吴茱萸碱抑制破骨细胞分化延缓骨丢失的研究

目的探讨吴茱萸碱对破骨细胞分化与骨吸收功能的调控及对骨质疏松症的治疗作用。 方法取小鼠原代骨髓来源巨噬细胞分别给予0、10、20、50、100、200 μmol/L吴茱萸碱处理,CCK8检测细胞活力;然后利用原代骨髓来源巨噬细胞给予小鼠重组可溶性核因子κB受体活化因子配体与集落刺激因子行破骨细胞分化诱导,分别给予20与50 μmol/L吴茱萸碱干预。抗酒石酸酸性磷酸酶（TRAP）染色检测破骨细胞形成能力,荧光定量PCR分析破骨细胞分化相关基因表达,免疫荧光检测F肌动蛋白（F-actin）形成,扫描电镜观察破骨细胞骨吸收能力。7月龄C57BL/6小鼠灌胃给予100与200 mg/kg吴茱萸碱,给药3个月后Micro-CT检测小鼠骨密度与骨质量。采用单因素方差分析和t检验进行统计学分析。 结果CCK8结果显示,与对照组相比,给予10、20、50、100 μmol/L吴茱萸碱处理后细胞活力无明显变化,差异无统计学意义（P > 0.05）;而给予200 μmol/L吴茱萸碱的细胞活力下降（100.64±0.18比47.54±5.58）,差异具有统计学意义（P < 0.01）。与对照组相比,20 μmol/L吴茱萸碱的TRAP染色阳性细胞数[（200.57±28.35）比（142.29±19.21）个]、Trap （1.00±0.13比0.55±0.16）、组织蛋白酶K（Ctsk） （1.01±0.17比0.59±0.11）mRNA水平、骨吸收面积比（1.00±0.15比0.79±0.19）均减少,差异有统计学意义（P < 0.05）。与对照组相比,50 μmol/L吴茱萸碱的TRAP阳性细胞数[（200.57±28.35）比（112.71±12.18）个]、Trap （1.00±0.13比0.46±0.17）、Ctsk（1.01±0.17比0.49±0.12）、树突状细胞-特异性跨膜蛋白（DC- Stamp） （1.00±0.10比0.55±0.14）、c-Fos （1.01±0.10比0.58±0.14）、活化T细胞核因子c1 （Nfatc1） （1.00±0.10比0.59±0.14）、H+转运ATP酶v0亚基d2 （Atp6v0d2）的mRNA表达（1.00±0.10比0.59±0.18）、F-actin数量[（165.00± 18.50）比（98.33±21.15）个]和骨吸收面积比（1.00±0.15比0.62±0.10）均降低,差异有统计学意义（P < 0.05）。Micro-CT结果显示,与生理盐水组相比,100 mg/kg吴茱萸碱组小鼠骨密度有一定升高[（0.19±0.03）比（0.21±0.01）g/cm³],但差异无统计学意义（P > 0.05）;与生理盐水组相比,200 mg/kg吴茱萸碱组小鼠胫骨的骨密度[（0.19±0.03）比（0.23±0.01）g/cm³]、骨体积比[（9.79±1.39）﹪比（11.62±1.18）﹪]、骨小梁数量[（2.43±0.29）比（3.08±0.43）/mm]上升,骨小梁分离度[（0.44±0.06）比（0.27±0.05）mm]下降,差异具有统计学意义（P < 0.05）。 结论吴茱萸碱通过抑制破骨细胞分化与骨吸收功能延缓小鼠骨量丢失。     

Apoptotic osteocytes regulate osteoclast precursor recruitment and differentiation in vitro

Fatigue loading causes a spatial distribution of osteocyte apoptosis co-localized with bone resorption spaces peaking around microdamage sites. Since osteocytes have been shown to regulate osteoclast formation and activity, we hypothesize that osteocyte apoptosis regulates osteoclastogenesis. In this study, we used serum-starvation to mimic reduced nutrient transport in microdamaged bone and induce apoptosis in MLO-Y4 osteocyte-like cells; conditioned medium was used to apply soluble factors released by apoptotic osteocytes (aOCY) to healthy non-apoptotic MLO-Y4 cells. Osteoclast precursor (RAW264.7 monocyte) migration and differentiation were assessed in the presence of conditioned media (CM) from: (A) aOCY, (B) osteocytes treated with apoptosis conditioned medium (i.e., healthy osteocytes in the presence of apoptosis cues; apoptosis CM-treated osteocytes (atOCY)), and (C) osteocytes treated with non-apoptosis conditioned medium (i.e., healthy osteocytes in the absence of apoptosis cues; non-apoptosis CM-treated osteocytes (natOCY)). Receptor activator for nuclear factor-κB ligand (RANKL), macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEGF) and osteoprotegerin (OPG) mRNA, and protein expression were measured. Our findings indicate that soluble factors released by aOCY and atOCY promoted osteoclast precursor migration (up to 64% and 24% increase, respectively) and osteoclast formation (up to 450% and 265% increase, respectively). Osteoclast size increased up to 233% in the presence of aOCY and atOCY CM. Recruitment, formation and size were unaltered by natOCY. RANKL mRNA and protein expression were upregulated only in aOCY, while M-CSF and VEGF increased in atOCY. Addition of RANKL-blocking antibody abolished aOCY-induced osteoclast precursor migration and osteoclast formation. VEGF and M-CSF blocking antibodies abolished atOCY-induced osteoclastogenesis. These findings suggest that aOCY directly and indirectly (through atOCY) initiate targeted bone resorption by regulating osteoclast precursor recruitment and differentiation.     

Vascular endothelial growth factor receptors in osteoclast differentiation and function

Osteoclasts are derived from haematopoietic stem cell precursors of the monocyte/macrophage cell lineage, through interaction with factors that are believed to include M-CSF and RANKL. VEGF is a proangiogenic cytokine that has been shown to promote osteoclast differentiation and survival. In this study, we assessed the role of VEGF and its receptors in osteoclastogenesis, in vitro, by culturing osteoclast precursors in the presence of VEGF, VEGF receptor-specific ligands, and blocking antibodies to VEGF receptors. Activation of VEGFR1 in the presence of RANKL induces osteoclast differentiation. Stimulating the receptors individually induced increased resorption by osteoclasts compared to controls but not to the level observed when stimulating both receptors simultaneously. We have shown that VEGF induces osteoclast differentiation through its action on VEGFR1. The way in which VEGF mediates its effect on mature osteoclast activity, however, may be through its interaction with both receptor subtypes.     

The effects of luteolin on osteoclast differentiation, function in vitro and ovariectomy-induced bone loss

Flavonoids, a group of polyphenolic compounds abundant in plants, are known to prevent bone loss in ovariectomized (OVX) animal models. Inhibition of osteoclast differentiation and bone resorption is considered as an effective therapeutic approach in the treatment of postmenopausal bone loss. Luteolin, a plant flavonoid, has potent anti-inflammatory properties both in vivo and vitro. In this study, we found that luteolin markedly decreased the differentiation of both bone marrow mononuclear cells and Raw264.7 cells into osteoclasts. Luteolin also inhibited the bone resorptive activity of differentiated osteoclasts. We further investigated the effects of luteolin on ovariectomy-induced bone loss using micro-computed tomography, biomechanical tests and serum markers assay for bone remodeling. Oral administration of luteolin (5 and 20 mg/kg per day) to OVX mice caused significant increase in bone mineral density and bone mineral content of trabecular and cortical bones in the femur as compared to those of OVX controls, and prevented decreases of bone strength indexes induced by OVX surgery. Serum biochemical markers assays revealed that luteolin prevents OVX-induced increases in bone turnover. These data strongly suggest that luteolin has the potential for prevention of bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function.     

Flavonoids isolated from Tridax procumbens (TPF) inhibit osteoclasts differentiation and bone resorption

Background

The Tridax procumbens flavonoids (TPF), are well known for their medicinal properties among local natives. The TPF are traditionally used for dropsy, anaemia, arthritis, gout, asthma, ulcer, piles, and urinary problems. It also used in treating gastric problems, body pain, and rheumatic pains of joints. The TPF have been reported to increase osteogenic functioning in mesenchymal stem cells. However, their effects on osteoclastogenesis remain unclear. The TPF isolated from T. procumbens and investigated the effects of the TPF inhibit on osteoclast differentiation and bone resorption activities using primary osteoclastic cells. Osteoclast formation was assessed by counting the number of tartrate resistant acid phosphatase (TRAP) positive multinucleated cells and by measuring both TRAP activities.

Results

The TPF significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in primary osteoclastic cells. The TPF also decreased the expression of mRNAs related to osteoclast differentiation, including Trap, Cathepsin K, Mmp-9, and Mmp-13 in primary osteoclastic cells. The treatment of primary osteoclastic cells with the TPF decreased Cathepsin K, Mmp-9, and Mmp-13 proteins expression in primary osteoclastic cells.

Conclusion

These results indicated that TPF inhibit osteoclastogenesis and pits formation activities. Our results suggest that the TPF could be a potential anti-bone resorptic agent to treat patients with bone loss-associated diseases such as osteoporosis.     

Osteoclast receptors and signaling

Osteoclasts are bone-resorbing cells derived from hematopoietic precursors of the monocyte-macrophage lineage. Besides the well known Receptor Activator of Nuclear factor-κB (RANK), RANK ligand and osteoprotegerin axis, a variety of factors tightly regulate osteoclast formation, adhesion, polarization, motility, resorbing activity and life span, maintaining bone resorption within physiological ranges. Receptor-mediated osteoclast regulation is rather complex. Nuclear receptors, cell surface receptors, integrin receptors and cell death receptors work together to control osteoclast activity and prevent both reduced or increased bone resorption. Here we will discuss the signal transduction pathways activated by the main osteoclast receptors, integrating their function and mechanisms of action.     

Cajaninstilbene acid inhibits osteoporosis through suppressing osteoclast formation and RANKL-induced signaling pathways

Osteoporosis is a form of osteolytic disease caused by an imbalance in bone homeostasis, with reductions in osteoblast bone formation, and augmented osteoclast formation and resorption resulting in reduced bone mass. Cajaninstilbene acid (CSA) is a natural compound derived from pigeon pea leaves. CSA possesses beneficial properties as an anti-inflammatory, antibacterial, antihepatitis, and anticancer agent; however, its potential to modulate bone homeostasis and osteoporosis has not been studied. We observed that CSA has the ability to suppress RANKL-mediated osteoclastogenesis, osteoclast marker gene expression, and bone resorption in a dose-dependent manner. Mechanistically, it was revealed that CSA attenuates RANKL-activated NF-κB and nuclear factor of activated T-cell pathways and inhibited phosphorylation of key signaling mediators c-Fos, V-ATPase-d2, and ERK. Moreover, in osteoclasts, CSA blocked RANKL-induced ROS activity as well as calcium oscillations. We further evaluated the therapeutic effect of CSA in a preclinical mouse model and showed that in vivo treatment of ovariectomized C57BL/6 mice with CSA protects the mice from osteoporotic bone loss. Thus, this study demonstrates that osteolytic bone diseases can potentially be treated by CSA.     

Thyroid-stimulating hormone maintains bone mass and strength by suppressing osteoclast differentiation

It has been suggested that pituitary hormone might be associated with bone metabolism. To investigate the role of thyroid-stimulating hormone (TSH) in bone metabolism, we designed the present study as follows. After weaning, TSH receptor (TSHR) null mice (Tshr^−/−) were randomly divided into a thyroxine treatment group (n=10) or non-treatment group (n=10); the treatment group received a dose of desiccated thyroid extract at 100 ppm daily for 5 weeks. Age-matched wild-type (Tshr^+/+, n=10) and heterozygote mice (Tshr^+/−, n=10) served as controls. After 5 weeks, the animals were sacrificed, and the femurs were collected for histomorphometrical and biomechanical analyses. In addition, the effect of TSH on osteoclastogenesis was examined in the RAW264.7 osteoclast cell line. We found that compared with Tshr^+/+ mice, Tshr^−/− and Tshr^+/− mice had lower bone strength. The histomorphometric results showed that trabecular bone volume, osteoid surface, osteoid thickness and osteoblast surface were significantly decreased, whereas the osteoclast surface was significantly increased in both Tshr^−/− and Tshr^+/− mice compared with Tshr^+/+ mice. Bone resorption and formation in Tshr^−/− mice were further enhanced by thyroxine replacement. bTSH inhibited osteoclast differentiation in vitro, as demonstrated by reduced development of TRAP-positive cells and down-regulation of differentiation markers, including tartrate-resistant acid phosphatase, matrix metallo-proteinase-9 and cathepsin K in RAW264.7 cells. Our results confirm that TSH increased bone volume and improved bone microarchitecture and strength at least partly by inhibiting osteoclastogenesis.     

The 1,2,3-triazole derivative KP-A021 suppresses osteoclast differentiation and function by inhibiting RANKL-mediated MEK-ERK signaling pathway

The triazole family of compounds has been implicated in modulating various biological processes such as inflammation, tumorigenesis, and infection. To our knowledge, this is the first study to demonstrate the effects of 1,2,3-triazole substituted biarylacrylonitrile compounds, including KP-A021, on the differentiation and function of osteoclasts. KP-A021 and its triazole derivatives, at a concentration that does not cause a cytotoxic response in bone marrow macrophages (BMMs), significantly inhibited osteoclast differentiation induced by receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) as assessed by tartrate-resistant acid phosphatase (TRAP) staining. KP-A021 also dramatically inhibited the expression of marker genes associated with osteoclast differentiation, such as TRAP, cathepsin K (Cat K), dendritic cell-specific transmembrane protein (DC-STAMP), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). Furthermore, KP-A021 inhibited actin ring formation in osteoclasts as well as resorption pit formation induced by osteoclasts. Analysis of the signaling pathway for KP-A021 indicated that this triazole compound inhibited the RANKL-induced activation of extracellular signal-regulated kinase (ERK) and its upstream signaling molecule, mitogen-activated protein kinase kinase1/2 (MEK1/2). Taken together, these results demonstrate that KP-A021 has an inhibitory effect on the differentiation and function of osteoclasts via modulation of the RANKL-induced activation of the MEK-ERK pathway.     

Fibroblast growth factor receptor 1 regulates the differentiation and activation of osteoclasts through Erk1/2 pathway

To elucidate the direct role and mechanism of FGFR1 signaling in the differentiation and activation of osteoclasts, we conditionally inactivated FGFR1 in bone marrow monocytes and mature osteoclasts of mice. Mice deficient in FGFR1 (Fgfr1^−/−) exhibited misregulated bone remodeling with reduced osteoclast number and impaired osteoclast function. In vitro assay demonstrated that the number of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts derived from bone marrow monocytes of Fgfr1^−/− mice was significantly diminished. The bone resorption activity of mature osteoclasts derived from Fgfr1^−/− mice was also suppressed. Further analysis showed that the osteoclasts with FGFR1 deficiency exhibited downregulated expression of genes related to osteoclastic activity including TRAP and MMP-9. The phosphorylation of Erk1/2 mitogen-activated protein (MAP) kinase was also decreased. Our results suggest that FGFR1 is indispensable for complete differentiation and activation of osteoclasts in mice.     

Hippuric acid and 3-(3-hydroxyphenyl) propionic acid inhibit murine osteoclastogenesis through RANKL-RANK independent pathway

Nutritional factors influence bone development. Previous studies demonstrated that bone mass significantly increased with suppressed bone resorption in early life of rats fed with AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB) powder for 2 weeks. However, the effects of increased phenolic acids in animal serum due to this diet on bone and bone resorption were unclear. This in vitro and in ex vivo study examined the effects of phenolic hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on osteoclastic cell differentiation and bone resorption. We cultured murine osteoclast (macrophage) cell line, RAW 264.7 cells, and hematopoietic osteoclast progenitor cells (isolated from 4-week-old C57BL6/J mice) with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL). Morphologic studies showed decreased osteoclast number with treatment of 2.5% mouse serum from BB diet–fed animals compared with those treated with serum from standard casein diet–fed mice in both RAW 264.7 cell and primary cell cultures. HA and 3-3-PPA, but not 3–4-PPA, had dose-dependent suppressive effects on osteoclastogenesis and osteoclast resorptive activity in Corning osteo-assay plates. Signaling pathway analysis showed that after pretreatment with HA or 3-3-PPA, RANKL-stimulated increase of osteoclastogenic markers, such as nuclear factor of activated T-cells, cytoplasmic 1 and matrix metallopeptidase 9 gene/protein expression were blunted. Inhibitory effects of HA and 3-3-PPA on osteoclastogenesis utilized RANKL/RANK independent mediators. The study revealed that HA and 3-3-PPA significantly inhibited osteoclastogenesis and bone osteoclastic resorptive activity.     

Ebselen Is a Potential Anti-Osteoporosis Agent by Suppressing Receptor Activator of Nuclear Factor Kappa-B Ligand-Induced Osteoclast Differentiation In vitro and Lipopolysaccharide-Induced Inflammatory Bone Destruction In vivo

Ebselen is a non-toxic seleno-organic drug with anti-inflammatory and antioxidant properties that is currently being examined in clinical trials to prevent and treat various diseases, including atherosclerosis, stroke, and cancer. However, no reports are available for verifying the pharmacological effects of ebselen on major metabolic bone diseases such as osteoporosis. In this study, we observed that ebselen suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in an osteoblast/osteoclast co-culture by regulating the ratio of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin secreted by osteoblasts. In addition, ebselen treatment in the early stage of osteoclast differentiation inhibited RANKL-dependent osteoclastogenesis by decreasing the phosphorylation of IκB, PI3K, and Akt in early signaling pathways and by subsequently inducing c-Fos and nuclear factor of activated T-cells c1. Further, ebselen induced apoptosis of osteoclasts in the late stage of osteoclast differentiation. In addition, ebselen treatment suppressed filamentous actin ring formation and bone resorption activity of mature osteoclasts. Reflecting these in vitro effects, administration of ebselen recovered bone loss and its µ-CT parameters in lipopolysaccharide-mediated mouse model. Histological analysis confirmed that ebselen prevented trabecular bone matrix degradation and osteoclast formation in the bone tissues. Finally, it was proved that the anti-osteoclastogenic action of ebselen is achieved through targeting N-methyl-D-aspartate (NMDA) receptor. These results indicate that ebselen is a potentially safe drug for treating metabolic bone diseases such as osteoporosis.     

Protein tyrosine phosphatases in osteoclast differentiation, adhesion, and bone resorption

Osteoclasts are large cells derived from the monocyte-macrophage hematopoietic cell lineage. Their primary function is to degrade bone in various physiological contexts. Osteoclasts adhere to bone via podosomes, specialized adhesion structures whose structure and subcellular organization are affected by mechanical contact of the cell with bone matrix. Ample evidence indicates that reversible tyrosine phosphorylation of podosomal proteins plays a major role in determining the organization and dynamics of podosomes. Although roles of several tyrosine kinases are known in detail in this respect, little is known concerning the roles of protein tyrosine phosphatases (PTPs) in regulating osteoclast adhesion. Here we summarize available information concerning the known and hypothesized roles of the best-researched PTPs in osteoclasts - PTPRO, PTP epsilon, SHP-1, and PTP-PEST. Of these, PTPRO, PTP epsilon, and PTP-PEST appear to support osteoclast activity while SHP-1 inhibits it. Additional studies are required to provide full molecular details of the roles of these PTPs in regulating osteoclast adhesion, and to uncover additional PTPs that participate in this process.     

Jolkinolide B inhibits RANKL-induced osteoclastogenesis by suppressing the activation NF-κB and MAPK signaling pathways

Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. The unique function and ability of osteoclasts to resorb bone makes them critical in both normal bone homeostasis and pathologic bone diseases such as osteoporosis and rheumatoid arthritis. Thus, new compounds that may inhibit osteoclastogenesis and osteoclast function may be of great value in the treatment of osteoclast-related diseases. In the present study, we examined the effect of jolkinolide B (JB), isolated from the root of Euphorbia fischeriana Steud on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. We found that JB inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages (BMMs) without cytotoxicity. Furthermore, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), and calcitonin receptor (CTR), was significantly inhibited. JB inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκBα degradation. Moreover, JB inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases (p38, JNK, and ERK). This study thus identifies JB as an inhibitor of osteoclast formation and provides evidence that JB might be an alternative medicine for preventing and treating osteolysis.     

Excessive retinoic acid inhibit mouse embryonic palate mesenchymal cell growth through involvement of Smad signaling

All-trans retinoic acid (atRA), the oxidative metabolite of retinoic acid (RA), is essential for palatogenesis. Overdose RA is capable of inducing cleft palate in mice and humans. Normal embryonic palatal mesenchymal (EPM) cell growth is crucial for shelf growth. Smad signaling is involved in many biological processes. However, it is not much clear if atRA could affect Smad signaling during EPM cells growth. In this study, the timed pregnant mice with maternal administration of 100?mg/kg body weight of RA by gastric intubation were cervical dislocation executed to evaluate growth changes of palatal shelves by hematoxylin and eosin (H&E) staining. At the same time, a primary mouse EPM (MEPM) cell culture model was also established. MEPM cells were treated with atRA (0.1, 0.5, 1, 5 and 10?μM) for 24, 48 and 72?h. The results indicated that the sizes of the shelves were smaller than those in control. AtRA inhibited MEPM cell growth with both increasing concentration and increasing incubation time, especially at 72?h in vitro. Moreover, atRA significantly increased the mRNA and protein expression levels of Smad7 (P?<?.05), but the mRNA and protein expression levels of PCNA were reduced (P?<?.05). We also found atRA inhibited phosphorylation of Smad2 compared with untreated group (P?<?.05). However, the protein and mRNA levels of Smad2 did not change both in atRA-treated and untreated group (P?>?.05). We demonstrated that RA induced inhibition of MEPM cell growth that could cause cleft palate partly by down-regulation of Smad pathway.     

Inhibitory effect of organotin compounds on rat neuronal nitric oxide synthase through interaction with calmodulin

Organotin compounds, triphenyltin (TPT), tributyltin, dibutyltin, and monobutyltin (MBT), showed potent inhibitory effects on both L-arginine oxidation to nitric oxide and L-citrulline, and cytochrome c reduction catalyzed by recombinant rat neuronal nitric oxide synthase (nNOS). The two inhibitory effects were almost parallel. MBT and TPT showed the highest inhibitory effects, followed by tributyltin and dibutyltin; TPT and MBT showed inhibition constant (IC(50)) values of around 10microM. Cytochrome c reduction activity was markedly decreased by removal of calmodulin (CaM) from the complete mixture, and the decrease was similar to the extent of inhibition by TPT and MBT. The inhibitory effect of MBT on the cytochrome c reducing activity was rapidly attenuated upon dilution of the inhibitor, and addition of a high concentration of CaM reactivated the cytochrome c reduction activity inhibited by MBT. However, other cofactors such as FAD, FMN or tetrahydrobiopterin had no such ability. The inhibitory effect of organotin compounds (100microM) on L-arginine oxidation of nNOS almost vanished when the amount of CaM was sufficiently increased (150-300microM). It was confirmed by CaM-agarose column chromatography that the dissociation of nNOS-CaM complex was induced by organotin compounds. These results indicate that organotin compounds disturb the interaction between CaM and nNOS, thereby inhibiting electron transfer from the reductase domain to cytochrome c and the oxygenase domain.