• Journal of Microbiology and Biotechnology
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• 제32권2호
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• pp.141-148
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• 2022

Many bone diseases such as osteolysis, osteomyelitis, and septic arthritis are caused by gram-negative bacterial infection, and lipopolysaccharide (LPS), a bacterial product, plays an essential role in this process. Drugs that inhibit LPS-induced osteoclastogenesis are urgently needed to prevent bone destruction in infective bone diseases. Marein, a major bioactive compound of Coreopsis tinctoria, possesses anti-oxidative, anti-inflammatory, anti-hypertensive, anti-hyperlipidemic, and anti-diabetic effects. In this study, we measured the effect of marein on RAW264.7 cells by CCK-8 assay and used TRAP staining to determine osteoclastogenesis. The levels of osteoclast-related genes and NF-κB-related proteins were then analyzed by western blot, and the levels of pro-inflammatory cytokines were quantified by ELISA. Our results showed that marein inhibited LPS-induced osteoclast formation by osteoclast precursor RAW264.7 cells. The effect of marein was related to its inhibitory function on expressions of pro-inflammatory cytokines and osteoclast-related genes containing RANK, TRAF6, MMP-9, CK, and CAII. Additionally, marein leads to markedly inhibited NF-κB signaling pathway activation in LPS-induced RAW264.7 cells. Concurrently, when the NF-κB signaling pathway was inhibited, osteoclast formation and pro-inflammatory cytokine expression were decreased. Collectively, marein could inhibit LPS-induced osteoclast formation in RAW264.7 cells via regulating the NF-κB signaling pathway. Our data demonstrate that marein might be a potential drug for bacteria-induced bone destruction disease. Our findings provide new insights into LPS-induced bone disease.

• Journal of Veterinary Science
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• 제23권4호
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• pp.47.1-47.11
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• 2022

Background: In lipopolysaccharide-induced RAW264.7 cells, Aster tataricus (AT) inhibits the nuclear factor kappa-light-chain-enhancer of activated B cells and MAPKs pathways and critical pathways of osteoclast development and bone resorption. Objectives: This study examined how aster saponin A2 (AS-A2) isolated from AT affects the processes and function of osteoclastogenesis induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in RAW264.7 cells and bone marrow macrophages (BMMs). Methods: The cell viability, tartrate-resistant acid phosphatase staining, pit formation assay, polymerase chain reaction, and western blot were carried out to determine the effects of AS-A2 on osteoclastogenesis. Results: In RAW264.7 and BMMs, AS-A2 decreased RANKL-initiated osteoclast differentiation in a concentration-dependent manner. In AS-A2-treated cells, the phosphorylation of ERK1/2, JNK, and p38 protein expression were reduced considerably compared to the control cells. In RAW264.7 cells, AS-A2 suppressed the RANKL-induced activation of osteoclast-related genes. During osteoclast differentiation, AS-A2 suppressed the transcriptional and translational expression of NFATc1 and c-Fos. AS-A2 inhibited osteoclast development, reducing the size of the bone resorption pit area. Conclusion: AS-A2 isolated from AT appears to be a viable therapeutic therapy for osteolytic illnesses, such as osteoporosis, Paget's disease, and osteogenesis imperfecta.

• 대한본초학회지
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• 제35권3호
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• pp.1-8
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• 2020

Objectives : In this study, we examined the estrogenic activities and anti-osteo clastogenesis effects of PCE17, a mixture of PE (an extract of Puerariae Flos), and CE (an extract of Citri Unshius Pericarpium). Methods : The estrogenic effect of PCE17, PE and CE were examined by ER-β/ERE reporter gene assay and proliferation assay in 293 T and MCF-7 cells. The expression of estrogen-responsive gene and protein were checked by Real Time-PCR (RT-PCR) and Western blotting in MCF-7 cells. Inhibitory effect of PCE17, PE and CE on RANKL-induced osteoclast differentiation were evaluated by TRAP staining and RT-PCR in primary osteoclast precursors from rat bone marrow cells. Results : PCE17 and PE bind to ERs (estrogen receptors) and show estrogenic activities in 293T cells. They also stimulated the proliferation of MCF-7 cells and increased the expression of ER response gene, pS2. Tectorigenin, an active ingredient of PE, shows similar estrogenic activities in MCF-7 cells. PCE17 and CE inhibited RANKL-induced osteoclastogenesis in rat primary osteoclast precursor cells and down-regulated the osteoclast-specific genes of Nfatc1, Ctsk, and Acp5. Conclusions : In conclusion, PCE17 may have therapeutic potential in cases of menopause and osteoporosis.

• 대한본초학회지
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• 제37권5호
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• pp.1-8
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• 2022

Objectives : This study aimed to investigate the synergistic effect of combining Cynanchi Wilfordii Radix extract with Humuli Lupuli Flos extract on estrogenic and anti-osteoclastogenic activity. Methods : Estrogenic effect of a mixture of Cynanchi Wilfordii Radix extract and Humuli Lupuli Flos extract (CWHL), Cynanchi Wilfordii Radix extract, Humuli Lupuli Flos extract, caudatin (an active ingredient of Cynanchi wilfordii Radix extract) and 8-prenylnaringenin (an active ingredient of Humuli Lupuli Flos extract) were examined by proliferation E-screen assay and expression of estrogen inducible gene, pS2 via Real Time-PCR (RT-PCR) in MCF-7 estrogen responsive cells. And their estrogenic activities were investigated how to modulate Estrogen receptor 𝛽 by binding affinity assay. Inhibitory effect of CWHL, Cynanchi Wilfordii Radix extract, Humuli Lupuli Flos extract, caudatin and 8-prenylnaringenin on RANKL-induced osteoclast differentiation were tested by TRAP (Tartrate-resistant acid phosphatase) staining in osteoclastogenic RAW 264.7 cells. Results : CWHL, Humuli Lupuli Flos extract and 8-prenylnaringenin accelerated the proliferation of MCF-7 and the expression of pS2 in MCF-7. CWHL, Cynanchi Wilfordii Radix extract, Humuli Lupuli Flos extract, caudatin and 8-prenylnaringenin bind to estrogen receptor 𝛽. CWHL, Cynanchi Wilfordii Radix extract, Humuli Lupuli Flos extract, caudatin and 8-prenylnaringenin inhibited RANKL-induced osteoclastogenesis in osteoclastogenic RAW 264.7. CWHL is more effective for all markers than Cynanchi Wilfordii Radix extract or Humuli Lupuli Flos extract alone. Conclusions : CWHL may a potential therapeutic agent for menopause and osteoporosis as a natural food resource. CWHL as a natural food source has therapeutic potential in cases of menopause and osteoporosis.

• 대한화학회지
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• 제67권2호
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• pp.120-128
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• 2023

Osteoporosis is a disease that causes the weakening of bone by increasing porosity, which often results in fractures. Osteoporosis treatment measures include the use of Bisphosphonates and estrogen. However, these treatments cannot be used in the long term as these treatments have adverse side effects. Therefore, there is a need to identify better and safer treatment options. For this, 63 plant extracts were screened and among them, six extracts showed high anti-osteoclastic activity with low cytotoxicity. Of these six extracts, three extracts, Cudrania tricuspidata (P371), Ulmus davidiana var. japonica (P401), and Torilis japonica (P411), showed more than 50 percent osteoclast inhibition. While the remaining, Stewartia pseudocamellia extracts I and II (P370, P397) and Cuscuta chinensis (P418), showed moderate or between 40-50 percent osteoclast inhibition. Among all the extracts, Torilis japonica (P411) showed the highest inhibitory action against osteoclast development. Torilis japonica (P411) primary components include Kaempferol, Quercetin, and Luteolin, all proven to inhibit osteoclastogenesis. Stewartia pseudocamellia extracts I and II (P370 and P397) showed moderate or 44% osteoclast inhibition. Stewartia pseudocamellia extract II (P397) enhanced the growth of RAW 264.7 cells by 19%. Torilis japonica (P411) and Stewartia pseudocamellia extract II (P397) suppressed the expression of osteoclast-specific genes in RANKL-induced osteoclastogenesis in RAW 246.7 cells. Torilis japonica (P411) extracts even increased osteoblast-specific RUNX2 gene expression. This results provide that six extracts could be used as a potential treatment option for osteoporosis disease with the extracts of Torilis japonica (P411) and Stewartia pseudocamellia (P397) as an ideal candidates. However, the combination of the extract with higher osteoclastic inhibition and less toxic effects with further analysis should be recommended.

• Bulletin of the Korean Chemical Society
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• 제32권11호
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• pp.4137-4140
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• 2011

• 대한한방부인과학회지
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• 제26권2호
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• pp.1-16
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• 2013

Objectives: This study was performed to evaluate the effect of Dokhwalgisaengtang-gami water extract(DGG) on osteoporosis. Methods: The osteoclastogenesis and gene expression were determined in RANKL-stimulated RAW 264.7 cell. And osteoblastogenesis was also determined in rat calvarial cell. Results: The results were summarized as followes. 1. DGG decreased the number of TRAP positive cell in RANKL-stimulated RAW 264.7 cell. 2. DGG inhibited TRAP activity in RANKL-stimulated RAW 264.7 cell. 3. DGG decreased the expression of NAFTc1, MITF in RANKL-stimulated RAW 264.7 cell. 4. DGG increased the expression of iNOS, COX-2, IL-6 in RANKL-stimulated RAW 264.7 cell. 5. DGG decreased the expression of cathepsin K, MMP-9, TRAP in RANKL-stimulated RAW 264.7 cell. 6. DGG increased cell proliferation of rat calvarial cell. 7. DGG increased ALP activity in rat calvarial cell 8. DGG increased bone matrix protein, collagen synthesis and nodule formation in rat calvarial cell. Conclusions: It is concluded that DGG might decrease the bone resorption resulted from decrease of osteoclast differentiation and it's related gene expression. And DGG might increase the bone formation resulted from increase of osteoblast function.

• 약학회지
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• 제51권4호
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• pp.235-238
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• 2007

To determine the regulatory roles of phosphodiesterase (PDE) inhibitors on $PGE_2$-induced osteoclastogenesis, we investigated the effect of PDE inhibitors on osteoclast formation in the presence of $PGE_2$. Among PDE isozyme specific inhibitors, milrinone, a selective PDE3 inhibitor, and rolipram, a specific PDE4 inhibitor, increased $PGE_2$-induced osteoclast formation in cocultures of mouse bone marrow cells and osteoblasts. To verify that whether the PDE3 and PDE4 inhibitors act indirectly on osteoblasts, we measured the concentration of intracellular cAMP in osteoblasts. Treatment of milrinone or rolipram increased $PGE_2$-stimulated cAMP levels in osteoblasts. Furthermore, northern blot analysis revealed that the PDE3 and PDE4 inhibitors works synergistically with $PGE_2$ to increase the expression of TRANCE mRNA in osteoblasts. On the contrary, the PDE3 and PDE4 inhibitors did not augment the number of osteoclasts differentiated from bone marrow cells by $PGE_2$. In conclusion, the stimulation of $PGE_2$-induced osteoclast formation by the PDE3 and PDE4 inhibitors are attributable to their indirect effect on osteoblasts, not to their direct effect on bone marrow-derived osteoclast precursors.

• 대한예방한의학회지
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• 제19권2호
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• pp.135-144
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• 2015

Objective : Saengmaek-san (SM) has been commonly used in Korea to treat various diseases that involve the respiratory and cardiovascular system. However, to date, the mechanism underlying the effects of osteoclasts differentiation of SM has not been clearly understood. Method : To evaluate the effect of SM on osteoclast differentiation, we induced RAW 264.7 cells to be differentiated to osteoclasts by RANKL, and we performed RT-PCR to measure gene expression. Results : SM decreased the number of TRAP(+) MNCs in RANKL-induced osteoclast. SM decreased the expression of MMP-9, cathepsin K1, TRAP, NFATc1, MITF, and COX-2 in the osteoclast. But SM increased the expression of iNOS, $TNF-{\alpha}$ and IL-6 in osteoclast. Conclusion : It is concluded that SM might decrease the bone resorption resulted from decrease of osteoclast differentiation and it's related gene expression.

• Molecules and Cells
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• 제25권3호
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• pp.352-357
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• 2008

Osteoprotegerin (OPG) is a soluble decoy receptor that inhibits osteoclastogenesis and is closely associated with bone resorption processes. We have designed and determined the solution structures of potent OPG analogue peptides, derived from sequences of the cysteine-rich domain of OPG. The inhibitory effects of the peptides on osteoclastogenesis are dose-dependent ($10^{-6}M-10^{-4}M$), and the activity of the linear peptide at $10^{-4}M$ is ten-fold higher than that of the cyclic OPG peptide. Both linear and cyclic peptides have a ${\beta}$-turn-like conformation and the cyclic peptide has a rigid conformation, suggesting that structural flexibility is an important factor for receptor binding. Based on structural and biochemical information about RANKL and the OPG peptides, we suggest that complex formation between the peptide and RANKL is mediated by both hydrophobic and hydrogen bonding interactions. These results provide structural insights that should aid in the design of peptidyl-mimetic inhibitors for treating metabolic bone diseases caused by abnormal osteoclast recruitment.