Osteoclastogenesis In RAW264 Research Articles

Dihydrodaidzein and 6-hydroxydaidzein mediate the fermentation-induced increase of antiosteoporotic effect of soybeans in ovariectomized mice.

The consumption of soybeans is known to have beneficial effects on osteoporosis in postmenopausal women. However, the effects of soybean fermentation on the bioavailability and the antiosteoporotic effect have not yet been elucidated. To address this question, we fed ovariectomized C57BL/6J mice with a 5% nonfermented raw soybean (RS)- or fermented soybean (FS)-supplemented diet. After 18 wk of treatment, microcomputed tomography showed that FSs significantly increased bone mineral density compared with RSs. This was because of the up-regulation of bone morphogenic protein 2 (Bmp2) and its downstream target osteopontin in bone tissues. We analyzed isoflavone metabolite profiles in the sera of RS- or FS-fed mice and observed that the levels of 19 isoflavone metabolites were significantly increased in the sera of FS-fed mice. Among these metabolites, we observed that both dihydrodaidzein (DHD) and 6-hydroxydaidzein (6-HD) increased osteogenesis via Bmp2 signaling pathway in MC3T3-E1 cells and reduced receptor activator of nuclear factor κ-B ligand-induced osteoclastogenesis in RAW264.7 cells through the inhibition of NF-κB activation and MAPK phosphorylation. These data suggest that improved bioavailability of FSs resulted from the production of active metabolites such as DHD and 6-HD after consumption. DHD and 6-HD can be used as potential therapeutics for the amelioration of osteoporotic bone loss.-Kim, J.-S., Lee, H., Nirmala, F. S., Jung, C. H., Kim, M. J., Jang, Y.-J., Ha, T. Y., Ahn, J. Dihydrodaidzein and 6-hydroxydaidzein mediate the fermentation-induced increase of anti-osteoporotic effect of soybeans in ovariectomized mice.

The expression of Semaphorin 5A in patients with rheumatoid arthritis and its effect on osteoclastogenesis

Objective: To investigate the clinical significance of soluble Semaphorin 5A(Sema 5A) in patients of rheumatoid arthritis(RA) and the effect of Sema 5A on osteoclastogenesis in RAW264.7 cells. Methods: (1)Soluble Sema 5A was detected in the serum of 62 RA patients, 30 osteoarthritis(OA) patients and 48 healthy controls(HC) by enzyme-linked immunosorbent assay(ELISA).The relationships between serum Sema 5A and disease activity, radiographic severity and laboratory parameters were investigated in RA patients.(2)RAW264.7 cells were treated with different concentrations of Sema 5A(0,0.5,1,2.5,5 μg/ml).After 7 days, tartrate-resistant acid phosphate(TRAP) staining was performed. The mRNA levels of TRAP, cathepsin-K and matrix metallopeptidase 9(MMP-9) were tested using real-time polymerase chain reaction (RT-PCR).(3)Bone resorption area of dentine slides cultured with RAW264.7 cells was calculated after Sema 5A(5μg/ml) treatment. Results: (1)The serum Sema 5A in RA patients[(5.24±0.59)μg/L] was significantly higher than those in healthy controls[(2.93±0.34)μg/L,P<0.01] and OA patients[(2.68±0.47)μg/L,P<0.05]. The Sema 5A level in RA patients was positively correlated with disease activity score with 28 joint using C-reactive protein(DAS28-CRP), clinical disease activity index (CDAI),C-reactive protein(CRP) and sharp scores(P<0.05 or P<0.01).In addition,the serum Sema 5A in RA patients with positive anti-cyclic citrullinated peptide(CCP) antibody was significantly greater than that of anti-CCP antibody-negative patients(P<0.05).(2)After RAW264.7 cells were treated with Sema 5A, the number of TRAP positive osteoclasts increased according to the increase of Sema 5A concentration with maximal effect at 5 μg/ml. Meanwhile, Sema 5A promoted mRNA expression of TRAP,Cathepsin-K and MMP-9.(3)Bone resorption area increased when RAW264.7 cells were treated with Sema 5A(5 μg/ml). Conclusions: Serum Sema 5A is elevated in RA patients and correlated with disease activity and radiographic severity. Sema 5A promotes osteoclastogenesis of RAW264.7 cells.

Thymol inhibits RANKL-induced osteoclastogenesis in RAW264.7 and BMM cells and LPS-induced bone loss in mice

Thymol was identified as one of key compounds contributing to the aroma of thyme leaves. We investigated the effects of thymol on receptor activator NF-κB ligand (RANKL)-induced osteoclastogenesis in murine macrophage RAW264.7 cells and bone marrow derived macrophage (BMMs) cells and lipopolysaccharide (LPS)-induced bone loss in vivo. Thymol markedly reduced RANKL-stimulated osteoclast formation and differentiation in RAW264.7 cells and BMMs cells without any cytotoxic effects. The in vitro and in vivo osteoclastogenesis inhibitory effect of thymol was assessed by calculating the quantity of TRAP (+) multinucleated cells and its inhibitory effects on the resorbing capacity were examined on calcium phosphate-coated plates. Moreover, the inhibitory effects of thymol resulted in a reduction of RANK, cathepsin K, matrix metalloproteinase-9 (MMP-9), dendritic cell-specific transmembrane protein (DC-STAMP), c-terminal myc kinase (C-MYC), C-terminal Src kinase (C-SRC), GRB2-associated-binding protein 2 (GAB2), microphthalmia-associated transcription factor (MITF), and carbonic anhydrase II genes. Similarly, activities of ERK, JNK and AKT and protein expressions of NFATc1, C-FOS, MMP-9 and cathepsin K were downregulated by thymol. More importantly, the application of thymol significantly reduced LPS-induced inflammatory bone loss in mice. In conclusion, these findings identified that thymol could be a useful therapeutic agent for the prevention of bone destructive diseases.

Inhibitory Effects of Curcuma xanthorrhiza Supercritical Extract and Xanthorrhizol on LPS-Induced Inflammation in HGF-1 Cells and RANKL-Induced Osteoclastogenesis in RAW264.7 Cells.

Periodontal disease is triggered by the host immune response to pathogens in the microbial biofilm. Worsening of periodontal disease destroys the tooth-supporting tissues and alveolar bone. As oral inflammation can induce systemic diseases in humans, it is important to prevent periodontal disease. In this study, we demonstrated that Curcuma xanthorrhiza supercritical extract (CXS) and its active compound, xanthorrhizol (XAN), exhibit anti-inflammatory effects on lipopolysaccharide (LPS)-treated human gingival fibroblast-1 cells and anti-osteoclastic effects on receptor activator of nuclear factor kappa B ligand (RANKL)-treated RAW264.7 cells. LPS-upregulated inflammatory factors, such as nuclear factor kappa B p65 and interleukin-1β, were prominently reduced by CXS and XAN. In addition, RANKL-induced osteoclastic factors, such as nuclear factor of activated T-cells c1, tartrate-resistant acid phosphatase, and cathepsin K, were decreased in the presence of CXS and XAN. CXS and XAN inhibited the mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling pathway. Collectively, these results provide evidence that CXS and XAN suppress LPS-induced inflammation and RANKL-induced osteoclastogenesis by suppressing the MAPK/AP-1 pathway.

Increase in anti-inflammatory activities of radical-degraded porphyrans isolated from discolored nori (Pyropia yezoensis)

The anti-inflammatory properties of porphyrans (D1–D4) obtained from four discolored nori (Pyropia yezoensis) with different growth backgrounds were studied to examine possible variations in their bioactivities. Elution profiles of the porphyrans on Sepharose 4B indicated that D2-porphyran had relatively lower-molecular-size porphyrans than the other porphyrans. Inhibitory activities of the four porphyrans against nitric oxide (NO) and tumor necrosis factor-α (TNF-α) secretion by lipopolysaccharide (LPS)-stimulated RAW264.7 cells were different, whereas no significant differences were observed in the sulfate and anhydrogalactose levels. D2-porphyran showed the highest inhibitory activity against NO and TNF-α secretion by LPS-stimulated RAW264.7 cells, whereas D3- and D4-porphyrans had almost no activity. All porphyrans were efficiently degraded by free radical generated with ascorbate and hydrogen peroxide. The free-radical degradation resulted in a significant increase in the inhibitory activities of the four porphyrans against NO and TNF-α secretion, with varying rates depending on the porphyrans. The ability of D2-porphyran to suppress the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells was also significantly enhanced after degradation. Our results suggest that molecular size is an important factor affecting the anti-inflammatory activity of porphyrans, and radical degradation might be a promising procedure to obtain active low-molecular-size porphyrans.

Effect of TRPV1 Agonist Capsaicin on Osteoclastogenesis in RAW 264.7 Derived Osteoclasts.

Effect of TRPV1 Agonist Capsaicin on Osteoclastogenesis in RAW 264.7 Derived Osteoclasts.

Anti-bacterial zinc-doped calcium silicate cements: Bone filler

A bone-filler is expected to accomplish diverse purposes such as osteoblastogenesis, osteoclastogenesis and anti-bacterial activity. Taken into consideration of the potent antibacterial activity of certain trace elements, in present study, the effect of Zn-doping in tricalcium silicate cement is explored by means of its biological function. Cement setting reaction is slowed down with Zn addition, while the compressive strength of the doped cement increases. In stimulating body fluid, Zn is incorporated slowly into developing apatite nuclei; it results in restricted apatite crystallization and formation of amorphous apatite layer. The slow release of zinc ions into culture medium, greatly enhances the proliferation and alkaline phosphatase activity of rat bone marrow stromal cells. Released Zn2+ also takes part in bone-remodeling; facilitates osteoclastogenesis in RAW 264.7 (in presence of RANKL). Furthermore, the accumulation of Zn2+ at bacterial membrane impairs membrane permeability, causing death of both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterias. The data indicate a gradual release of Zn ions from the bioactive injectable doped calcium silicate; these ions modulate bone metabolism induce concentration-dependent cell proliferation, render osteo-inductivity to self-curing cement. Zn also imparts anti-bacterial property to C3S; perquisite for medical bone implants.

Tea polysaccharide inhibits RANKL-induced osteoclastogenesis in RAW264.7 cells and ameliorates ovariectomy-induced osteoporosis in rats

Background and purposeTea drinking has positive effects on bone health and may prevent and treat osteoporosis, especially in older and postmenopausal women. Tea polysaccharide (TPS) is a major bioactive constituent in tea. Despite its profound effects on human health, whether TPS has anti-osteoporotic effects remains largely unknown. As such, we investigated the anti-osteoporotic effects of TPS. MethodsIn vitro, TPS effects on osteoclastogenesis were examined using osteoclast precursor RAW264.7 cells. TPS effects on osteoclastogenesis-related expression of marker genes and proteins were determined by gene expression and immunoblotting analyses, respectively. For in vivo studies, 12-week-old female Wistar rats were divided randomly into a sham-operated group (sham) and four ovariectomized (OVX) subgroups: OVX with vehicle (model) and OVX with low-, medium-, and high-dose TPS (0.32, 0.64 and 1.28 g/kg body weight/day, respectively). TPS was administered intragastrically to rats for 13 weeks. Body weight, blood biochemical parameters, organ weight, organ coefficients, femoral length, bone mineral density (BMD), biomechanical properties, and bone microarchitecture were documented. ResultsTPS inhibited osteoclast differentiation significantly and dose-dependently, and its inhibitory effect was not due to toxicity to RAW264.7 cells. TPS suppressed expression of osteoclastogenesis-related marker genes and proteins significantly. In in vivo studies, medium-dose TPS treatment ameliorated OVX-induced calcium loss significantly. Low-dose TPS treatment decreased the activity of acid phosphatase (ACP) in OVX rats significantly. In addition, TPS treatment improved other blood biochemical parameters and femoral biomechanical properties to a certain extent. More importantly, TPS treatment ameliorated bone microarchitecture in OVX rats strikingly because of increased cortical bone thickness and trabecular bone area in the femur. ConclusionTPS can inhibit receptor activator nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells and ameliorate ovariectomy-induced osteoporosis in rats.

Combined treatment with Dendrobium candidum and black tea extract promotes osteoprotective activity in ovariectomized estrogen deficient rats and osteoclast formation

AimsDendrobium candidum (DC) and black tea, are traditional chinese drinks, which contain multiple active ingredients. However, whether or not the combination of these two ingredients can improve osteoporosis remains unknown. This study therefore aimed to examine the effects of the combination of DC and black tea extract (BTE) on osteoporosis. Main methodsOvariectomy (OVX)-induced osteoporosis in vivo as well as receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in vitro was selected. Key findingsResults showed that OVX rats that were treated orally with a DC and BTE combination for 12 weeks maintained their calcium (Ca) and phosphorus (P) homeostasis and exhibited significantly enhanced estradiol (E2) and OPG levels. This combination treatment also simultaneously reduced levels of interleukin (IL)-1β, IL-6 and improved the organ coefficients of the uterus and femur as well as BMD and BMC in OVX rats. In addition, this DC and BTE combination suppressed osteoclast differentiation in the RANKL-stimulated osteoclastogenesis of RAW 264.7 cells and effectively inhibited the expression of osteoclast-associated genes and proteins. The results of this study further highlight the fact that a combination of DC and BTE improved ovariectomy-induced osteoporosis in rats and suppressed RANKL-stimulated osteoclastogenesis in RAW 264.7 cells. SignificanceThis combination also significantly alleviated osteoporosis when compared to the alternative sole treatments above, due to synergistic effects among components. One partial mechanism of this combination might be the inhibition of osteoclast proliferation and the regulation of NFATC1/c-Fos expression.

Synthesis of some novel orcinol based coumarin triazole hybrids with capabilities to inhibit RANKL-induced osteoclastogenesis through NF-κB signaling pathway

A total of twenty-two novel coumarin triazole hybrids (4a-4k and 6a-6k) were synthesized from orcinol in good to excellent yields of 70–94%. The structures of all the synthesized compounds were elucidated by spectroscopic techniques such as 1H NMR, 13C NMR, and HRMS. The anti-inflammatory potential of synthesized compounds was investigated against the proinflammatory cytokine, TNF-α on U937 cell line and compounds 4d, 4j, and 6j were found to exhibit promising anti-inflammatory activity. These three compounds were further screened against TNF-α on LPS-stimulated RAW 264.7 cells, which confirm their anti-inflammatory potential. Furthermore, the above said active compounds were tested for their inhibitory effect on RANKL-induced osteoclastogenesis in RAW 264.7 cells by using tartrate resistant acid phosphatase (TRAP) staining assay at 10 µM. Molecular mechanism studies demonstrated that compound 4d exhibited dose dependent inhibition of RANKL-induced osteoclastogenesis by suppression of the NF-kB pathway. Thus, compound 4d is a promising candidate for further optimization to develop as a potent anti-osteoporotic agent.

Psoralen inhibits RAW264.7 differentiation into osteoclasts and bone resorption by regulating CD4+T cell differentiation

This paper aimed to investigate whether psoralen inhibits the differentiation and bone resorption by regulating CD4+T cell differentiation in RANKL-induced osteoclastogenesis in RAW264.7 cells, and elucidate its mechanism for osteoporosis. CD4+T cells were isolated from spleen cells of Balb/c mice by immunomagnetic separation method. The cells were divided into blank control group and psoralen group. The cells were cultured in 24-well plates and cultured for 3 days, and then they were collected for co-culture experiments after 4 days. Co-culture experiments were divided into RAW264.7 cell group, psoralen+RAW264.7 cell group, without psoralen treatment of CD4+T cells+RAW264.7 cell group, psoralen treatment of CD4+T cells+RAW264.7 cell group. After 5 days of co-culture, TRAP staining was used to detect the number of osteoclasts, and after 8 days of co-culture, bone resorption was evaluated by toluidine blue staining. The expressions of RORγt, Foxp3, IL-17, TNF-α, TGF-β and IL-10 in CD4+T cells and osteoclast differentiation-related genes MMP-9, TRAP and Cat-K were detected by Real-time polymerase chain reaction (RT-PCR); ELISA kit was used to detect IL-17, TNF-α, TGF-β and IL-10 and other cytokines levels. Our data confirmed that the psoralen significantly promoted the expression of Foxp3, TGF-β and IL-10 in CD4+T, and inhibited the expression of RORγt, IL-17 and TNF-α in CD4+T, the CD4+T cells without treatment by psoralen can significantly promote RANKL-induced differentiation of RAW264.7 to osteoclasts, and psoralen treatment of CD4+T can significantly inhibit RANKL-induced RAW264.7 osteoclast differentiation and bone resorption. Taken together, psoralen inhibits the differentiation and bone resorption of RAW264.7 into osteoclasts by promoting the development of CD4+ CD25+ Treg/Th17 balance in CD4+T cells to CD4+CD25+T.

Inhibitory effect of sulphated polysaccharide porphyran (isolated from Porphyra yezoensis) on RANKL-induced differentiation of RAW264.7 cells into osteoclasts.

Safe and efficient therapeutic agents for bone diseases are required in natural sources. We previously found that edible seaweed-derived polysaccharide porphyran exhibited anti-inflammatory effects through the down regulation of nuclear factor-κB. The aim of this study was to investigate the availability of porphyran as a therapeutic agent for bone diseases. The effects of porphyran on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells were examined. Porphyran suppressed RANKL-induced osteoclast formation in a concentration-dependent manner (6.25-50μg/ml) without any cytotoxic effects. Furthermore, real-time polymerase chain reaction analyses indicated that porphyran at 50μg/ml significantly attenuated the RANKL-induced increase in the mRNA levels of osteoclastogenesis-related marker genes such as nuclear factor of activated T cells, tartrate-resistant acid phosphatase, cathepsin K, and matrix metalloproteinase-9 in RAW264.7 cells. To our knowledge, this is the first report showing that edible-seaweed-derived polysaccharide porphyran can suppress RANKL-induced osteoclastogenesis. Our results suggest that porphyran can be used as a safe therapeutic agent to improve osteoclast-related pathological conditions.

The function of miR-218 and miR-618 in postmenopausal osteoporosis.

Postmenopausal osteoporosis (POMP) is a serious disorder with significant physical, psychosocial, and financial consequences, which greatly reduce the postmenopausal women's life quality. The related issues of postmenopausal osteoporosis are increasingly concerned by society. Past researches have shown that miRNAs play an important role in the occurrence and development of postmenopausal osteoporosis. However, the role of miR-218 and miR-618 in the osteoporosis regulation is still unclear. First of all, we investigated the alteration of miR-218 and miR-618 during osteoclastogenesis of RAW264.7 cells. Next, we transfected RAW264.7 cells with miR-218 or miR-618 mimics and inhibitors to explore the influences of miR-218 and miR-618 on osteoclast differentiation. Then, we conducted bioinformatics analysis and luciferase reporter assay to identify and test the target gene of miR-218 and miR-618. MiR-218 and miR-618 were down-regulated when RAW264.7 cells differentiated into osteoclasts. In addition, overexpression of miR-218 or miR-618 attenuated RAW264.7 cells differentiated into osteoclasts in vitro, whereas inhibition of miR-218 or miR-618 promoted this progress. This was demonstrated by increased expression of osteoclast-specific genes and TRAP staining. TLR-4 was confirmed to be the direct target of miR-218 and miR-618 by bioinformatics and luciferase reporter assay. These results suggested that miR-218 and miR-618 play an important role in osteoclastogenesis via TLR-4/MyD88/NF-κB signaling pathway. Thus, targeting miR-218 and miR-618 promise a therapeutic potential in the treatment of osteoporosis.

I‑BET151 inhibits osteoclastogenesis via the RANKL signaling pathway in RAW264.7 macrophages.

Excessive bone resorption mediated by osteoclasts may lead to the risk of various lytic bone diseases. In the present study, the effects of I‑BET151, a bromodomain and extra terminal domain protein inhibitor, on osteoclastogenesis in RAW264.7 cells and the underlying mechanism of this process was investigated. Cells were divided into 6 groups, including the control group, receptor activator of nuclear factor‑κB ligand (RANKL) group and 4 other groups containing RANKL and I‑BET151 at different concentrations. Tartrate‑resistant acid phosphatase (TRACP) staining was used to observe the effect of I‑BET151 on osteoclastogenesis and the number of TRACP positive multinucleated cells was calculated. Western blotting was used to evaluate the expression of tumor necrosis factor receptor associated factor (TRAF6), nuclear factor of activated T‑cells cytoplasmic 1 (NFATcl), transcription factor p65 (p65), nuclear factor of κ light polypeptide gene enhancer in B‑cells inhibitor‑α (IκB‑α), extracellular signal‑regulated kinase, Jun N‑terminal kinase (JNK) and p38. mRNA expression levels of osteoclast specific genes TRACP, matrix metalloproteinase‑9 (MMP9), cathepsin K (CtsK) and proto‑oncogene tyrosine‑protein kinase Src (c‑Src) were measured using the reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). TRACP staining results demonstrated that I‑BET151 inhibited osteoclastogenesis induced by RANKL and the inhibition was dose dependent. TRACP multinucleated positive cells were significantly decreased when treated with I‑BET151 compared with the RANKL group. The inhibitory effect on TRAF6 was significant when concentrations of 100 and 200nM I‑BET151 were used, and NFATcl was significantly inhibited when a concentration of 200nM was used compared with the RANKL group, in a dose-dependent manner. Nuclear translocation of p65 was significantly inhibited by I‑BET151 at all concentrations. The degradation of IκB‑α, and phosphorylation of JNK and p38 were also significantly inhibited by I‑BET151, with the exception of the expression of IκB‑α following treatment with 50nM I‑BET151. The RT‑qPCR results revealed that osteoclast‑specific genes TRACP, MMP9, CtsK and c‑Src were all dose‑dependently inhibited by I‑BET151, except for CtsK. In conclusion, I‑BET151 may significantly suppress the osteoclastogenesis of RAW264.7 cells via the RANKL signaling pathway.

Lipoxin A4 suppresses osteoclastogenesis in RAW264.7 cells and prevents ovariectomy-induced bone loss

Lipoxin A4 (LXA4; 5S, 6R, 15Strihydroxy- 7,9,13-trans-11-eicosatetraenoic acid) is a metabolic product of arachidonic acid under the action of lipoxidase. This lipid molecule plays important roles in several biological functions, especially inflammatory processes. In vivo, LXA4 regulates the inflammatory response through several signaling pathways. Its mechanism suggests that it might have an effect on osteoclastogenesis and bone loss. Using both in vitro and in vivo studies, it was here observed that LXA4 could significantly inhibit the formation and function of osteoclasts and these effects could be blocked by Boc-2, the specific inhibitor of FPR2/ALX (the receptor of LXA4). Meanwhile, LXA4 reduce the amount of ovariectomy-induced bone loss. These protective effects was found to be associated with inhibition of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), PI3K-AKT, and p-38, ERK, and JNK in MAPKs. The expression of the receptor activator of the NF-κB ligand RANKL:osteoprotegerin ratio and serum levels of TNF-α, IL-1β, and IL-6 were decreased by LXA4. Moreover, LXA4 prevented the production of reactive oxygen species (ROS), the expression of osteoclast-specific genes, including tartrate-resistant acid phosphatase (TRAP), cathepsin K (CK), matrix metalloproteinase (MMP)-9, RANK, and osteoclastic related transcription factors of c-Fos, NFATc1 could also be significantly inhibited by LXA4 in a dose-dependent manner. Studies have demonstrated that LXA4 can inhibit the formation and function of osteoclasts through modulation of several pathways both upstream and downstream of RANKL signaling and FPR2/ALX wasinvolved in the procedures. This shows that LXA4 may be used as a new strategy for the treatment of osteoclast-related diseases.

Glycated-proteins modulate RANKL-induced osteoclastogenesis in RAW264.7 cells.

Glycated-proteins modulate RANKL-induced osteoclastogenesis in RAW264.7 cells.

Glycated-HSA inhibits osteoclastogenesis in RAW264.7 cells depending on the glycating agents via downregulating RANKL-signaling

Glycated-HSA inhibits osteoclastogenesis in RAW264.7 cells depending on the glycating agents via downregulating RANKL-signaling

Fructus Ligustri Lucidi ethanol extract inhibits osteoclastogenesis in RAW264.7 cells via the RANKL signaling pathway.

Fructus ligustri Lucidi (FLL) is the fruit of Ligustrumlucidum Ait and a traditional Chinese medicine, primarily known for its role in osteoporosis prevention and treatment. The present study aimed to elucidate the effect and underlying mechanism of action of ethanol extract of FLL on osteoclast differentiation and bone resorption, and to identify the active compounds within it. RAW264.7 murine monocyte/macrophage cells were stimulated with the receptor activator of nuclear factor κB ligand (RANKL) to induce osteoclast differentiation invitro. The present study demosntrated that FLL extract and its two primary components, oleanolic acid (OA) and ursolic acid (UA), significantly suppressed RANKL‑induced tartrate resistant acid phosphatase (TRAP) activity and multinucleate osteoclast formation without inducing cytotoxicity; however, no effect was observed on the apoptosis of mature osteoclasts. Additionally, RANKL‑induced mRNA expression levels of the key transcription factors, tumor necrosis factor receptor associated factor‑6, nuclear factor of activated Tcell‑c1 and c‑Fos, and the osteoclast markers, TRAP, cathepsin K and matrix metalloproteinase‑9 were suppressed by FLL, OA and UA. However, no effect was observed on RANKL‑induced mRNA expression levels of Src. These results demonstrated that FLL may inhibit osteoclastogenesis in RAW264.7 cells via RANKL signaling pathways. OA and UA are active compounds in inducing this effect; however, their specific roles remain to be elucidated.

Interleukin-21 promotes osteoclastogenesis in RAW264.7 cells through the PI3K/AKT signaling pathway independently of RANKL

Cytokines play a key role in the bone destruction of rheumatoid arthritis (RA). Interleukin-21 (IL-21) promotes osteoclastogenesis in RA in a receptor activator of nuclear factor-κB ligand (RANKL)-dependent way. Whether IL-21 is capable of promoting osteoclastogenesis directly in the absence of RANKL remains unknown. In the present study, we examined the osteoclastogenic activity of IL-21 in RAW264.7 cells in the absence of RANKL. We found that IL-21 enhanced osteoclastogenesis and this was demonstrated by increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive stained, multinucleated cells compared with the negative control. Western blot analysis and immunocytochemistry showed the positive expression of calcitonin receptor (CTR) in the IL-21 group. RT-PCR and RT-qPCR also verified the increased mRNA expression of CTR and cathepsin K in the IL-21 group compared with the negative control. The scanning electronic microscope images showed a few resorption pits on the bone slices cultured with IL-21. The phosphoinositide 3-kinase (PI3K)/AKT pathway inhibitor LY294002 significantly suppressed IL-21-induced osteoclastogenesis. Taken together, these findings suggest that IL-21 has direct osteoclastogenic potential independently of RANKL. IL-21 may promote osteoclastogenesis through the PI3K/AKT signaling pathway. Therapy targeting IL-21 may be of value in preventing bone erosions in patients with RA.

Iguratimod (T-614) suppresses RANKL-induced osteoclast differentiation and migration in RAW264.7 cells via NF-κB and MAPK pathways

IntroductionIguratimod (T-614) has been confirmed as a highly efficacious and safe novel disease-modifying anti-rheumatic drug (DMARD) for rheumatoid arthritis therapy in China and Japan due to its potent anti-inflammation effect. Here, we investigate the effects of Iguratimod on osteoclast differentiation, migration and function. MethodsThe effect of Iguratimod on osteoclastogenesis, migration and bone resorption were assessed by TRAP staining, transwell migration assay and osteologic discs, respectively. Relative expressions of osteoclastic related genes, chemokines and transcription factors were assessed by reverse transcription polymerase chain reaction (RT-PCR) and signaling pathways were analyzed by western blotting. ResultsIguratimod significantly inhibits osteoclast differentiation, migration and bone resorption in RANKL-induced RAW264.7 cell in a dose-dependent manner. The expressions of osteoclastic related genes including TRAP, CTSK and CTR were increased in RAW264.7 cell upon RANKL stimulation but were obviously suppressed in the presence of Iguratimod. RANKL induced the expression of chemokines including CCL7, CCL4 and CCL12 and osteoclastic related transcription factors of c-Fos, c-Jun and NFATc1 could be significantly inhibited by Iguratimod in a dose dependent manner. Western blotting indicated Iguratimod could suppress the activation of MAPKs and NF-κB pathway in RANKL induced osteoclastogenesis in RAW264.7. ConclusionsThese findings revealed a directly inhibitory role of Iguratimod on osteoclast formation and function, which is distinct from previous report, suggesting Iguratimod provide a unique therapeutic strategy for RA and especially in light of preventing bone destruction.