Anti-inflammatory agent

Abstract

[Problem] To indicate that when DHMBA is added for culture, proliferation of mouse inflammatory macrophage RAW264.7 cells can be inhibited, cell death can be promoted, and the number of cells can be reduced; it has been found that DHMBA can inhibit the increase in the production of inflammatory cytokines in RAW264.7 cells cultured with LPS; particularly, the formation of osteoclasts of RAW264.7 cells caused by LPS (Lipopolysaccharide) stimulation can be inhibited by DHMBA (docosahexaenoic acid) treatment; thus, the present invention provides a useful therapeutic means for inflammatory diseases using DHMBA. [Solution] The present invention is characterized by having 3, 5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an active ingredient and having an anti-inflammatory effect.

Description

TECHNICAL FIELD

[0001] The present application relates to an anti-inflammatory agent. BACKGROUND

[0002] Inflammation is involved in the etiology of many diseases. Inflammatory cytokines are produced by macrophages by stimulation of lipopolysaccharide (LPS) and are used clinically as biomarkers associated with various conditions.

[0003] 3,5-Dihydroxy-4-methoxybenzyl alcohol (hereinafter sometimes referred to as DHMBA), which is a marine factor, is found in Pacific oysters (Crassostrea gigas).

[0004] DHMBA has the properties of reducing oxidative stress, scavenging free radicals, and inducing antioxidant proteins in cells. However, the pharmacological effects of DHMBA are almost unknown.

[0005] The present application was conducted to verify whether DHMBA inhibits the proliferation of inflammatory mouse macrophage RAW264.7 cells, the production of cytokines, and the formation of osteoclasts.

[0006] For cell culture using DHMBA (1 to 1000 μM), it is known that it inhibits the proliferation of inflammatory mouse macrophage RAW264.7 cells in vitro, induces cell death, and reduces the number of cells. It is known that DHMBA reduces the expression levels of signal transduction factors that promote cell proliferation, such as Ras, PI3K, Akt, MAPK, phospho-MAPK, mTOR, and increases the expression levels of p53, p21, Rb, Regucalcin, which inhibit cell proliferation. Further, by DHMBA treatment, the levels of caspase-3 and cleaved caspase-3 increase, which suggests an induction mechanism of cell death.

[0007] The production of inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, interleukin-1β, or prostaglandin E2 is increased by LPS stimulation. These increases are prevented by culturing the cells with DHMBA. It should be noted that the level of NF-κB p65 is increased by LPS treatment, and this increase is inhibited by DHMBA treatment. Further, in relation to these, osteoclastogenesis of RAW264.7 cells is enhanced by LPS treatment, and this stimulation is blocked by DHMBA treatment. In this way, DHMBA is invented to suppress the activity of inflammatory macrophages in vitro, showing usefulness for the treatment of inflammatory diseases.

[0008] (ABSTRACT)

[0009] Inflammation is a complex biological response of body tissues to an injurious agent, and is a coordinated biological defense response of immune cells, blood vessels, and molecular mediators. Inflammatory cytokines such as interleukin (IL), tumor necrosis factor (TNF)-α are considered to be biomarkers in chronic human muscle pain, rheumatoid arthritis. These cytokines are produced by macrophages in an inflammatory state. In particular, inflammatory macrophages are attracting attention because of their potential to promote cancer cell progression, metastasis, angiogenesis.

[0010] RAW264.7 cells are monocyte / macrophage-like cell lines. This cell line has characteristics of macrophage-mediated immunity, metabolism, phagocytosis. RAW264.7 cells are used as model cells of macrophages in an inflammatory state in vitro, and this cell line is also attracting attention as a model cell for osteoclastogenesis studies. Osteoclasts are cells differentiated from monocyte-macrophage cell lines. Lipopolysaccharide (LPS) is a core antigen of Gram-negative bacteria and can activate the host's innate immune system. LPS is an endotoxin that produces a persistent inflammatory stimulus to tissues. LPS induces osteoclastogenesis of RAW264.7 cells by regulating NF-κB-related signaling pathways and transcriptional activity.

[0011] 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a novel phenolic antioxidant substance, was originally identified from Crassostrea gigas. DHMBA has properties of reducing oxidative stress, scavenging free radicals, and inducing antioxidant proteins in cells. DHMBA was shown to scavenge hydroxyl radicals. In addition, it has been reported that DHMBA has a preventive effect on glutamate neuron hyperactivity in rats and mice. DHMBA has the potential to participate in the regulation of cell functions as an antioxidant substance. Further, recent studies have confirmed that DHMBA inhibits the proliferation of metastatic prostate cancer cells, providing a new strategy for the treatment of prostate cancer based on DHMBA. Further, it is considered that elucidation of the pharmacological effects of DHMBA is important in the treatment of various diseases.

[0012] The present application was carried out to explore whether DHMBA can be used as a therapeutic agent for inflammatory diseases and to confirm whether it has an effect on the activity of inflammatory macrophages in vitro. As a result, in the present application, it was confirmed that when cultured with the addition of DHMBA, the proliferation of mouse inflammatory macrophage RAW264.7 cells was inhibited, cell death was promoted, and the number of cells was reduced.

[0013] It was further found that DHMBA inhibits the increased production of inflammatory cytokines in RAW264.7 cells cultured with LPS. In particular, it was confirmed that osteoclast formation in RAW264.7 cells caused by LPS stimulation was inhibited by DHMBA treatment. In this way, the novel marine factor DHMBA has the potential to exert a pharmacological effect on inflammatory pathologies caused by macrophages. The present application provides a therapeutic means for inflammatory diseases using DHMBA.

[0014] Prior Art Documents

[0015] Patent Documents

[0016] Patent Document 1: Japanese Patent Application Publication No. 2017-132753 SUMMARY

[0017] Technical Problem to be Solved by the Invention

[0018] The present application was carried out to explore whether DHMBA can be used as a therapeutic agent for inflammatory diseases and to confirm whether it has an effect on the activity of inflammatory macrophages in vitro. As a result, in the present application, it was confirmed that when cultured with the addition of DHMBA, the proliferation of mouse inflammatory macrophage RAW264.7 cells was inhibited, cell death was promoted, and the number of cells was reduced.

[0019] Further, it was found that DHMBA inhibits the enhancement of inflammatory cytokine production in RAW264.7 cells cultured with LPS. In particular, it was confirmed that osteoclast formation in RAW264.7 cells caused by LPS stimulation was inhibited by DHMBA treatment. Thus, the novel marine factor DHMBA has the possibility of exerting a pharmacological effect on inflammatory pathologies caused by macrophages. The present application provides a therapeutic means for inflammatory diseases using DHMBA.

[0020] Means for solving the technical problem

[0021] The present application is characterized by 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient, and has an anti-inflammatory effect;

[0022] or 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient, and has an effect of inhibiting inflammatory macrophage activity;

[0023] or 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient, and has an effect of inhibiting RAW264.7 cell proliferation;

[0024] or 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient, and has an effect of inhibiting RAW264.7 cell proliferation cultured with LPS;

[0025] or 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient, and has an effect of promoting RAW264.7 cell death;

[0026] or 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient, and has an effect of inhibiting inflammatory cytokine production in RAW264.7 cells;

[0027] or 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient, and has an effect of inhibiting inflammatory cytokine production in RAW264.7 cells;

[0028] or 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient, and has an effect of inhibiting osteoclast formation in RAW264.7 cells.

[0029] Effects of the Invention

[0030] The present application was implemented to explore whether DHMBA can be used as a therapeutic drug for inflammatory diseases and to clarify whether it has an effect on the activity of inflammatory macrophages in vitro. As a result, in the present application, it was confirmed that when cultured with the addition of DHMBA, the proliferation of mouse inflammatory macrophage RAW264.7 cells was inhibited, cell death was promoted, and the number of cells was reduced.

[0031] It was further found that DHMBA inhibits the increase in the production of inflammatory cytokines in RAW264.7 cells cultured with LPS. In particular, it was confirmed that osteoclast formation in RAW264.7 cells caused by LPS stimulation was inhibited by DHMBA treatment. In this way, the novel marine factor DHMBA has the potential to exert a pharmacological effect on inflammatory pathologies caused by macrophages. The present application exerts an effect capable of providing a therapeutic means for inflammatory diseases using DHMBA. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is an explanatory diagram showing the chemical structure of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA). The molecular formula of DHMBA is C8H 10 O4, and the molecular weight is 170.164.

[0033] Figure 2 is an explanatory diagram showing that the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits the proliferation of mouse macrophage RAW264.7 cells. RAW264.7 cells (1 x 10 5 cells / ml per 1 well in a 24-well plate) were cultured in DMEM medium containing 10% FBS (fetal bovine serum), 1% P / S (penicillin / streptomycin), and 1% fungizone for 1 (A in the figure), 2 (B in the figure), 3 (C in the figure), and 4 (D in the figure) days in a control group (containing 1% ethanol as a final concentration, DHMBA 0 μM) or a DHMBA (0.1, 1, 10, 100, 1000 μM) addition group. After culture, the number of cells attached to the culture dish was counted. Data were obtained from a total of 8 wells of 2 plates using different cell preparations, and are expressed as the mean ± SD (standard deviation). *: Comparison of the control group (gray bar) and the DHMBA addition group, p < 0.001 is significantly different. Statistical processing used 1-way ANOVA, Tukey-Kramer post-test.

[0034] Figure 3Figure 1 is a bar graph showing the levels of various proteins related to the proliferation of mouse macrophage RAW264.7 cells modulated by marine agent 3,5-dihydroxy-4- methoxybenzyl alcohol (DHMBA). RAW264.7 cells (1 x 10 6 cells / 10 ml medium) were cultured in DMEM medium containing 10% FBS, 1% P / S, 1% amphotericin B for 3 days in control (containing 1% ethanol, DHMBA 0 μM as final concentration) or DHMBA (10 μM) addition groups. After culture, cells were removed from the culture dish using cell scrapers in cell lysis buffer containing protease inhibitors. Forty micrograms of supernatant protein per well was added to SDS-PAGE (12%), subjected to electrophoretic separation, transferred to a nylon membrane, and subjected to western blotting using antibodies against various proteins. Figure 3 (A) shows representative data. Figure 3 The bands of (B) of Figure 1 are shown in fold of control. Data were obtained from 4 culture dishes using different cell preparations, and are expressed as mean ± SD. *: Comparison of control and DHMBA addition groups, p < 0.001 indicates a significant difference. Statistical treatment used one-way ANOVA, Tukey-Kramer post-hoc test.

[0035] Figure 4 Figure 2 is a bar graph showing the death of mouse macrophage RAW264.7 cells stimulated by marine agent 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA). RAW264.7 cells (1 x 10 5 cells / ml / well) were cultured in DMEM containing 10% FBS, 1% P / S, and 1% amphotericin B for 3 days to reach subconfluence, and further added to control (containing 1% ethanol, DHMBA 0 μM as final concentration) or DHMBA (0.1, 1, 10, 100, 1000 μM) for 24 Figure 4 (A) hours or 48 Figure 4 (B) hours. Figure 4C) involves adding sub-convergent RAW264.7 cells, along with a caspase-3 inhibitor (10 μM), to either the control group (1% ethanol, 0 μM DHMBA as the final concentration) or DHMBA (1 or 10 μM), and then cultured for an additional 48 hours. After culture, the number of cells attached to the culture dish was counted. Data were obtained from a total of 8 wells in two plates using different cell preparations and are expressed as mean ± SD. (D) In ​​this case, to determine the expression levels of caspase-3 or cleaved caspase-3, PC-3 cells (1 × 10⁻⁶ cells in 100 mm culture dishes) were added. 6 Cells / 10ml of culture medium were added to either the control group (final concentration containing 1% ethanol and 0 μM DHMBA) or DHMBA (10 μM) and cultured in DMEM medium for 3 days. After culture, cell lysates were obtained for Western blotting assays. Figure 4 D) shows representative data. (E) bands in the figure are shown as multiples of the control. Data were obtained from four culture dishes using different cell preparations and are expressed as mean ± SD. *: The control group was compared with the DHMBA-added group, and p < 0.001 was considered statistically significant. Statistical analysis was performed using one-way ANOVA and Tukey-Kramer post-hoc test.

[0036] Figure 5 This is an illustration of the effects of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the proliferation and death of mouse macrophage RAW264.7 cells, regardless of the presence or absence of lipopolysaccharide (LPS) stimulation. Figure 5 A) To investigate the effect on cell proliferation, RAW264.7 cells (1×10⁻⁶) were subjected to... 5 / ml / well), in DMEM culture medium containing 10% FBS, 1% P / S and 1% amphotericin B, added to the control group (as the final concentration containing 1% ethanol) or LPS (1, 10, 50, 100, 500 ng / ml), and cultured for 3 days. Figure 5 B) is to use cells (1×10 5 / ml / well), in DMEM medium containing 10% FBS, 1% P / S and 1% amphotericin B, in the presence of DHMBA (1 or 10 μM), with or without the addition of LPS (100 ng / ml), and cultured together for 3 days. Figure 5 (C) To investigate the effect on cell death, sub-confluenced cells cultured for 3 days were further cultured for 48 hours in either a control group (containing 1% ethanol) or a lipopolysaccharide (LPS) supplementation group (1, 10, 50, 100, or 500 ng / ml). Figure 5D) is the number of cells that reached subconfluence, cultured for 48 hours in the presence of DHMBA (1 or 10 μΜ) with or without LPS (100 ng / ml). After incubation, the number of cells adhered to the culture dish was counted. Data were obtained from a total of 8 wells from 2 plates using different cell preparations and expressed as mean ± SD. *: significant difference compared to control (gray bar) without addition of LPS (A) and DHMBA (B) or with addition of LPS (C) and DHMBA (D) by one-way ANOVA, Tukey-Kramer post-test. Figure 5 A and Figure 5 C) (gray bar) or without addition of DHMBA Figure 5 B and Figure 5 D) (gray bar) with p < 0.01. Statistical treatment used one-way ANOVA, Tukey-Kramer post-test.

[0037] Figure 6 is a graphical representation illustrating the effect of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the number of mouse macrophage RAW264.7 cells with or without stimulation by lipopolysaccharide (LPS). RAW264.7 cells (1 x 10 5 / ml / well) were incubated in DMEM medium containing 10% FBS, 1% P / S and 1% amphotericin B. DHMBA (0.1, 1, 10, 100, 1000 μΜ) or control (1% ethanol as final concentration, DHMBA 0 μΜ) was added for 5 hours (A). After incubation, the number of cells adhered to the culture dish was counted. Data were obtained from a total of 8 wells from 2 plates using different cell preparations and expressed as mean ± SD. No significant change in the number of cells was observed by DHMBA and / or LPS treatment compared to control (gray bar). No significant change was observed by one-way ANOVA, Tukey-Kramer post-test and / or LPS treatment compared to control (gray bar). Statistical treatment used one-way ANOVA, Tukey-Kramer post-test. Figure 6 A). RAW264.7 cells (1 x 10 5 / ml / well) were incubated in DMEM medium containing 10% FBS, 1% P / S and 1% amphotericin B with LPS (100 ng / ml added to the total medium). DHMBA (0.1, 1, 10, 100, 1000 μΜ) or control (1% ethanol as final concentration, DHMBA 0 μΜ) was added for 5 hours (B). After incubation, the number of cells adhered to the culture dish was counted. Data were obtained from a total of 8 wells from 2 plates using different cell preparations and expressed as mean ± SD. No significant change in the number of cells was observed by DHMBA and / or LPS treatment compared to control (gray bar). No significant change was observed by one-way ANOVA, Tukey-Kramer post-test and / or LPS treatment compared to control (gray bar). Statistical treatment used one-way ANOVA, Tukey-Kramer post-test. Figure 6 B). After incubation, the number of cells adhered to the culture dish was counted. Data were obtained from a total of 8 wells from 2 plates using different cell preparations and expressed as mean ± SD. No significant change in the number of cells was observed by DHMBA and / or LPS treatment compared to control (gray bar). No significant change was observed by one-way ANOVA, Tukey-Kramer post-test and / or LPS treatment compared to control (gray bar). Statistical treatment used one-way ANOVA, Tukey-Kramer post-test.

[0038] Figure 7This diagram illustrates how the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibited the production of TNF-α, IL-6, IL-1β, and PGE2 in mouse RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS). RAW264.7 cells were placed in 24-well plates, with 1 × 10⁶ cells per well. 5 Cells were cultured in DMEM medium without DHMBA for 3 days to achieve sub-convergence. Subsequently, the control group (containing 1% ethanol and 0 μM DHMBA) and the DHMBA-added groups (0.1, 1, 10, 100, or 1000 μM) were further incubated in LPS (100 ng / ml) or LPS-free medium for 5 hours. After incubation, the culture medium was recovered, and TNF-α levels in the culture medium were measured using an ELISA kit. Figure 7 A), IL-6 ( Figure 7 IL-1β) Figure 7 C) and PGE2 ( Figure 7 Cytokine concentrations (D) were measured. Data were obtained from a total of 8 wells in two plates using different cell preparations and are presented as mean ± SD. *: For the control group (white bar) without DHMBA and LPS, P < 0.001 was considered statistically significant. #: For the control group containing LPS but without DHMBA, P < 0.001 was considered statistically significant. Statistical analysis was performed using one-way ANOVA and Tukey-Kramer post-hoc test.

[0039] Figure 8 This is an illustrative diagram illustrating the effect of marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) on the expression levels of COX-1, COX-2, MAPK, phosphorylated MAPK, NF-κB p65, and STAT3 in mouse macrophage RAW264.7 cells stimulated by lipopolysaccharide (LPS). Cells (1×10⁻⁶) 6 Cells / 10ml of culture medium were cultured in DMEM for 3 days to achieve sub-confluence. Afterwards, cells were cultured for another 5 hours with either no LPS or with LPS (100 ng / ml) added to the control group (containing 1% ethanol and 0 μM DHMBA) or DHMBA (10 μM). Following culture, if... Figure 3 As described, cell lysates were obtained for Western blot analysis using specific antibodies against COX-1, COX-2, MAPK, phosphorylated MAPK, NF-κB p65, STAT3, and β-actin. Representative data are presented. Figure 8 A). The strip used LPS-free ( Figure 8 B) Contains LPS alone (100 ng / ml)Figure 8 C) and those containing LPS (100 ng / ml) and DHMBA (10 μM) Figure 8 The data are presented as fold increases compared to the control group (D). Data were obtained from four culture dishes using different cell preparations and are expressed as mean ± SD. *: p < 0.01 was considered statistically significant compared to the control group without LPS. Figure 8 *: Compared with the control group containing LPS but not DHMBA, p<0.01 is considered statistically significant. Figure 8 (D). Statistical analysis was performed using one-way ANOVA and Tukey-Kramer post-hoc tests.

[0040] Figure 9 This is an illustrative diagram illustrating how the marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) inhibits the enhanced osteoclast formation in mouse RAW264.7 macrophage cells stimulated by lipopolysaccharide (LPS). Cells (in 24-well plates, 1×10⁶ cells) 5 Cells / 1ml / well were cultured for 3 days in DMEM containing 10% FBS, 1% P / S, and 1% amphotericin B in the presence of control (as a final concentration) or LPS (100 ng / ml), with or without the addition of DHMBA (0.1, 1, 10, 100 μM), and without the addition of DHMBA. The culture was then replaced with freshly prepared culture medium (0.5 ml) containing LPS and / or DHMBA for an additional 3 days. Figure 9 A). Cells (in a 24-well plate, 1 × 10⁶) 5 After culturing cells / 1ml / well in the above-mentioned DMEM medium containing LPS (100 ng / ml) without DHMBA for 3 days, replace 0.5 ml of the old medium with fresh medium (0.5 ml) containing LPS (100 ng / ml) and / or DHMBA (0.1, 1, 10, or 100 μM) and culture for an additional 3 days. Figure 9 (B) After culture, cells attached to the plates were fixed and stained with tartrate-resistant acid phosphatase (TRACP), an enzyme that serves as a marker for osteoclasts. TRACP-positive multinucleated cells (MNCs) (with more than three nuclei) were counted as osteoclast-like cells under a microscope (40x). Data were obtained from a total of 8 wells of 2 plates per data group using different cell preparations and are expressed as mean ± SD. *: p < 0.001 was considered statistically significant compared to the control group (white bars). #: p < 0.001 was considered statistically significant compared to LPS (gray bars). Statistical analysis was performed using one-way ANOVA and Tukey-Kramer post-hoc test. Detailed Implementation

[0041] First, the present inventors cultured cells in the presence of DHMBA. It was then found that the aforementioned cultured cells hindered the proliferation of mouse inflammatory macrophage RAW264.7 cells, promoted cell death, and reduced the number of cells.

[0042] It was further found that DHMBA inhibited the enhancement of inflammatory cytokine production by RAW264.7 cells cultured with LPS. In particular, it was found that osteoclast formation by RAW264.7 cells caused by LPS stimulation was inhibited by DHMBA treatment. In this way, the novel marine factor DHMBA has the potential to exert a pharmacological effect on inflammatory pathologies caused by macrophages. It is believed that the present application can provide a therapeutic means for inflammatory diseases using DHMBA.

[0043] (EXPERIMENTAL EXAMPLE)

[0044] MATERIALS AND METHODS

[0045] REAGENTS

[0046] Dulbecco's Modification of Eagle's Medium (DMEM) containing 4.5 g / L glucose, L-glutamine and sodium pyruvate, and antibiotics (100 units / mL penicillin and 100 pg / mL streptomycin; 1% P / S) were purchased from Corning (Mediach, Inc. Manassas, VA, USA).

[0047] Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Fungizone, caspase-3 inhibitor (CAS 169332-60-9-Calbiochem), lipopolysaccharide (LPS), and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

[0048] Caspase-3 inhibitor was diluted in sterile phosphate buffered saline (PBS). Other reagents were dissolved in 100% ethanol and stored at -20°C until use.

[0049] 3,5-Dihydroxy-4-methoxybenzyl alcohol:

[0050] 3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA) is a novel amphiphilic phenolic compound, for example, isolated from Pacific oysters (Crassostrea gigas) as a substance characterized by its antioxidant properties. However, this invention uses synthetic DHMBA. Even synthetic DHMBA has equivalent effects to DHMBA extracted from the aforementioned Pacific oyster (Crassostrea gigas).

[0051] The chemical structural formula of DHMBA is as follows: Figure 1 As shown.

[0052] It should be noted that the synthesized DHMBA is 100% pure. The DHMBA is then dissolved in, for example, 100% ethanol and stored at -20°C until use.

[0053] RAW264.7 cells

[0054] RAW264.7 cells are a mononuclear globulin / macrophage-like cell line derived from an Alperson's leukemia virus-transformed cell line in BALB / c mice. The mouse macrophage RAW264.7 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). RAW264.7 cells were cultured in Durbeco Modified Eagle Medium (DMEM) containing 10% FBS, 1% P / S, and 1% amphotericin B.

[0055] Analysis of cell proliferation

[0056] RAW264.7 cells (1 × 10⁶ cells per well) were cultured in a 24-well plate. 5 Cells were cultured for 3 days in DMEM medium containing 10% FBS, 1% P / S, and 1% amphotericin B ( / ml / well). Subconfluenced cells were added to the control group (as the final concentration, containing PBS or 1% ethanol and 0 μM DHMBA) or DHMBA (containing 0.1, 1, 10, 100, or 1000 μM) and cultured for another 24 or 48 hours.

[0057] In other experiments, as described above, cells that reached subfusion after 3 days of culture (1×10⁻⁶ cells) were... 5 / ml / well, with or without caspase-3 inhibitor (10μM), further cultured for 48 hours in either the control group (as the final concentration containing 1% ethanol, 0μM DHMBA) or the DHMBA (1 or 10μM) addition group.

[0058] Furthermore, in additional experiments, in order to establish the effect of DHMBA on RAW264.7 cell death, RAW264.7 cells were cultured in the presence of LPS, and the cells (1 x 10 5 / ml / well, 24-well plate) were cultured in DMEM medium (containing 10% FBS, 1% P / S, and 1% amphotericin B) for 3 days to reach sub-confluence. Further, the cells were cultured for 48 hours in the presence of DHMBA (containing 1 or 10 μM) or in the absence of DHMBA, in any one of a control group (containing 1% ethanol as a final concentration) or LPS (1, 10, 50, 100, or 500 ng / ml added to the medium). After the culture, the detachment of the cells was performed by adding 0.05% trypsin + EDTA solution (0.1 ml) in PBS without Ca 2+ / Mg 2+ , and the cell number was counted as described below in the item of "Measurement of cell number".

[0059] Measurement of cell number

[0060] After the culture, in order to detach the cells on each well, 0.05% trypsin + EDTA solution (0.1 ml) in PBS without Ca 2+ / Mg 2+ was spread in the culture dish, and incubated at 37°C for 2 minutes, and the cells were detached by adding DMEM containing 10% FBS and 1% P / S (0.9 ml) as described in the previous study.

[0061] The number of living cells was counted using a microscope (Olympus MTV-3) using a hemocytometer (Sigma-Aldrich) with a cell counter (Line Seiki H-102P, Tokyo, Japan).

[0062] For each culture dish, the average value of the two counts was calculated. The cell number was expressed as the value per well.

[0063] Measurement of cytokine production amount

[0064] RAW264.7 cells (1 x 10 5cells / 10ml) in DMEM medium containing 10% FBS and 1% P / S for 3 days to reach subconfluent state, and further incubated for 5 hours with or without LPS (100 ng / ml) under the condition of control group (containing 1% ethanol, DHMBA 0 μM as final concentration) or DHMBA (containing 0.1, 1, 10, 100, 1000 μM). After incubation, to measure cytokines, the culture medium was recovered. Thereafter, the cells were peeled off from each culture dish, and the number of cells was measured according to the method described in "Measurement of cell number". The concentration of TNF-α, IL-6, IL-1β or PGE2 in the culture medium was measured using an ELISA kit. TNF-α (cat. no. BM, KHC301) and IL-1β (cat. no. BMS6002) were measured using a material purchased from Thermo Fisher Scientific (Waltham, MA, USA), and in addition, IL-6 (cat. no. 583371) and PGE2 (cat. no. 514010) were measured using a material purchased from Cayman Chemical (Ann Arbor, MI, USA). The production amount of each cytokine was expressed as a pictogram (pg) secreted in the culture medium (ml).

[0065] In addition, to measure the expression level of Cox-1, Cox-2, NF-κB p65, STAT3 involved in cytokine signaling, RAW264.7 cells (1 x 10 6 cells / 10ml) in DMEM medium containing 10% FBS and 1% P / S for 3 days to reach subconfluent state, and further incubated for 5 hours with or without LPS (100 ng / ml) under the condition of control group (containing 1% ethanol, DHMBA 0 μM as final concentration) or DHMBA (containing 10 μM) added group. After incubation, to measure cytokines, the culture medium was recovered. Thereafter, the cells were peeled off from each culture dish, and the number of cells was measured according to the method described in "Measurement of cell number". The concentration of TNF-α, IL-6, IL-1β or PGE2 in the culture medium was measured using an ELISA kit. TNF-α (cat. no. BM, KHC301) and IL-1β (cat. no. BMS6002) were measured using a material purchased from Thermo Fisher Scientific (Waltham, MA, USA), and in addition, IL-6 (cat. no. 583371) and PGE2 (cat. no. 514010) were measured using a material purchased from Cayman Chemical (Ann Arbor, MI, USA). The production amount of each cytokine was expressed as a pictogram (pg) secreted in the culture medium (ml).

[0066] Western blotting

[0067] RAW264.7 cells (1 x 10 6Cells were cultured for 3 days in any one of the following media: DMEM (Gibco, Cat. No. 11995-065) containing 10% FBS, 1% P / S, 1% ethanol or DHMBA (10 μΜ). After incubation, the culture dishes were washed with cold PBS (10 ml) to remove floating and dead cells, and cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease and protein phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA) was added to recover the cell lysate of adherent cells as described in previous studies. The cell lysate was centrifuged at 4°C, 17,000 x g for 10 min. The protein concentration in the supernatant was determined using Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin as a standard. The cell lysate was stored at -80°C until use. The supernatant protein (40 μg / well) was separated by SDS polyacrylamide gel electrophoresis (12% SDS-PAGE) and transferred to a PVDF membrane. The transferred membrane was immunoblotted with specific antibodies for various proteins purchased from Cell Signaling Technology (Danvers, MA, USA) for Ras (cat. no. 3339, rabbit), Akt (cat. no. 9272, rabbit), mitogen-activated protein kinase (MAPK, cat. no. 4695, rabbit), phosphorylated MAPK (cat. no. 4370, rabbit), mammalian target of rapamycin (mTOR, cat. no. 4517, mouse), Rb (cat. no. 9309, mouse), p21 (cat. no. 2947, rabbit), STAT3 (cat. no. 12640, rabbit), COX-1 (cat. no. 48415), COX-2 (cat. no. 4842), and β-actin (cat. no. 3700, mouse). On the other hand, specific antibodies for p53 (cat. no. sc-126, mouse) and NF-κΒ p65 (cat. no. sc-109, rabbit) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Further, rabbit anti-calmodulin antibody was purchased from Sigma-Aldrich (cat. no. HPA029103, rabbit). The target proteins were incubated with one of the primary antibodies (1 : 1,000) at 4°C for one night as described above.After incubation, the membranes were further incubated in secondary antibody against horseradish peroxidase (Santa Cruz Biotechnology, mouse; cat. no. sc-2005 or rabbit sc-2305; diluted 1 :2000) for 60 min at room temperature and protein bands were detected using chemiluminescence substrate on X-ray films (cat. no. 34577, Thermo Scientific, Rockford, IL, USA). Using the respective membranes, a total of 3 or 4 X-ray films obtained from 4 independent experiments were scanned using an Epson Perfection 1660 Photo scanner and bands were quantified using ImageJ2 software (National Institutes of Health, Bethesda, MD, USA). Further, for immunoblots using additional antibodies, the attached chemiluminescence substrate (Thermo Scientific) was removed using restore Western blot stripping buffer (cat. no. 21059; Thermo Scientific, Rockford, IL, USA) by incubation for 30 min at room temperature and the membranes were further used for determination of other protein molecules.

[0068] Determination of osteoclastogenesis

[0069] RAW264.7 cells (1 x 10 5 cells / 1 ml / well) were incubated for 3 days in DMEM medium containing 10% FBS, 1% P / S, 1% amphotericin B with or without DHMBA (0.1, 1, 10 or 100 μΜ) or control (1% ethanol as final concentration in culture medium) or LPS (100 ng / ml in culture medium) and the old medium (0.5 ml) was replaced with fresh medium containing LPS or DHMBA and the cells were further incubated for 3 days. In another experiment, RAW264.7 cells (1 x 10 5RAW264.7 cells (5 x 104cells / ml / well) were cultured for 3 days in DMEM containing 10% FBS, 1% P / S and 1% amphotericin B with control group (1% ethanol as final concentration in culture medium) or LPS (100 ng / ml) addition group. Further, 0.5 ml of old medium was replaced with new medium (0.5 ml) containing either control group (1% ethanol as final concentration) or LPS (100 ng / ml) addition group with DHMBA (0.1, 1, 10 or 100 μM as final concentration) for 3 days. RAW264.7 cells adhered to the plate were fixed and stained for tartrate-resistant acid phosphatase (TRACP), a marker enzyme for osteoclasts. Staining was performed after culture, washing the cells with PBS solution, and fixing with 10% neutral formalin-phosphate (pH 7.2) for 1 min. The fixed cells were incubated at room temperature for 10 h in the presence of 10 mM sodium tartrate in acetate buffer (pH 5.0) containing stained naphthol AS-MX phosphate (Sigma-Aldrich) as a reaction product. TRACP-positive multinucleated cells (MNCs) containing more than 3 nuclei were counted as osteoclast-like cells under a microscope (40x) (Olympus MTV-3; Olympus Corporation, Tokyo, Japan). To evaluate the number of osteoclast-like TRACP-positive MNCs, one field per well was photographed and measured using ImageJ2 software. The number of osteoclast-like TRACP-positive MNCs was counted randomly in 5 fields under an optical microscope (40x) (Olympus MTV-3; Olympus Corporation, Tokyo, Japan), and the mean value was calculated.

[0070] Statistical analysis

[0071] Statistical significance was estimated using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc. La Jolla, CA). Data are expressed as mean ± standard deviation (SD). To calculate statistically significant differences between two groups, Student-t-test was used. Multiple comparisons on parametric data were performed by one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison post-test, as indicated. A p value < 0.05 was considered statistically significant.

[0072] Results

[0073] DHMBA inhibited the proliferation of RAW264.7 cells.

[0074] First, we investigated whether DHMBA affects the proliferation of mouse macrophage RAW264.7 cells. Figure 2 RAW264.7 cells were cultured for 1, 2, 3, and 4 days in either a control group (with a final concentration of 1% ethanol and 0 μM DHMBA) or groups supplemented with DHMBA (0.1, 1, 10, 100, or 1000 μM). Culture for 1–4 days with DHMBA (1, 10, 100, or 1000 μM) inhibited the proliferation of RAW264.7 cells. Thus, DHMBA was found to inhibit the proliferation of RAW264.7 cells in vitro.

[0075] To better understand the fundamental mechanism by which DHMBA inhibits the proliferation of RAW264.7 cells, this study investigated whether DHMBA controls the expression of key proteins associated with the proliferation of RAW264.7 cells. Figure 3 When cells were cultured with DHMBA (10 μM), the expression levels of Ras, PI3 kinase, Akt, MAPK, phosphorylated MAPK, and mTOR, which are involved in promoting RAW264.7 cell proliferation, decreased. Furthermore, an increase in calmodulin expression, which is closely related to cell proliferation inhibition, was observed. These results suggest that the mechanism by which DHMBA inhibits RAW264.7 cell proliferation is related to changes in signaling proteins and cell proliferation inhibitory factors.

[0076] DHMBA promotes the death of RAW264.7 cells.

[0077] Furthermore, this study aimed to clarify whether DHMBA affects the death of mouse macrophage RAW264.7 cells. Cells were cultured for 3 days at the subconvergence timepoint, and then further cultured for 24 or 48 hours in either the control group (final concentration of 1% ethanol, 0 μM DHMBA) or the DHMBA (0.1, 1, 10, 100, 1000 μM) supplementation groups. Cells were cultured for 24 hours in DHMBA (1, 10, 100, or 1000 μM) to determine the cell death rate. Figure 4 A) or 48 hours Figure 4 B) can promote the death of RAW264.7 cells. The stimulatory effect of DHMBA (1 or 10 μM) on the death of RAW264.7 cells was blocked in the presence of caspase-3 inhibitor (10 μM). Figure 4 The results of Western blotting showed that, by culturing with DHMBA (10 μM), the expression levels of intracellular caspase-3 and cleaved caspase-3 were significantly reduced. Figure 4 The D) increased. These results show that DHMBA stimulates apoptotic cell death in mouse macrophage RAW264.7 cells.

[0078] The effect of DHMBA on RAW264.7 cells cultured with LPS

[0079] Next, the inhibitory effect of DHMBA on LPS-treated inflammatory macrophages RAW264.7 cells was investigated. LPS is known to enhance the inflammatory activity of mouse macrophages RAW264.7 cells. For RAW264.7 cells cultured in the presence of LPS, the study investigated whether DHMBA attenuated the inhibitory effect on proliferation or the promoting effect on cell death in vitro. Figure 5 RAW264.7 cells were cultured for 3 days in the presence of LPS (containing culture medium of 1, 10, 50, 100, and 500 ng / ml). Even with the addition of LPS (1, 10, 50, and 100 ng / ml), the proliferation of RAW264.7 cells remained unchanged, but proliferation was inhibited when cultured at a higher concentration of LPS (500 ng / ml). Figure 5 (A). In the presence of LPS (100 ng / ml), DHMBA (1 or 10 μM) also inhibited the proliferation of RAW264.7 cells. Figure 5 (B).

[0080] In this way, even in the presence of LPS, DHMBA exhibits an inhibitory effect on cell proliferation.

[0081] To investigate the effect of DHMBA on cell death induced by LPS, RAW264.7 cells that had reached subconfluence after 3 days of culture were further encapsulated in LPS (1, 10, 50, 100, 500 ng / ml) medium and cultured for an additional 48 hours. Figure 5 The addition of LPS (1, 10, 50, 100 ng / ml) to cultures had no significant effect on cell number, but the addition of higher concentrations of LPS (500 ng / ml) promoted RAW264.7 cell death. In the presence of LPS (100 ng / ml), DHMBA (1 or 10 μM) was found to have an effect on RAW264.7 cell death. Figure 5 These results show that DHMBA retains its activity in reducing the number of RAW264.7 cells in the presence of LPS (100 ng / ml).

[0082] DHMBA inhibits the production of inflammatory cytokines in RAW264.7 cells.

[0083] Furthermore, this study investigated whether DHMBA affects the production of inflammatory cytokines in mouse macrophage RAW264.7 cells cultured in the presence of LPS (100 ng / ml) in vitro.

[0084] RAW264.7 cells were cultured for 3 days to reach sub-confluence, and then incubated for 5 hours with or without DHMBA (0.1, 1, 10, 100, 1000 μM) after further addition of LPS (100 ng / ml). The number of RAW264.7 cells did not change in the absence of LPS (A), and in the presence of LPS (100 ng / ml) added to the culture medium (B). Figure 6 Figure 6 The number of RAW264.7 cells did not change in the absence of LPS (A), and in the presence of LPS (100 ng / ml) added to the culture medium (B).

[0085] The production of major inflammatory cytokines, such as TNF-α (A), IL-6 (B), IL-1β (C), or PGE2 (D), in the culture medium obtained from cultured RAW264.7 cells was measured under the same culture conditions in which no change in the number of cells occurred. The production of IL-6 or IL-1β in RAW264.7 cells was inhibited by the addition of DHMBA (100 or 1000 μM) in the absence of LPS (B and C). Notably, the production of TNF-α (A), IL-6 (B), IL-1β (C), or PGE2 (D) was significantly increased by LPS treatment. These increases were inhibited by the addition of DHMBA (1, 10, 100, or 1000 μM). Thus, it was determined that the production of inflammatory cytokines in RAW264.7 cells was inhibited by DHMBA. Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 7 Figure 8

[0086] It was elucidated whether DHMBA is involved in the production of cytokines, and regulates the expression level of proteins related to intracellular signal transduction processes of cytokines (A). Figure 8

[0087] RAW264.7 cells were incubated in vitro for 5 hours in the presence of LPS (100 ng / ml) with or without the addition of DHMBA (10 μM). In the culture of RAW264.7 cells without LPS, the levels of COX-1, COX-2, MAPK, phosphorylated-MAPK, NF-κB p65, and STAT3 did not change by DHMBA-based treatment compared to the control (A and B). Figure 8 Figure 8 ​​​​​​​​​​​​​The levels of MAPK, phosphorylated MAPK, NF-κB p65, and STAT3 in RAW264.7 cells were increased by culturing with LPS (100 ng / ml). Figure 8 A and Figure 8 These increases were inhibited by DHMBA (10 μM) treatment. Figure 8 A and Figure 9 (D). These results show that DHMBA treatment suppresses the levels of signaling factors involved in cytokine production in inflammatory RAW264.7 cells.

[0088] DHMBA inhibits osteoclast formation in RAW264.7 cells.

[0089] RAW264.7 cells are commonly used as a model cell for osteoclast formation studies. Osteoclasts are cells differentiated from a mononuclear globulin-macrophage lineage. It has been shown that LPS can stimulate osteoclast formation in RAW264.7 cells by activating the NF-κB signaling pathway. Therefore, this study investigated whether DHMBA affects osteoclast formation in RAW264.7 cells cultured in LPS. RAW264.7 cells (1 × 10⁶ cells per well) were cultured in 24-well plates. 5 Cells / 1ml / well were cultured for 3 days in DMEM medium containing 10% FBS, 1% P / S, and 1% amphotericin B. In the control group (final concentration containing 1% ethanol) or the LPS (100 ng / ml) addition group, after culturing for 3 days with or without DHMBA (0.1, 1, 10, 100 μM), 0.5 ml of the old medium was replaced with 0.5 ml of fresh medium containing LPS (100 ng / ml) or DHMBA (0.1, 1, 10, or 100 μM), and cultured for another 3 days. When both LPS and DHMBA were added and cultured for 6 days, ( Figure 9 A and Figure 9 LPS (C) induces increased osteoclast formation in RAW264.7 cells. This increased formation is inhibited when cultured with DHMBA (0.1, 1, 10, 100 μM). Figure 9 A and Figure 9 (C).

[0090] In another experiment, RAW267.4 cells were cultured for 3 days in the presence of LPS (100 ng / ml) without DHMBA, then exchanged for fresh medium containing LPS (100 ng / ml), and DHMBA (0.1, 1, 10, 100 μM) was added and cultured for 3 days. DHMBA exerted an inhibitory effect even in the later stages of LPS-enhanced osteoclastogenesis. Figure 9 B andFigure 9 D). These results support that DHMBA inhibits osteoclast formation of RAW264.7 cells caused by LPS stimulation (Figs. 2A, 2B, 2C, 2D). Figure 9 B and ​ D).

[0091] Investigation

[0092] 3,5-Dihydroxy-4-methoxybenzyl alcohol (DHMBA), a novel phenolic antioxidant, has both the properties of scavenging intracellular free radicals and reducing oxidative stress. DHMBA has the potential to participate in the regulation of cell functions as an antioxidant. Previous studies have shown that DHMBA targets various signaling pathways to inhibit the growth and activity of metastatic prostate cancer cells, providing a new strategy for the treatment of prostate cancer. Further, the present inventors used mouse macrophage RAW264.7 cells in vitro to investigate whether DHMBA showed anti-inflammatory effects. The results showed that DHMBA showed anti-inflammatory effects in RAW264.7 cells, such as reducing the number of inflammatory macrophages, inhibiting the production of inflammatory cytokines, and inhibiting osteoclast formation. It was confirmed that DHMBA is useful for the treatment of inflammatory diseases.

[0093] Based on the culture of DHMBA, the proliferation of RAW264.7 cells was inhibited, cell death was promoted, and the number of macrophages was reduced. It is known that DHMBA can promote the proliferation of RAW264.7 cells, reduce the expression levels of Ras, PI3 kinase, Akt, MAPK, phosphorylated-MAPK, mTOR, and further inhibit cell proliferation, and increase the expression levels of p53, Rb, and p21. It is believed that the decrease in the levels of these cell signal-related proteins and the increase in cell proliferation inhibitors caused by DHMBA contribute to the basic mechanism of the compound hindering the proliferation of RAW264.7 cells.

[0094] Therefore, DHMBA has the potential to regulate the expression of various proteins related to cell signaling and transcriptional activity. Further, the levels of caspase-3 and cleaved caspase-3, which are involved in cell death by apoptosis by DHMBA treatment, were increased. These have the potential to activate the activation of nuclear DNA fragmentation that induces cell death by apoptosis. In this way, DHMBA has the potential to affect the levels of various proteins related to the control of cell number.

[0095] In addition, under LPS treatment that stimulates inflammatory activity of macrophage RAW264.7 cells, it was found that DHMBA strongly inhibits production of inflammatory cytokines such as TNF-α, IL-6, IL-1β, or PGE2. These reductions were observed under culture conditions in which RAW264.7 cells were cultured with both LPS and DHMBA, but the number of RAW264.7 cells was not reduced. Note that it was confirmed that DHMBA inhibits cytokine production independently of changes in the number of RAW264.7 cells. It was shown that TNF-α, IL-6, IL-1β, or PGE2 production by RAW264.7 cells can be enhanced by LPS treatment.

[0096] The present inventors et al. found that the production of these cytokines enhanced by LPS treatment is inhibited by in vitro culture of RAW264.7 cells using DHMBA. It was learned that DHMBA treatment can be a useful tool for inhibiting cytokine production under inflammatory conditions.

[0097] Further, in order to understand the basic mechanism by which DHMBA inhibits cytokine production by inflammatory RAW264.7 cells caused by LPS stimulation, further studies were conducted. LPS binds to Toll-like receptor 4 (TLR4) on the cell membrane of macrophage RAW264.7 cells, and the signal of the LPS / TLR4 pathway is transmitted into the cell. In particular, TLR4 signals activate NF-κB and MAPK signals in RAW264.7 cells. In addition, it has been found in an increasing number of studies that LPS binds to intracellular proteins and receptors. It is believed that TNF-α, IL-6, IL-1β, or PGE2 production caused by LPS treatment is related to intracellular signaling NF-κB p65 and MAPK in RAW264.7 cells. COX-1 and COX-2 are involved in PGE2 production in RAW264.7 cells. It was confirmed that the levels of NF-κB p65, MAPK, and phosphorylated MAPK increase when RAW264.7 cells are cultured with LPS. These increases are inhibited by DHMBA treatment. It is believed that this inhibition is related to the inhibition of inflammatory cytokine production by LPS in RAW264.7 cells. Further, DHMBA has the potential to hinder the binding of LPS to TLR4 and intracellular receptor proteins and to reduce the production of inflammatory cytokines in RAW264.7 cells. In addition, it is speculated that DHMBA modulates transcriptional activity related to the production of inflammatory cytokines in RAW264.7 cells. Further, DHMBA also has the potential to participate in the inhibition of cytokine production as an antioxidant substance.

[0098] Interestingly, the level of STAT3 was increased by LPS treatment, and this increase was inhibited by co-culturing RAW264.7 cells with DHMBA. STAT3 is involved in IL-6 intracellular signaling. RAW264.7 cells produce IL-6 and IL-1β by LPS stimulation. These cytokines have the possibility to affect the activity of RAW264.7 cells by autocrine action. By DHMBA treatment, there is the possibility to block the signal transduction process mediated by IL-6 and IL-1β produced by RAW264.7 cells.

[0099] LPS stimulates osteoclastogenesis in RAW264.7 cells via activation of NF-κB signaling. By LPS treatment, TLR4 signaling of macrophages is enhanced, and NF-κB-related signaling systems are activated. Osteoclastogenesis enhanced by culture in LPS was found to be inhibited by the administration of DHMBA to RAW264.7 cells. This inhibition was observed in both the early and late stages of osteoclastogenesis by DHMBA treatment. It is presumed that the inhibitory effect of DHMBA on osteoclastogenesis is related to the inhibition of NF-κB signaling involved in the decrease in NF-κB p65 levels by DHMBA treatment.

[0100] CONCLUSION

[0101] The present application demonstrates that DHMBA reduces the number of inflammatory macrophages by inhibiting the proliferation of RAW264.7 cells and promoting cell death. In addition, it is demonstrated that DHMBA inhibits the production of inflammatory cytokines. Further, DHMBA treatment inhibits osteoclastogenesis of RAW264.7 cells by LPS stimulation. Although it has not been clarified whether DHMBA shows an anti-inflammatory effect in vivo, according to the present application, it is found that DHMBA exerts an anti-inflammatory effect in mouse macrophage RAW264.7 cells in an in vitro model. DHMBA is a functional factor with very low toxicity. It is considered that DHMBA provides a new strategy for the prevention and treatment of inflammation and has pharmaceutical importance in the treatment of inflammatory pathologies.

Claims

1. An anti-inflammatory agent, characterized in that, An anti-inflammatory agent comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient.

2. An inhibitor of the activity of inflammatory macrophages, characterized in that, An agent for inhibiting inflammatory macrophage activity comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient.

3. A RAW264.7 cell proliferation inhibitor, characterized by, An agent for inhibiting proliferation of RAW264.7 cells comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient.

4. A proliferation inhibitor for RAW264.7 cells cultured with LPS, characterized by, An agent for inhibiting proliferation of RAW264.7 cells cultured with LPS comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient.

5. A death promoter of RAW264.7 cells, characterized by, An agent for promoting death of RAW264.7 cells comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient.

6. An inhibitor of inflammatory cytokine production in RAW264.7 cells, characterized by, An agent for inhibiting production of inflammatory cytokines in RAW264.7 cells comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient.

7. An inhibitor of inflammatory cytokine production in RAW264.7 cells, characterized by, An agent for inhibiting production of inflammatory cytokines in RAW264.7 cells comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient.

8. An inhibitor of osteoclastogenesis of RAW264.7 cells, characterized by, An agent for inhibiting osteoclast formation of RAW264.7 cells comprising 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) as an effective ingredient.

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