The extract of Trachelospermum jasminoides (Lindl.) Lem. vines inhibits osteoclast differentiation through the NF-κB, MAPK and AKT signaling pathways.

Osteoclasts are the only cells in the body with a bone-resorption function. The identification of anti-osteoclastogenic agents is important in managing bone loss diseases. The dried vines of Trachelospermum jasminoides (Lindl.) Lem. have been used as a herbal medicine to treat musculoskeletal soreness in East Asia for hundreds of years. In the present study, we focused on the effect of Trachelospermum jasminoides (Lindl.) Lem. extract (TJE) on osteoclast differentiation. As indicated by tartrate-resistant acid phosphatase (TRAP) staining, TJE inhibited osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand from bone marrow-derived monocytes/macrophages without showing any cytotoxicity. In addition, TJE effectively suppressed F-actin ring formation and the bone-resorption function of osteoclasts. The subsequent studies such as network pharmacology and molecular investigation, revealed that TJE inhibited osteoclastogenesis-related genes in a dose- and time-dependent manner through NF-κB, MAPK and AKT-mediated mechanism followed by the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)/c-Fos pathway. Our study could potentially explain the underlying molecular pharmacology of TJE in osteoclast-related diseases. What's more, it suggested that network pharmacology could help the modernization of traditional Chinese medicine.

A natural compound (LCA) isolated from Litsea cubeba inhibits RANKL-induced osteoclast differentiation by suppressing Akt and MAPK pathways in mouse bone marrow macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Litsea cubeba (Lour.) Pers. has been traditionally used as a folk prescription for treating rheumatic diseases in China. AIM OF THE STUDY: This study aimed to investigate the effects and underlying mechanism of LCA, a new type of dibenzyl butane lignin compound extracted from L. cubeba, on macrophage colony stimulating factor (M-CSF) plus receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation in mouse-derived bone marrow macrophages (BMMs). MATERIAL AND METHODS: TRAP staining, TRAP enzyme activity assay and actin ring staining were applied to identify the effects of LCA on osteoclast differentiation. Protein expression of NFATc1, c-Fos and MMP-9, and phosphorylation of p65, Akt, JNK, ERK and p38 in RANKL-induced osteoclasts was determined using western blotting to investigate the underlying mechanism. RESULTS: LCA significantly suppressed RANKL-induced osteoclast differentiation by inhibiting TRAP activity, decreasing the number of TRAP+ multinuclear osteoclasts and reducing the formation of F-actin ring without obvious cytotoxicity in BMMs. Moreover, LCA treatment strongly reduced protein expression of NFATc1, c-Fos and MMP-9, and attenuated the phosphorylation of p65, Akt, JNK, ERK and p38 in RANKL-stimulated BMMs. CONCLUSIONS: LCA ameliorated RANKL-induced osteoclast differentiation via inhibition of Akt and MAPK signalings in BMMs, and may serve as a potential pro-drug for bone destruction prevention.

Deferoxamine inhibits iron-uptake stimulated osteoclast differentiation by suppressing electron transport chain and MAPKs signaling.

Iron overload causes osteoporosis by enhancing osteoclastic bone resorption. During differentiation, osteoclasts demand high energy and contain abundant mitochondria. In mitochondria, iron is used for the synthesis of Fe-S clusters to support mitochondria biogenesis and electron transport chain. Moreover, mitochondrial reactive oxygen species (ROS) play an important role in osteoclastogenesis. Activation of MAPKs (ERK1/2, JNK, and p38) by ROS is essential and contribute to osteoclast differentiation. How iron chelation impairs electron transport chain and ROS dependent MAPKs activation during osteoclast differentiation is unknown. This study aimed to determine the direct effects of iron chelation on osteoclast differentiation, electron transport chain and MAPKs activation. In the present study, we found that when iron chelator, deferoxamine (DFO), was added, a dose-dependent inhibition of osteoclast differentiation and bone resorption was observed. Supplementation of transferrin-bound iron recovered osteoclastogenesis. Iron chelation resulted in a marked decrease in ferritin level, and increased expression of transferrin receptor 1 and ferroportin. As an iron chelator, DFO negatively affected mitochondrial function through decreasing activities of all the complexes. Expressions of mitochondrial subunits encoded both by mitochondrial and nuclear DNA were decreased. DFO augmented production of mitochondrial ROS, but inhibited the phosphorylation of ERK1/2, JNK, and p38, even in the presence of hydrogen peroxide. These results suggest that iron chelation directly inhibits iron-uptake stimulated osteoclast differentiation and suppresses electron transport chain. Iron chelation negatively regulates MAPKs activation, and this negative regulation is independent on ROS stimulation.

Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase.

Tetrandrine, a bisbenzylisoquinoline alkaloid, was previously demonstrated to attenuate inflammation and cartilage destruction in the ankles of mice with collagen-induced arthritis (CIA). Here, we explored the underlying mechanism by which tetrandrine prevented arthritis-induced bone erosion by focusing on the differentiation and function of osteoclasts. We found that daily administration of tetrandrine (30 mg/kg) markedly reduced the bone damage and decreased the number of osteoclasts in CIA rats. In vitro, tetrandrine inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis at the early stage and reduced the expressions of osteoclast-related marker genes. In bone marrow-derived macrophages and RAW264.7 cells, tetrandrine inhibited RANKL-induced translocation of NF-κB-p65 and nuclear factor of activated T cell 1 (NFATc1) through suppressing spleen tyrosine kinase (Syk)-Bruton's tyrosine kinase-PLCγ2-Ca2+ signaling. Of interest, tetrandrine did not affect the phosphorylation of immunoreceptor tyrosine-based activation motifs, the conventional upstream of Syk, but it inhibited the activity of Syk by enhancing its ubiquitination and degradation. The anti-osteoclastogenesis effect of tetrandrine nearly disappeared when it was used in combination with the Syk inhibitor piceatannol or in constitutively activated Syk-overexpressing cells. Taken together, tetrandrine attenuated CIA-induced bone destruction by inhibiting osteoclastogenesis through hindering the translocation of NF-κB-p65 and NFATc1 via reducing the activation of Syk.-Jia, Y., Miao, Y., Yue, M., Shu, M., Wei, Z., Dai, Y. Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase.

Curcumin improves bone microarchitecture in glucocorticoid-induced secondary osteoporosis mice through the activation of microRNA-365 via regulating MMP-9.

The present study aimed to investigate bone microarchitecture of the proximal tibia in glucocorticoid-induced osteoporosis (GIOP) mice, and the underlying molecular mechanisms of curcumin in DXM-induced osteoporosis were performed. DXM-treated facilitated to induce hypercalciuria in mice, and curcumin-treated showed a decrease in urine calcium. Curcumin reversed DXM-induced bone resorption, including an increase in serum OCN and a decrease in bone resorption markers CTX and TRAP-5b. H&E staining showed the increased disconnections and separation in trabecular bone network as well as the reduction of trabecular thickness throughout the proximal metaphysis of tibia in GIOP group. Importantly, curcumin reversed DXM-induced trabecular deleterious effects and stimulated bone remodeling. The further evidence showed that curcumin supplement significantly decreased the TRAP-positive stained area and inhibited the activity of OPG/RANKL/RANK signaling in the GIOP mice. Moreover, bioinformatics analysis suggested that miR-365 was a regulator of MMP9. The levels of miR-365 were markedly suppressed; however, curcumin treatment could reverse the downregulation of miR-365 in the tibia of GIOP mice. Simultaneously, the results demonstrated that the mRNA and protein expression of MMP-9 were significantly increased in GIOP mice compared with that of the control group. Curcumin treatment could suppress the expression of MMP-9 in the tibia of GIOP mice. The present study demonstrated the protective effects of curcumin against bone deteriorations in the experimentally DIOP mice, and the underlying mechanism was mediated, at least partially, through the activation of microRNA-365 via suppressing MMP9.

Spatholobus suberectus inhibits osteoclastogenesis and stimulates chondrogenesis.

This study was carried out to investigate the effect of Spatholobus suberectus Dunn (SS) on the protection of chondral defect and inhibition of osteoclastogenesis. To examine these effects, we measured the matrix metalloproteinase (MMP) levels in SW1353 chondrosarcoma cells and performed tartrate-resistant acid phosphatase (TRAP) staining in bone marrow macrophage (BMM)-derived osteoclasts. To investigate the anti-osteoarthritis (OA) effects, we assessed TNF-α-induced MMP-1, -3, -9 and tissue inhibitors of matrix metalloproteinase (TIMP) expression levels in SW1353 cells. We observed that SS extract significantly inhibited MMP and TIMP expression in SW1353 cells. Also, SS extract inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. These results suggest that SS extract may have a potential in the treatment of bone loss and chondral defect by suppressing osteoclast differentiation and decreasing the expression of OA factors. Therefore, clarification of the mechanism of the action of SS extract and its active components is needed.

Antiosteoclastic activity of milk thistle extract after ovariectomy to suppress estrogen deficiency-induced osteoporosis.

Bone integrity abnormality and imbalance between bone formation by osteoblasts and bone resorption by osteoclasts are known to result in metabolic bone diseases such as osteoporosis. Silymarin-rich milk thistle extract (MTE) and its component silibinin enhanced alkaline phosphatase activity of osteoblasts but reduced tartrate-resistant acid phosphatase (TRAP) activity of osteoclasts. The osteoprotective effects of MTE were comparable to those of estrogenic isoflavone. Low-dose combination of MTE and isoflavone had a pharmacological synergy that may be useful for osteogenic activity. This study attempted to reveal the suppressive effects of MTE on bone loss. C57BL/6 female mice were ovariectomized (OVX) as a model for postmenopausal osteopenia and orally administered 10 mg/kg MTE or silibinin for 8 weeks. The sham-operated mice served as estrogen controls. The treatment of ovariectomized mice with nontoxic MTE and silibinin improved femoral bone mineral density and serum receptor activator of nuclear factor- κB ligand/osteoprotegerin ratio, an index of osteoclastogenic stimulus. In addition, the administration of MTE or silibinin inhibited femoral bone loss induced by ovariectomy and suppressed femoral TRAP activity and cathepsin K induction responsible for osteoclastogenesis and bone resorption. Collectively, oral dosage of MTE containing silibinin in the preclinical setting is effective in preventing estrogen deficiency-induced bone loss.

Comparison of the ability of chondroitin sulfate derived from bovine, fish and pigs to suppress human osteoclast activity in vitro.

Chondroitin sulfate (CS) compounds are commonly used to manage OA symptoms. Recent literature has indicated that abnormal subchondral bone metabolism may have a role in the pathogenesis of OA. The aim of this study was to access the effects of chondroitin sulfate obtained from bovine, fish and porcine sources on human osteoclast formation and activity in vitro. Human osteoclasts were generated from blood mononuclear cells. Cells were cultured over 17 days with the addition of macrophage colony stimulating factor (M-CSF) and then stimulated with receptor activator of nuclear factor kappa B ligand from day 7. Cells were treated with the CS commencing from day 7 onwards. To assess effects on osteoclasts, tartrate resistant acid phosphatate (TRAP) expression and resorption of whale dentine assays were used. Bovine-derived CS consistently suppressed osteoclast activity at concentrations as low as 1 µg/ml. Fish and porcine CS was less consistent in their effects varying with different donor cells. All CS compounds had little effect on TRAP activity. mRNA analysis using real-time PCR of bovine CS treated cells indicated that the inhibition of activity was not due to inhibition of the late stage NFATc1 transcription factor (p > 0.05). These results are consistent with CS inhibition of mature osteoclast activity rather than the formation of mature osteoclasts. It would appear that there are differences in activity of the different CS compounds with bovine-derived CS being the most consistently effective inhibitor of osteoclast resorption, but the results need to be confirmed.

NAMPT/PBEF1 enzymatic activity is indispensable for myeloma cell growth and osteoclast activity.

Multiple myeloma (MM) cells typically grow in focal lesions, stimulating osteoclasts that destroy bone and support MM. Osteoclasts and MM cells are hypermetabolic. The coenzyme nicotinamide adenine dinucleotide (NAD(+)) is not only essential for cellular metabolism; it also affects activity of NAD-dependent enzymes, such as PARP-1 and SIRT-1. Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin, encoded by PBEF1) is a rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide. Coculture of primary MM cells with osteoclasts induced PBEF1 upregulation in both cell types. PBEF1 expression was higher in experimental myelomatous bones than in nonmyelomatous bone and higher in MM patients' plasma cells than in healthy donors' counterparts. APO866 is a specific PBEF1 inhibitor known to deplete cellular NAD(+). APO866 at low nanomolar concentrations inhibited growth of primary MM cells or MM cell lines cultured alone or cocultured with osteoclasts and induced apoptosis in these cells. PBEF1 activity and NAD(+) content were reduced in MM cells by APO866, resulting in lower activity of PARP-1 and SIRT-1. The inhibitory effect of APO866 on MM cell growth was abrogated by supplementation of extracellular NAD(+) or NAM. APO866 inhibited NF-κB activity in osteoclast precursors and suppressed osteoclast formation and activity. PBEF1 knockdown similarly inhibited MM cell growth and osteoclast formation. In the SCID-rab model, APO866 inhibited growth of primary MM and H929 cells and prevented bone disease. These findings indicate that MM cells and osteoclasts are highly sensitive to NAD(+) depletion and that PBEF1 inhibition represents a novel approach to target cellular metabolism and inhibit PARP-1 and bone disease in MM.

Effect of oviductus ranae and oviductus ranae eggs on bone metabolism and osteoporosis.

OBJECTIVE: To evaluate the roles or effects of oviductus ranae (OR) or oviductus ranae eggs (ORE) in preventing and treating postmenopausal osteoporosis. METHODS: In vivo experiment: Sixty female adult Wistar rats were randomly divided into 5 groups of 12. To provide an osteoporosis model 4 groups of rats were ovariectomized (OVX), with the 5th being sham operated. Medication commenced 7 days after the operation and lasted continuously for 12 weeks. Sham operated and OVX groups were given equivalent volumes of 5% Tween-80. The other three groups intragastrically received conjugated estrogens (CE), OR or ORE of the corresponding doses. At the 12th week, serum estrogen, bone gla protein (BGP), serum calcium, phosphorus, and alkaline phosphatase (ALP) were assayed; bone mineral densities (BMD) were measured and bone scanning was conducted; uteri were weighed, and weight, volume and length of the femoral bones were determined; and cortical thickness of femoral heads and area of bone trabecula were measured by image analyzer. In vitro experiment: Eighty 10-month old SD rats, with equal numbers of males and females, were randomly divided into 8 groups. Osteoblasts were isolated from neonatal rat calvariae, and the cells were exposed to various concentrations of serum from OR and ORE groups to study the impact of these sera on osteoblastic proliferation, ALP activity and mineralization. Osteoclastic numbers were determined using tartrate resistant acid phosphatase (TRAP). RESULTS: In vivo experiment: The body weight of the four OVX groups increased significantly (P<0.01). Uterine weight of the CE group was the highest (P<0.01); Compared with the model group, estrogen level, BMD, bone scanning/bone imaging index weight of the femoral bones, cortical thickness of femoral heads in the OR and ORE groups increased significantly (P<0.05, P<0.01); femoral volume in the ORE group increased significantly (P<0.05); and the content of osteocalcin, phosphorus, and ALP in serum decreased significantly (P<0.05, P<0.01). In vitro experiment: Sera from OR and ORE groups had notable effects on the proliferation of osteoblasts (P<0.05 and P<0.01, repsectively) and stimulated the formation of calcium nodes (P<0.05, P<0.01), while the enhancement of ALP activity in osteoblasts was significant (P<0.05, P<0.01). The number of TRAP-positive cells was significantly reduced as well (P<0.01). CONCLUSIONS: OR and its eggs could effectively suppress OVX-induced osteoporosis in rats, and increase bone turnover possibly by both an increase in osteoblastic activity and a decrease in osteoclastic activity. The present study provides evidence that OR and its eggs could be considered a complementary and alternative medicine for the treatment of postmenopausal osteoporosis.

Grape-seed proanthocyanidin extract as suppressors of bone destruction in inflammatory autoimmune arthritis.

Chronic autoimmune inflammation, which is commonly observed in rheumatoid arthritis (RA), disrupts the delicate balance between bone resorption and formation causing thedestruction of the bone and joints. We undertook this study to verify the effects of natural grape-seed proanthocyanidin extract (GSPE), an antioxidant, on chronic inflammation and bone destruction. GSPE administration ameliorated the arthritic symptoms of collagen-induced arthritis (CIA), which are representative of cartilage and bone destruction. GSPE treatment reduced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and osteoclast activity and increased differentiation of mature osteoblasts. Receptor activator of NFκB ligand expression in fibroblasts from RA patients was abrogated with GSPE treatment. GSPE blocked human peripheral blood mononuclear cell-derived osteoclastogenesis and acted as an antioxidant. GSPE improved the arthritic manifestations of CIA mice by simultaneously suppressing osteoclast differentiation and promoting osteoblast differentiation. Our results suggest that GSPE may be beneficial for the treatment of inflammation-associated bone destruction.

V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption.

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.

Inhibition of epidermal growth factor receptor tyrosine kinase ameliorates collagen-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.

Piper sarmentosum prevents glucocorticoid-induced osteoporotic bone resorption by increasing 11ß-hydroxysteroid dehydrogenase type 1 activity.

AIMS: Osteoporosis is a proven complication of long-term glucocorticoid therapy. Concern on glucocorticoid induced osteoporosis has increased dramatically in recent years with the widespread use of synthetic glucocorticoids. Glucocorticoid action in bone depends upon the activity of 11ßhydroxysteroid dehydrogenase type 1 enzyme (11ßHSD1). This enzyme plays an important role in regulating corticosteroids by locally interconverting cortisone into active cortisol. This has been demonstrated in primary cultures of human, mouse or rat osteoblasts. Therefore, inhibition of this enzyme may reduce bone resorption markers. Piper sarmentosum (Ps) is a potent inhibitor of 11ßHSD1 in liver and adipose tissue. In this study we determined the effect of Ps on 11ßHSD1 activity in bones of glucocorticoid-induced osteoporotic rats. MATERIALS AND METHODS: Three-month old male Sprague-Dawley rats were adrenalectomised to remove the main source of circulating glucocorticoids. The animals were administered with dexamethasone 120 µg/kg body weight/day. Treatment with Ps 125 mg/kg body weight and glycirrhizic acid (GCA) 120 mg/kg body weight were given simultaneously. RESULTS: The results showed that Ps extract reduced plasma corticosterone concentration (1.05+0.02 µg/ml) and induced 11ßHSD1 dehydrogenase activity in bone (87.69+1.41%). Consequently, it also reduced the bone resorption marker, pyridinoline, in dexamethasone-treated adrenalectomised rats (2.07+0.62/L). Despite that, our data showed an inverse relationship between the plasma corticosterone level and the dehydrogenase activity of 11ßHSD1 in the bone. CONCLUSIONS: This suggests that 11ßHSD1 acts as the local regulator of glucocorticoid and its activity in bone was not correlated to systemic corticosterone level.

Elevated cytokine production restores bone resorption by human Btk-deficient osteoclasts.

Mutations in Bruton's tyrosine kinase (Btk) cause the B-cell disorder X-linked agammaglobulinaemia (XLA) in humans, but the effect of Btk deficiency in human bone health has not been investigated previously. In this study, we show that human Btk-deficient osteoclasts are defective at resorption activity in vitro owing to a dysregulation of the actin cytoskeletal function. Contrary to expectation, XLA patients did not exhibit increased bone density or alterations in serum markers of bone turnover, indicating that a potential compensation mechanism normalizes bone homeostasis. In contrast to the bone turnover markers, the levels of inflammatory cytokines interleukin 6 (IL-6), IL-1ß, and tumor necrosis factor α (TNF-α) were significantly elevated in XLA patients' serum compared with control individuals. Supplementation of osteoclast cultures from normal and XLA subjects with serum from XLA patients or recombinant inflammatory cytokines IL-6, IL-1ß, and TNF-α resulted in a stimulation of osteoclast activity in vitro, whereas the addition of cytokine-neutralizing antibodies inhibited this stimulatory effect, confirming that elevated inflammatory cytokines in XLA serum heightened osteoclast activity in vitro. This study provides novel evidence that Btk signaling is crucial for optimal actin cytoskeletal organization and lacunar resorption in isolated osteoclasts. In XLA patients, however, these inherent osteoclast defects are corrected by increased inflammatory cytokine levels, restoring osteoclast activity and leading to the normalization of bone density.

Dietary alpha lipoic acid supplementation prevents synovial inflammation and bone destruction in collagen-induced arthritic mice.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by chronic inflammation and joint destruction. In this study, we investigated whether dietary supplementation with alpha lipoic acid (ALA) suppresses collagen-induced arthritis (CIA) in mice. Mice were randomly divided into three groups: (1) a control CIA group was fed a normal diet, (2) a CIA group was fed a 0.1% ALA diet (average ALA intake of 160 mg/kg/day), and (3) a CIA group was fed a 0.5% ALA diet (average ALA intake of 800 mg/kg/day). The ALA-fed mice showed a decreased incidence and severity of arthritis compared to the normal diet group. Radiographic findings revealed a dramatic decrease in bone destruction, and histological findings showed extensively suppressed pathological changes in the ALA-fed mice. The ALA-fed mice exhibited inhibited generation of tartrate resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Additionally, ALA-fed mice reduced production of various proinflammatory cytokines and the soluble receptor activator of NF-κB ligand (sRANKL) in the joint tissues and the sera. In conclusion, dietary supplementation with ALA attenuated inflammatory responses and bone destruction in CIA mice.

Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogen-activated protein kinase: preclinical-to-clinical translation.

Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.

Role of MKP-1 in osteoclasts and bone homeostasis.

Bone mass is maintained through the complementary activities of osteoblasts and osteoclasts; yet differentiation of either osteoblasts and osteoclasts engages the mitogen-activated protein kinase (MAPK) pathway. The MAPKs are negatively regulated by a family of dual-specificity phosphatases known as the MAPK phosphatases (MKPs). MKP-1 is a stress-responsive MKP that inactivates the MAPKs and plays a central role in macrophages; however, whether MKP-1 plays a role in the maintenance of bone mass has yet to be investigated. We show here, using a genetic approach, that mkp-1(-/-) female mice exhibited slightly reduced bone mass. We found that mkp-1(+/+) and mkp-1(-/-) mice had equivalent levels of bone loss after ovariectomy despite mkp-1(-/-) mice having fewer osteoclasts, suggesting that mkp-1(-/-) osteoclasts are hyperactive. Indeed, deletion of MKP1 led to a profound activation of osteoclasts in vivo in response to local lipopolysaccharide (LPS) injection. These results suggest a role for MKP-1 in osteoclasts, which originate from the fusion of macrophages. In support of these observations, receptor activator for nuclear factor-kappaB ligand induced the expression for MKP-1, and osteoclasts derived from mkp-1(-/-) mice had increased resorptive activity. Finally, receptor activator of nuclear factor-kappaB ligand-induced p38 MAPK and c-Jun NH2-terminal kinase activities were enhanced in osteoclasts derived from mkp-1(-/-) mice. Taken together, these results show that MKP-1 plays a role in the maintenance of bone mass and does so by negatively regulating MAPK-dependent osteoclast signaling.

Effects of low-level laser therapy on proliferation and differentiation of murine bone marrow cells into osteoblasts and osteoclasts.

BACKGROUND AND OBJECTIVE: Low-Level Laser Therapy (LLLT) has been suggested to improve bone tissue healing. The cellular and molecular mechanisms involved in this effect are still unclear but bone cell proliferation and differentiation alteration have been proposed. The aim of the present study was to investigate, in vitro, the effect of LLLT on bone cell proliferation, osteoblastic and osteoclastic differentiation, both involved in bone remodeling and regeneration. STUDY DESIGN/MATERIALS AND METHODS: Murine bone marrow cells, which contain both osteoblast and osteoclast progenitors, were cultured and induced to differentiate in the absence or in the presence of LLLT. Laser exposition parameters were determined using a powermeter and consisted in an 808 nm infrared wavelength laser light in continuous mode, with an energy density of 4 J/cm(2) administered three times a week. Cell proliferation and differentiation were assessed after specific staining and microscopic analysis of the cultures after various times, as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteoclast markers after nucleic acid extraction. RESULTS: The use of a powermeter revealed that the power emitted by the optical fiber of the laser device was markedly reduced compared to the displayed power. This allowed to adjust the LLLT parameters to a final energy density exposure of 4 J/cm(2). In these conditions, proliferation of bone marrow mesenchymal stem cells as well as osteoclast or osteoblast differentiation of the corresponding progenitors were found similar in control and LLLT conditions. CONCLUSION: Using the present experimental protocol, we concluded that an 808 nm wavelength infrared LLLT does not alter murine bone progenitor cell proliferation and differentiation. Moreover our results confirm the necessary use of a powermeter to fix LLLT protocol parameters.

Effects of the drug (BSZGC)-containing serum on proliferation of rat's osteoclasts and TRACP activity in vitro.

OBJECTIVE: To observe effects of the drug-containing serum of Bu Shen Zhuang Gu Capsule (BSZGC Capsule for Tonifying the Kidney to Strengthen the Bones) on proliferation of the rat's osteoclasts and tartrate-resistant acid phosphatase (TRACP) activity in vitro so as to delve into the mechanisms of its preventive and therapeutic actions on osteoporosis. METHODS: Forty female Sprague-Dawley rats aged three months were randomly divided into high dosage BSZGC group, medium dosage BSZGC group, low dosage BSZGC group, and the control group. BSZGC was orally administered into the rats of high, medium, and low dosage groups at different dosages for 12 days, and isometric normal saline was orally administered to the rats of the control group. The drug-containing serum and control serum were prepared. Osteoclasts isolated mechanically from the femur and tibia of Sprague-Dawley rats aged one week were cultured with medium added with different drug-containing sera and control serum. The growth of osteoclasts was observed under an inverted phase-contrast microscope, and optic density (OD) value of osteoclasts was determined by MTT colorimetric assay. TRACP activity was measured by the diazol method. RESULTS: OD value of osteoclasts in the high dosage drug-containing serum group, medium dosage drug-containing serum group, and low dosage drug-containing serum group was significantly lower than that in the control serum group (P < 0.05) with a dose-effect correlation. TRACP activity in high dosage drug-containing serum group, medium dosage drug-containing serum group, low dosage drug-containing serum group was significantly lower than that of the control serum group (P < 0.01), and no significant differences in TRACP activity were not found among the different dosages drug-containing serum groups. CONCLUSIONS: BSZGC can inhibit the proliferation of the osteoclasts and reduce TRACP activity, which may be one of the mechanisms of its preventive and therapeutic actions on osteoporosis.