasts (1x10 per well) were added to the wells. 5 Cells / ml) were cultured for 3 days to reach subconfluence, after which the medium was replaced with fresh medium containing either the reagent vehicle (1% ethanol as a final concentration) or DHMBA (1 or 10 μM). Then, a Transwell chamber was placed in each culture well, and RAW264.7 cells were added to the upper chamber in 0.5 ml (5 x 10 4 After further incubation for 48 h, the medium in the upper and lower compartments was aspirated and the filters were removed. The cells attached to the dishes (wells) were counted as described in the “Cell proliferation assay” section.

[0028] (Effect of conditioning 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) 5 When the cells reached subconfluence, they were cultured for 3 days in DMEM containing 10% FBS and 1% P / S with or without LPS (100 ng / ml in culture medium). After cell culture, the culture medium (conditioned medium) was collected. To determine the effect of conditioned medium on the proliferation of MC3T3-E1 cells, they were cultured for 3 days in α-MEM containing 10% FBS and 1% P / S, either in conditioned medium cultured without LPS (1 ml, control) or in conditioned medium cultured 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 conditioned medium per well).

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

[0030] (Assay for TNF-α production) RAW264.7 cells (1x10 per well) 5 RAW264.7 cells (100 ng / ml) were cultured in DMEM containing 10% FBS and 1% P / S for 3 days to reach subconfluence in 24-well plates. After 5 h of incubation with or without LPS (100 ng / ml medium), the culture medium was collected to assay cytokines, and cells were detached from each culture dish and counted as described in the “Assay for cell proliferation” section. Culture with LPS did not result in significant changes in the number of RAW264.7 cells. TNF-α concentration in the culture medium was analyzed using an ELISA kit for mouse TNF-α (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] (Effects of NF-κB signaling pathway inhibitors) We further investigated the effect of Bay11-7082, an NF-κB signaling inhibitor, on the proliferation or cell death of MC3T3-E1 cells. 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 in 100% PBS. 5MC3T3-E1 cells (1 ml / well) were cultured in 10% FBS, 1% P / S and reagent vehicle (final concentration 1% ethanol) or in the presence of Bay11-7082 (1, 10, 50, 100, or 1000 nM) for 3 days until subconfluent. MC3T3-E1 cells were then cultured in the presence or absence of Bay11-7082 (1 or 10 nM) and in the presence or absence of DHMBA (10 μM) in the presence of conditioned medium (0.5 ml per well, 1 ml total plus normal medium) for 3 days.

[0032] In other experiments, to evaluate the effect of Bay11-7082 on MC3T3-E1 cell death, cells (1x10 per well of a 24-well plate) were cultured in 100 wells. 5 Cells were cultured in α-MEM (1 ml) containing 10% FBS and 1% P / S. The cells were cultured for 3 days to reach subconfluence, after which the medium was replaced with α-MEM (1 ml) containing either normal medium or conditioned medium (0.5 ml per well, plus normal medium to a total of 1 ml). Cells were cultured for an additional 48 h 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 culture well and cell counts were determined 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) + TNF-α (10 ng / ml). After culture, dishes were washed three times with cold PBS (10 ml) and attached cells were removed from the dishes by scraping in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Forty microgram samples of protein obtained by cell lysis were loaded per lane and separated by SDS polyacrylamide gel electrophoresis (12% SDS-PAGE) as previously described. After electrophoresis, the membranes were incubated with primary antibodies (diluted 1:1,000) for the target proteins NF-κB p65 (cat. no. 3034, rabbit) and β-actin (cat. no. 3700, mouse) at 4°C overnight. After incubation, the membranes were further incubated in horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., mouse sc-2005 or rabbit sc-2305, diluted 1:1,000) at room temperature for 60 min. Protein bands were then detected using a chemiluminescent substrate on X-ray film (cat. no. 34577, Thermo Scientific, Rockford, IL, USA). 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) The NF-κB-responsive reporter luciferase assay (pNF-κB-Luc, BD Biosciences) was performed as we previously described. MC3T3-E1 cells (2 × 10 per 100 μl in 96-well plates) were cultured in 100-well plates. 4MC3T3-E1 cells (cells / well) were cultured for 24 h in α-MEM containing 10% FBS and 1% P / S. After culture, the medium was removed, washed twice with α-MEM without FBS and antibiotics, and α-MEM without FBS and antibiotics (100 μl) was added to the wells containing the cells. Reporter plasmids or empty vector control (pGL3-Basic) were transfected into MC3T3-E1 cells using Lipofectamine 2000 reagent (Invitrogen) in α-MEM without FBS and antibiotics. After 5 h, 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 h. 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, USA), 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, USA).

[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] (Result) Effects of the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on proliferation and cell death of osteoblastic MC3T3-E1 cells. To elucidate the effect of DHMBA on the proliferation and death of mouse osteoblastic MC3T3-E1 cells, cells (1x10 per well of a 24-well plate) were cultured in 100 wells. 5 MC3T3-E1 cells (cells / ml culture medium) were cultured in α-MEM medium containing 10% FBS and 1% P / S in the presence of reagent vehicle (1% ethanol as final concentration) or DHMBA (10 μM) for 1, 2, 3, 4, or 5 days. Five days of culture did not cause any significant changes in the proliferation of MC3T3-E1 cells in vitro (Figure 1A). To determine the effect of increasing concentrations of DHMBA, cells (1x10 per well) were cultured at 4°C for 1 h. 5 MC3T3-E1 cells (1000 cells / ml) were cultured for 3 days in the presence of reagent vehicle (final concentration 1% ethanol) or DHMBA (0.1, 1, 10, 100, or 1000 μM). The proliferation of MC3T3-E1 cells was not altered by incubation with DHMBA (0.1, 1, 10, or 100 μM) (Figure 1B). When the highest dose (1000 μM) was used, DHMBA caused a significant decrease in cell proliferation.

[0037] Furthermore, to evaluate the effect of DHMBA on cell death, cells (1x10 per well) were 5 MC3T3-E1 cells (0.1, 1, 10, 100, or 1000 μM) were cultured for 3 days until they reached subconfluence, and then 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 cell number was significantly decreased at the highest dose of DHMBA (1000 μM) (Figure 1C). Therefore, it was found that culture with a relatively low concentration of DHMBA did not cause any change in the number of MC3T3-E1 cells.

[0038] (DHMBA stimulates mineralization in osteoblastic MC3T3-E1 cells.) MC3T3-E1 cells (1x10 per well) 5MC3T3-E1 cells (1000 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 medium (1% ethanol as a final concentration) or DHMBA (1 or 10 μM) and was cultured for 18 days. After culture, the cells were stained with alizarin red solution. Culture with DHMBA (1 or 10 μM) enhanced the mineralization of MC3T3-E1 cells (Figure 2A and B). These results suggested that culture with DHMBA enhanced the 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 modulates the effect of TNF-α on osteoblasts in vitro. To measure the effect of TNF-α on cell proliferation, cells (1x10 per well) were cultured in 10 wells. 5 Cells (1000 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). Additionally, to examine the effect on cell death, cells (1x10 per well) were 5MC3T3-E1 cells (0.1, 1, or 10 μM) were cultured for 3 days to reach subconfluence, and then cultured for 48 h in culture medium containing TNF-α (1 or 10 ng / ml culture medium). 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 caused by TNF-α.

[0040] We next investigated whether DHMBA prevents the impaired mineralization of 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 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 in osteoblastic MC3T3-E1 cells co-cultured 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 elucidated whether inflammatory macrophages affect osteoblast proliferation and death by 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 (Figure 5A and 5B). MC3T3-E1 cells (1 × 10 per well) were cultured at 100°C for 1 h. 5 MC3T3-E1 cells (1000 cells / ml) were cultured in α-MEM containing 10% FBS in the presence or absence of LPS (1-1000 ng / ml in culture medium) for 3 days. LPS did not affect the proliferation of MC3T3-E1 cells (Figure 5A). Next, to evaluate the effect of LPS on cell death, subconfluent MC3T3-E1 cells were cultured in the presence of LPS (1-1000 ng / ml) for an additional 48 hours. LPS did not cause cell death (Figure 5B).

[0042] We next 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 co-culture with RAW264.7 cells suppressed proliferation of MC3T3-E1 cells (Figure 5C). This reduction was suppressed by the presence of DHMBA (1 or 10 μM).

[0043] To further investigate the effect of co-culture 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 and cultured for 48 h in the presence of LPS (100 ng / ml culture medium) (Figure 5D). Co-culture with RAW264.7 cells enhanced the death of MC3T3-E1 cells (Figure 5D). This enhancement was prevented by the presence of DHMBA (1 or 10 μM) (Figure 5D). These results suggested that inflammatory macrophages suppressed osteoblast proliferation and stimulated cell death, resulting in a decrease in cell number. Thus, DHMBA was found to suppress the effects of macrophages on osteoblasts in vitro.

[0044] (DHMBA protects against the effects of conditioned medium obtained from cultures 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 medium). MC3T3-E1 cells (1x10 per well) were cultured in 100% CO. 5 MC3T3-E1 cells (1000 cells / ml medium) were cultured in α-MEM containing 10% FBS and 1% P / S for 3 days and, after reaching subconfluence, were replaced with conditioned medium (containing 0.25 or 0.5 ml of conditioned medium, brought to 1 ml with normal medium) (Figure 6A). The proliferation 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 h in conditioned medium (containing 0.25 or 0.5 ml of conditioned medium, made up to 1 ml with normal medium) or in conditioned medium containing DHMBA (1 or 10 μM) (0.5 ml) (Figure 6B). Cell death was enhanced by incubation in conditioned medium (Figure 6B). This enhancement was suppressed by incubation in conditioned medium containing DHMBA (1 or 10 μM) (Figure 6B). Thus, conditioned medium incubated with LPS was found to inhibit proliferation, enhance cell death, and cause 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, by utilizing inhibition of NF-κB signaling, we investigated whether TNF-α and its signaling process are involved in the expression of the effect of conditioned medium in inhibiting proliferation and promoting cell death of MC3T3-E1 cells. Incubation with LPS (10 or 100 ng / ml) increased the production of TNF-α in RAW264.7 cells, but this increase was suppressed by the presence of DHMBA (1 or 10 μM) (Figure 7A). The number of MC3T3-E1 cells was not significantly decreased when cultured with Bay11-7082 (1, 10, or 50 nM), an inhibitor of NF-κB signaling (Figure 7B). Cultured with Bay11-7082 (1 or 10 nM) suppressed the effect of the conditioned medium in inhibiting proliferation (Figure 7C) and promoting cell death (Figure 7D) of MC3T3-E1 cells. These results suggested that TNF-α and its NF-κB signaling process are involved in the expression of the effect of the conditioned medium in inhibiting proliferation and promoting cell death of osteoblastic MC3T3-E1 cells.

[0047] We further elucidated whether DHMBA regulates the process of NF-κB signaling in MC3T3-E1 cells. When cultured with DHMBA (10 μM), the expression level of NF-κB p65 in MC3T3-E1 cells decreased (Figure 8A and B). The NF-κB p65 level was increased by culturing in the presence of TNF-α (10 ng / ml) (Figure 8A and B). This increase was suppressed by culturing with DHMBA (10 μM) (Figure 8A and B). Furthermore, the 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, it was found that the enhancement of 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 supported the notion that DHMBA suppresses gene promoter activity related to the transcription factor NF-κB in osteoblasts.

[0048] (Consideration) The marine factor phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) has been discovered as an excellent peroxyl radical scavenger, and its potency has been shown to be approximately 15 times, four orders of magnitude, superior to Trolox for that purpose in lipid and aqueous media, suggesting that it plays an important role in regulating cellular function and maintaining health. In recent years, it has been demonstrated that DHMBA exerts an inhibitory effect on the proliferation and metastatic activity of bone metastatic prostate cancer cells, suggesting that DHMBA plays a pharmacological role in preventing bone loss. Furthermore, in the present invention, it was invented that DHMBA stimulates osteoblastic mineralization and prevents the impairment of osteoblastic mineralization associated with the signal transduction of TNF-α produced by inflammatory macrophage RAW264.7 cells upon LPS stimulation. DHMBA was considered to play an important pharmacological role in repairing 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. There is a need to develop such factors. When osteoblastic MC3T3-E1 cells were cultured in vitro with DHMBA, it was found to promote osteoblast mineralization. DHMBA exerts a promoting effect on osteoblast mineralization and differentiation, suggesting that this may lead to the promotion of bone formation, and is therefore considered to be an important promoting factor for bone formation. It is well known that bone loss is caused by postmenopausal estrogen deficiency associated with an inflammatory state. The cytokine TNF-α, produced by inflammation, plays an important role in skeletal pathology, leading to bone loss. This cytokine inhibits osteoblast mineralization by activating NF-κB signaling in osteoblastic MC3T3-E1 cells.

[0050] In the present invention, we investigated whether incubation with DHMBA prevents the impairment of 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 suppressed the proliferation of osteoblastic MC3T3-E1 cells. This proliferation inhibition was caused by incubating osteoblasts with conditioned medium obtained from LPS-stimulated RAW264.7 cell cultures. Notably, the inhibitory effect of conditioned medium on the proliferation of MC3T3-E1 cells 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 supported the view that osteoblast mineralization is impaired by the activation of the TNF-α signaling process produced in inflammatory macrophages in the bone marrow microenvironment. Notably, incubation with DHMBA suppressed the production of TNF-α in inflammatory RAW264.7 cells. Furthermore, the inhibitory effect of the 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. Thus, DHMBA may be useful in preventing the impairment of osteoblast mineralization associated with inflammatory conditions.

[0052] We further investigated whether DHMBA affects the NF-κB signaling process in MC3T3-E1 cells. When cultured in the presence of DHMBA, the level of NF-κB p65 in osteoblasts was reduced. Furthermore, DHMBA was found to suppress the enhancement of reporter activity of NF-κB response elements in the promoter regions of nuclear genes caused by TNF-α stimulation in MC3T3-E1 cells. This is considered to be important as the underlying mechanism by which DHMBA suppresses the impairment of osteoblast mineralization caused by TNF-α. As summarized in Figure 9, inflammatory macrophages in the bone marrow microenvironment produce TNF-α, and this cytokine activates the process of NF-κB signaling in osteoblasts. DHMBA suppresses the production of TNF-α in inflammatory macrophages and controls the activation of its associated signaling process. In addition, DHMBA can translocate into the nucleus of osteoblasts. Once translocated into the nucleus, DHMBA can inhibit the binding of NF-κB to response elements in the promoter regions of genes. In addition, DHMBA can exert a direct regulatory effect on the transcriptional activity of promoter regions in the nucleus.

[0053] In conclusion, in this invention, it was confirmed that DHMBA stimulates the mineralization of mouse osteoblastic MC3T3-E1 cells in vitro. Furthermore, it was found that DHMBA controls the expression of osteoblast mineralization disorder, which is expressed by activating the signaling process of TNF-α produced by inflammatory macrophages in vitro. Thus, it is believed that DHMBA exerts its bone formation effect by preventing the impairment of osteoblast mineralization. The marine factor DHMBA may play a role in the prevention and repair of inflammation-associated bone loss. This study provides a new strategy for the treatment of impaired osteoblastic bone formation associated with inflammatory conditions in the bone marrow microenvironment.

Claims

1. The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), which has the effect of promoting the calcification of osteoblastic MC3T3-E1 cells. A mineralization promoter for osteoblast MC3T3-E1 cells, characterized in that

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

2. An agent for controlling the effect of TNF-α on the proliferation or cell death of osteoblastic MC3T3-E1 cells.

3. The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), which controls the effects of TNF-α on the proliferation or cell death of osteoblastic MC3T3-E1 cells and prevents TNF-α-induced calcification disorders in MC3T3-E1 cells. A method for preventing TNF-α-induced calcification damage in MC3T3-E1 cells, comprising:

4. The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), which has the effect of suppressing the proliferation and promoting cell death of osteoblastic cells MC3T3-E1 co-cultured with inflammatory macrophage cells RAW264.

7. An inhibitory regulator that inhibits proliferation and promotes cell death of osteoblastic cells MC3T3-E1 co-cultured with inflammatory macrophage cells RAW264.

7.

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

6. The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), which has the effect of suppressing gene promoter activity related to the transcription factor NF-κB in osteoblasts.

2. An inhibitor of gene promoter activity associated with the transcription factor NF-κB in osteoblasts.

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