• The Korea Journal of Herbology
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• v.29 no.2
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• pp.61-67
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• 2014

Objectives : Aconiti Lateralis Preparata Radix (Aconitum Carmichaeli, AC) and Cinnamomi Cortex (Cinnamomi Cortex, CC) have been treated to elderly for kidney yang enhancement in Korean traditional medicine. In this study, the effects of water extract of AC and CC on RANKL (Receptor Activator for Nuclear Factor ${\kappa}B$ Ligand)-induced osteoclast differentiation were evaluated in culture system. Methods : MTT assay was used to evaluate the potential cytotoxicity of AC and CC extracts in bone macrophage marrows (BMMs) stimulated with M-CSF. TRAP (tartrate-resistant acid phosphatase) staining and TRAP activity were performed to know the inhibitory effect on osteoclast differentiation. The protein expression levels of nuclear factors such as activated T cell(NFAT)c1, c-Fos, MAPKs and ${\beta}$-actin in cell lysates treated with AC and CC extracts were analysed by western blotting. Results : AC, CC extracts and their co-administration inhibited significantly RANKL-induced osteoclast differentiation in BMMs in a dose dependent manner without toxicity. Each AC and CC extracts inhibited the phosphorylation of p38. Also, AC and CC extracts, respectively, inhibited the protein expression of c-Fos and NFATc1 more than Co-administration of AC and CC even if all treatments did. It was observed that RANKL-induced degradation of I-${\kappa}B$ is significantly suppressed by all treatments. Conclusions : Taken together, It was concluded that AC and CC have beneficial effect on osteoporosis by inhibition of osteoclast differentiation. Thus, Atractylodis AC and CC could be a treatment option for osteoporosis.

• Journal of dental hygiene science
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• v.23 no.2
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• pp.169-175
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• 2023

Background: Although microbial infection is direct cause of periodontal disease, various environmental factors influence the disease severity. Aging is considered a risk factor for oral diseases, with the prevalence of periodontal diseases increasing with age. Moreover, senescence-associated secretory phenotype (SASP) expressed in age-related diseases is a key marker of chronic inflammation and aging phenotypes. Therefore, this study aimed to understand the relevance of senescent cells to periodontal health and disease, investigate the possibility of regulating the expression of aging- and osteolysis-related factors in gingival fibroblasts, and investigate the effect of senescence induction in gingival fibroblasts on osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). Methods: After stimulation with 400 nM hydrogen peroxidase, human gingival fibroblasts (HGFs) were examined for senescence-associated β-galactosidase. Western blot and enzyme-linked immunosorbent assays were performed to assess the expression of SASP. Osteoclast formation was assessed in BMMs using a conditioned medium (CM) from hydrogen peroxide-stimulated HGFs. Osteoclastic differentiation was investigated using tartrate-resistant acid phosphatase (TRAP) staining and activity. Data analysis was performed using SPSS version 25.0. Results: The expression of senescence-related molecules, including p53, p16, and p21, and the expression of osteolytic factors, including IL-6, IL-8, and IL-17, were found to be significantly higher in the hydrogen peroxide-stimulated HGF than in the control group. Regarding the indirect effects of senescent gingival cells, the number of osteoclasts and TRAP activity increased according to the differentiation of BMM cultured in CM. Conclusion: Our results on the of between osteolytic factors and cellular senescence in gingival fibroblast cells helped to reveal evidence of pathological aging mechanisms. Furthermore, our results suggest that the development of novel therapies that target specific SASP factors could be an effective treatment strategy for periodontal disease.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.4
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• pp.802-809
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• 2008

Gamijangsing-tang (GMJST) has been used for treatment of bone formation in traditional korean medicine. The purpose of this study is to examine effects of GMJST on bone metabolism. The effects on the osteoblasts were determined by measuring (1) cell proliferation, (2) alkaline phosphatase (ALP) activity, (3) osteoprotegerin (OPG) secretion. (4) The morphologic changes of cells were observed by light microscopy and electron microscopy. Mineralization of calcium was determined by quantitative alizarin red-S assay and mineralization of phosphate was observed by von kossa staining. The morphologic changes of mineralization on the cells were observed by transmission electron microscopy (TEM). The effects on the osteoclast were investigated by tartrate-resistant acid phosphatase (TRAP) staining. Following results were obtained: Celluar activity of osteoblastic cells (MG-63) was significantly increased in 10-5 of dilution of GMJST. ALP and OPG activity of osteoblastic cells were increased in GMJST than normal MG-63 cell. Mineralization of osteoblastic cells were increased in GMJST than normal MG-63 cell. The activity of osteoclast cells (RAW 264.7) was significantly decreased in GMJST than normal MG-63 cell. From the results, GMJST stimulated the proliferation and mineralization of bone-forming osteoblast and inhibited by bone- lysis osteoclast.

• Journal of Korean Medicine Rehabilitation
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• v.30 no.2
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• pp.19-35
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• 2020

Objectives The aim of this study is to evaluate the fracture healing effect of Jinmu-tang (JM) on femur fractured rats. Methods Rats were randomly divided into 5 groups (normal, control, positive control, JM extract with low concentration and JM extract with high concentration). All group except normal group went through both femur fracture. Normal and control group received no treatment at all. Positive control group were medicated with tramadol (20 mg/kg) once a day for 14 days. Experimental group was orally medicated with JM extract (10 mg/kg for low concentration, 50 mg/kg for high concentration) once a day for 14 days. In order to investigate fracture healing process, plasma and serum were obtained. Also, micro-computed tomography was conducted to see the frature site visually. Immunohistochemistry for transforming growth factor-β1, Ki67, alkaline phosphatase, runt-related transcription factor 2, receptor activator of nuclear factor kappa-β, tartrate resistant acid phosphatase was conducted to observe bone healing progress after 14 days since fracture occured. Aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen and creatinine levels were measured in plasma, for hepatotoxicity and nephrotoxicity of JM extract. Osteocalcin was measured to observe activity of osteoblast. Results Through Micro-CT, more fracture healing was observed on both experimental group than control and positive control group. Through Hematoxylin & Eosin and safranin O staining showed bone cell proliferation and bone formation in the experimental group. RANK was significantly increased in the experimental groups. JM with high concentration showed statistically significant of TGF-β and Osteocalcin. NO, TRAP and ALP were not significantly changed. Liver toxicity was not significantly observed. Creatinine significantly increased in both experimental groups after 28 days. Conclusions As described above, JM extract showed anti-inflammatory effect, promoted fracture healing by stimulating the bone regeneration factor, and showed little hepatotoxicity and nephrotoxicity. In conclusion, JM extract can promote fracture healing and it can be used clinically to patients with fracture.

• Molecules and Cells
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• v.43 no.1
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• pp.34-47
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• 2020

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erbα and Rev-erbβ, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Rev-erbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo. Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.

• International Journal of Molecular Medicine
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• v.44 no.3
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• pp.913-926
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• 2019

Leonurus sibiricus L. (LS) is a medicinal plant used in East Asia, Europe and the USA. LS is primarily used in the treatment of gynecological diseases, and recent studies have demonstrated that it exerts anti-inflammatory and antioxidant effects. To the best of our knowledge, the present study demonstrated for the first time that LS may promote osteoblast differentiation and suppress osteoclast differentiation in vitro, and that it inhibited lipopolysaccharide (LPS)-induced bone loss in a mouse model. LS was observed to promote the osteoblast differentiation of MC3T3-E1 cells and upregulate the expression of runt-related transcription factor 2 (RUNX2), a key gene involved in osteoblast differentiation. This resulted in the induction of the expression of various osteogenic genes, including alkaline phosphatase (ALP), osteonectin (OSN), osteopontin (OPN), type I collagen (COL1) and bone sialoprotein (BSP). LS was also observed to inhibit osteoclast differentiation and bone resorption. The expression levels of nuclear factor of activated T-cells 1 (NFATc1) and c-Fos were inhibited following LS treatment. NFATc1 and c-Fos are key markers of osteoclast differentiation that inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced mitogen-activated protein kinase (MAPKs) and nuclear factor (NF)-κB. As a result, LS suppressed the expression of osteoclast-associated genes, such as matrix metallopeptidase-9 (MMP-9), cathepsin K (Ctsk), tartrate-resistant acid phosphatase (TRAP), osteoclast-associated immunoglobulin-like receptor (OSCAR), c-src, c-myc, osteoclast stimulatory transmembrane protein (OC-STAMP) and ATPase H+ transporting V0 subunit d2 (ATP6v0d2). Consistent with the in vitro results, LS inhibited the reduction in bone mineral density and the bone volume/total volume ratio in a mouse model of LPS-induced osteoporosis. These results suggest that LS may be a valuable agent for the treatment of osteoporosis and additional bone metabolic diseases.

• Journal of Life Science
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• v.24 no.3
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• pp.297-303
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• 2014

The effects of Eisenia bicyclis extracts on osteoblast differentiation and osteoclast formation were investigated. The proliferation of MC3T3-E1 osteoblastic cells was tested in an MTT assay. Treatment with E. bicyclis ethanol extract increased cell proliferation by approximately 128% at a concentration of 10 ${\mu}g/ml$. The ALP activities in the MC3T3-E1 cells was 179% higher when the E. bicyclis ethanol extract was processed at a concentration of 50 ${\mu}g/ml$. The proliferation of RAW 264.7 osteoclastic cells decreased significantly in response to treatment with the E. bicyclis extracts. Moreover, the proliferation of the RAW 264.7 osteoclastic cells treated with E. bicyclis hot water extract decreased by nearly 80%. In addition, the E. bicyclis extract reduced the number of tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cells from osteoclastic RAW 264.7 cells. These results indicate that E. bicyclis extracts have an anabolic effect on bone through the promotion of osteoclast differentiation and suggest that the extracts could be used in the treatment of common metabolic bone diseases.

• Journal of Nutrition and Health
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• v.48 no.2
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• pp.157-166
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• 2015

Purpose: The effects of water extract and distillate from the mixture of black goat meat and medicinal herb on MG-63 osteoblast proliferation and mouse bone marrow derived osteoclast formation were investigated. Methods: Proximate composition, volatile basic nitrogen (VBN), mineral content, free amino acid composition and free fatty acid composition in black goat meat were determined. Water extract and distillate were prepared with three groups; goat meat only (BG-E, BG-D), six herbs added group (BG-E6, BG-D6), and eight herbs added group (BG-E8, BG-D8). Osteoblast proliferation, mineralization and calcium uptake activity of MG-63 cells were measured and tartrate resistant acid phosphatase activity of osteoclasts was analyzed. Results: Black goat meat had remarkably low fat and high level of calcium. Glutamic acid was the most abundant amino acid. Herbs added extract groups (BG-E6 and BG-E8) showed increased MG-63 cell proliferation in a concentration dependent manner, while all the distillates did not show the effect. All extracts and distillates showed significantly increased osteoblast mineralization depending on the concentration. In particular, herb added extract, BG-E6, increased 170.3% of control and the distillate of BG-D and BG-D6 increased up to 168.5% and 159.8%, respectively. Calcium uptake activities of all water extracts showed remarkable increase of BG-E6 and BG-E8 up to 615.5% and 628.1% of control, respectively. Ditillates had no effect except BG-D6. All water extracts significantly reduced the activity of tartrate-resistant acid phosphatase (TRAP) in osteoclasts derived from mouse bone marrow. Conclusion: Combination of black goat meat and medicinal herb increased the MG-63 cell proliferation and effectively inhibited osteoclast differentiation in both water extracts and distillate of them, which implies that they could be used as potent functional food materials for bone health.

• International Journal of Oral Biology
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• v.30 no.2
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• pp.47-57
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• 2005

Leptin, the product of the obese gene, is a circulating hormone secreted primarily from adipocytes. Several results suggest that leptin is important mediators of bone metabolism. The present study was undertaken to determine the effects of leptin on anti-osteoclastogenesis using murine precursors cultured on Ca-P coated plates and on the production of osteoprotegerin (OPG) in osteoblastic cells. Additionally, this study examined the possible involvement of prostaglandin $E_2\;(PGE_2)$/protein kinase C (PKC)-mediated signals on the effect of leptin on anti-osteoclastogenesis to various culture systems of osteoclast precursors. Osteoclast generation was determined by counting tartrate-resistant acid phosphatase positive [TRAP (+)] multinucleated cells (MNCs). Osteoclastic activity was determined by measuring area of resorption pits formed by osteoclasts on Ca-P coated plate. The number of 1,25-dihydroxycholecalciferol $(1,25[OH]_2D_3)$- or $PGE_2$-induced TRAP (+) MNCs in the mouse bone marrow cell culture decreased significantly after treatment with leptin. The number of receptor activator of NF-kB ligand (RANKL)-induced TRAP (+) MNCs in M-CSF dependent bone marrow macrophage (MDBM) cell or RAW264.7 cell culture decreased significantly with leptin treatment. Indomethacin inhibited osteoclast generation induced by $1,25[OH]_2D_3$ and dexamethasone, however, no significant differences were found in the leptin treated group when compared to the corresponding indomethacin group. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited osteoclast generation induced by $1,25[OH]_2D_3$. The number of TRAP (+) MNCs decreased significantly with treatment by PMA at concentrations of 0.01 and $0.1{\mu}M$ in culture. Leptin inhibited PMA-mediated osteoclast generation. Isoquinoline-5-sulfonic 2-methyl-1-piperazide dihydrochloride (H7) had no effect on osteoclast generation induced by $1,25[OH]_2D_3$. Cell culture treatment with leptin resulted in no significant differences in osteoclast generation compared to the corresponding H7 group. Indomethacin showed no significant effect on TRAP (+) MNCs formation from the RAW264.7 cell line. PMA inhibited TRAP (+) MNCs formation induced by RANKL in the RAW264.7 cell culture. H7 had no effect on osteoclast generation from the RAW264.7 cell line. There was no difference compared with the corresponding control group after treatment with leptin. $1,25[OH]_2D_3$- or $PGE_2$-induced osteoclastic activity decreased significantly with leptin treatment at a concentration of 100 ng/ml in mouse bone marrow cell culture. Indomethacin, PMA, and H7 significantly inhibited osteoclastic activity induced by $1,25[OH]_2D_3$ in mouse bone marrow cell culture. No significant differences were found between the leptin treated group and the corresponding control group. The secretion of OPG, a substance known to inhibit osteoclast formation, was detected from the osteoblasts. Treatment by leptin resulted in significant increases in OPG secretion by osteoblastic cells. Taken these results, leptin may be an important regulatory cytokines within the bone marrow microenvironment.

• Preventive Nutrition and Food Science
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• v.19 no.4
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• pp.353-357
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• 2014

Phytic acid (myo-inositol hexakisphosphate) or phytate is considered an anti-nutrient due to the formation of precipitated complexes that strongly reduces the absorption of essential dietary minerals. In this study, brown rice with reduced phytate was made by inoculation with Lactobacillus sakei Wikim001 having high phytase activity. The effects of brown rice extract treated with L. sakei Wikim001 (BR-WK) on osteoblast differentiation and osteoclast formation were investigated. The proliferation of SaOS-2 cells was measured by the MTT assay. Treatment with BR-WK increased cell proliferation by 136% at a concentration of $100{\mu}g/mL$. The Alkaline phosphate activity in SaOS-2 cells was 129% higher when BR-WK was processed at a concentration of $100{\mu}g/mL$. The proliferation of bone marrow macrophages decreased by nearly 60% in response to treatment with BR-WK. In addition, BR-WK reduced the number of tartrate-resistant acid phosphatase-positive ($TRAP^+$) multinucleated cells from bone marrow macrophages. These results indicate that BR-WK stimulates bone formation through its positive action on osteoblast differentiation and function and furthermore, decreases osteoclast differentiation.