Treatment Of Bone Loss Diseases Research Articles

Exploring the role and mechanism of Fuzi decoction in the treatment of osteoporosis by integrating network pharmacology and experimental verification

BackgroundFuzi decoction (FZD), a traditional Chinese medicine formula, was used to treat musculoskeletal diseases by warming channels, strengthening yang and dispelling pathogenic cold and dampness. In clinical practice, FZD has been used to treat rheumatoid arthritis and osteoarthritis. It alleviated osteoarticular disorders through ameliorating the degradation of cartilage and improving meniscal damage in osteoarthritis, while its roles and mechanisms in the treatment of bone loss diseases remain unclear. This study aims to investigate the underlying mechanisms of FZD in treating osteoporosis using an integrative method of network pharmacology and experimental study.MethodsIn this study, network pharmacology was used to predict the core targets and potential pathways of the bioactive ingredients of FZD to attenuate osteoporosis. Molecular docking was performed to evaluate the interactions between core compounds and key targets. In addition, both cell and animal experiments were carried out to validate the role and potential mechanism in treating osteoporosis.ResultsIn the present study, data revealed that kaempferol, beta-sitosterol, stigmasterol, fumarine, and (+)-catechin may be the primary bioactive ingredients of FZD in the treatment of osteoporosis, which were closely associated with the osteoporosis-related targets. And the KEGG results indicated that the NF-κB pathway was closely associated with the function of FZD in treating osteoporosis. In addition, in vivo demonstrated that FZD ameliorated osteoporosis. In vitro experiments showed that the pro-apoptotic factors indicators including CASP3 and BAX were decreased by FZD and the anti-apoptotic factor BCL2 was increased by FZD. In addition, FZD significantly suppressed the osteoclast differentiation in culture and the expression levels of osteoclast-related genes including TRAF6, CTSK, and MMP9. And the NF-κB pathway was confirmed, via in vitro experiment, to be involved in osteoclast differentiation.ConclusionsThis study demonstrated that FZD played a pivotal role in suppressing the osteoclast differentiation via regulating the NF-κB pathway, indicating that FZD could be a promising antiosteoporosis drug and deserve further investigation.

Dencichine prevents ovariectomy-induced bone loss and inhibits osteoclastogenesis by inhibiting RANKL-associated NF-κB and MAPK signaling pathways

AimsTo investigate the effect of dencichine on osteoclastogenesis in vivo and in vitro. MethodsRANKL-induced osteoclastogenesis were treated with different concentrations of dencichine. Pit forming assays were applied to evaluate the degree of bone resorption. Osteoclastogenic markers were detected by real-time quantitative PCR (RT-qPCR) and Western blot. Micro CT was conducted to investigate the effects of dencichine on osteoclastogenesis in ovariectomized (OVX) mice. ResultsDencichine suppressed osteoclastogenesis through the inhibition of phosphorylation of p65, p50 (NF-κB pathway), p38, ERK and JNK (MAPKs pathway) in vitro. Furthermore, dencichine inhibited the function of osteoclasts in a dose-dependent manner. In addition, the expression levels of the nuclear factor of activated T cells 1 (NFATc1) and osteoclastogenesis markers were decreased by dencichine, including MMP-9, Cathepsin K (CTSK), Tartrate-Resistant Acid Phosphatase (TRAP), C-FOS, dendritic cell specific transmembrane protein (DC-STAMP). In vivo data proved that dencichine alleviated ovariectomy-induced bone loss and osteoclastogenesis in mice. ConclusionOur results demonstrate that dencichine alleviates OVX-induced bone loss in mice and inhibits RANKL-mediated osteoclastogenesis via inhibition of NF-κB and MAPK pathways in vitro, suggesting that dencichine might serve as a promising candidate for treatment of bone loss diseases, including PMOP and rheumatoid arthritis.

Study of Intercellular Adhesion Molecule-1 (ICAM-1) in Bone Homeostasis.

Although studies have established the role of integrins in bone homeostasis, especially in osteoclastogenesis, and these molecules are novel and promising therapeutic drug targets for bone loss diseases, such as: osteolysis, the cellular mechanism still elusive. Bone homeostasis takes place through the interaction of bone cells (osteoblasts and osteoclast) via the activation of intercellular adhesion molecule-1 (ICAM-1), which is a critical submolecule of integrin. In the present study, we reviewed several novel studies on integrins and their submolecule, ICAM-1, in bone homeostasis. In order to demonstrated that ICAM-1 might exert dual effects on osteoclastogenesis by directly affecting the adhesive ability of mature osteoclasts and indirectly participating in RANKL/RANK induced osteoclastic precursors differentiation. Although these results still need to be verified in the future, the extending study about the role of ICAM-1 in osteoclastogenesis will cerntainly provide a promising therapeutic target for the treatment of bone loss diseases.

The effects of tranylcypromine on osteoclastogenesis in vitro and in vivo.

Identification of anti-osteoclastogenic agents is important for the treatment of bone loss diseases that feature excessive osteoclast (OC) activity and bone resorption. Tranylcypromine (TCP), an irreversible inhibitor of monoamine oxidase (MAO), has been used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders. TCP has been discovered to exert anabolic effect on osteoblasts, and MAO-A has also been verified as an important mediator in prostate cancer cells to accelerate osteoclastogenesis. In current study, we were focused on TCP and MAO-A effects on osteoclastogenesis. As illustrated by tartrate-resistant acid phosphatase staining, TCP was capable of inhibiting osteoclastogenesis induced by receptor activators of the NF-κB ligand (RANKL) in bone marrow-derived macrophage cells without any cytotoxicity. It was also shown to effectively suppress bone resorption of OCs. The subsequent study revealed that TCP inhibited osteoclastogenesis-related genes in a time-dependent manner through protein kinase B (AKT)-mediated mechanism followed by the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1)-c-fos pathway. And TCP could overcome the osteoclastogenic effects of AKT activator SC79. In addition, our results indicated that the expression and catalytic activity of MAO-A were up-regulated by RANKL stimulation and down-regulated by TCP in vitro and in vivo. Furthermore, the effects of MAO-A knockdown on OC differentiation indicated that MAO-A played an important role in osteoclastogenesis in vitro and might contribute to the inhibitory effects of TCP. And AKT, NFATc1, and c-fos were involved in the MAO-A pathway. Notably, our in vivo study reflected that TCPs were capable of restoring the bone loss in LPS-induced calvaria osteolysis and estrogen deficiency-induced osteoporosis models. Thus, our current work provided a potential option for the treatment of bone loss diseases and highlighted the important role of MAO-A in osteoclastogenesis as well.-Liu, Z., Yang, K., Yan, X., Wang, T., Jiang, T., Zhou, Q., Qi, J., Qian, N., Zhou, H., Chen, B., Huang, P., Guo, L., Zhang, X., Xu, X., Jiang, M., Deng, L. The effects of tranylcypromine on osteoclastogenesis in vitro and in vivo.

Pyk2 deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene.

Bone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17β-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor β (ERβ). As a consequence, E2 signaling via ERβ was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERβ signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases.

MicroRNA‐338‐3p Regulates Osteogenic Differentiation of Mouse Bone Marrow Stromal Stem Cells by Targeting Runx2 and Fgfr2

The elucidation of the molecular mechanisms that govern the differentiation of bone marrow stromal cells (BMSCs) could provide new insight into the treatment of bone loss diseases. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression during stem cell growth, proliferation, and differentiation. In the present study, we showed that miR-338-3p expression was significantly down-regulated during the osteoblastic differentiation of BMSCs. Additionally, miR-338-3p was up-regulated in ovariectomized (OVX) mice compared with sham mice. An in vitro analysis revealed that the over-expression of miR-338-3p can inhibit the expression of osteoblast differentiation markers such as Osterix (Osx), thus reducing osteoblast differentiation. Bioinformatic analysis and dual luciferase assays confirmed that miR-338-3p can repress gene expression by targeting Runx2 and Fgfr2. In the BMSCs derived from OVX mice, the inhibition of miR-338-3p partially rescued mineralization and osteoblast differentiation. Taken together, our data show that miR-338-3p plays an important role during osteoblast differentiation of BMSCs and serves as a potential modulator of osteoporosis via its effect on osteoblasts.

Comparative “in vitro” evaluation of the antiresorptive activity residing in four Ayurvedic medicinal plants. Hemidesmus indicus emerges for its potential in the treatment of bone loss diseases

Ethnopharmacological relevanceFour Indian plants, traditionally used in Ayurvedic medicine: Asparagus racemosus Willd., Emblica officinalis Gaertn., Hemidesmus indicus R. Br., and Rubia cordifolia L. were selected on the basis of their ethnobotanical use and of scientific evidence that suggests a potential efficacy in the treatment of bone-loss diseases. The antiresorptive properties of the four plants have been investigated. The aim was to provide adequate evidence for the exploitation of natural compounds as alternative therapeutics for the treatment of diseases caused by increased osteoclast activity. Materials and methodsDecoctions were prepared from dried plant material according to the traditional procedure and standardization by HPLC was performed using marker compounds for each species. Total polyphenols, flavonoids and radical scavenging activity of the decoctions were also determined. The bioactivity of the plant decoctions was evaluated in subsequent phases. (1) A cytotoxicity screening was performed on the mouse monocytic RAW 264.7 cell line to define the concentrations that could be utilized in the following step. (2) The antiresorptive properties of plant decoctions were compared with that of a “gold standard” drug (alendronate) by measuring osteoclastogenesis inhibition and osteoclast apoptosis. (3) The toxic effect on bone forming cells was excluded by evaluating the impact on the proliferation of osteogenic precursors (mesenchymal stem cells, MSC). ResultsAll the decoctions inhibited osteoclastogenesis similarly to alendronate at the highest doses, but Hemidesmus indicus and Rubia cordifolia were also effective at lower concentrations. Apoptosis increased significantly when cells were exposed to the highest concentration of Emblica officinalis, Hemidesmus indicus, and Rubia cordifolia. All concentrations of Emblica officinalis tested inhibited the proliferation of osteogenic precursors, while only the highest doses of Asparagus racemosus and Rubia cordifolia were toxic. On the contrary, Hemidesmus indicus did not affect osteogenic precursor growth at any concentration tested. ConclusionAmong the medicinal plants included in the study, Hemidesmus indicus showed the greatest antiosteoclastic activity without toxic effect on osteogenic precursors. Therefore, Hemidesmus indicus exhibits the properties of an antiresorptive drug and represents the ideal candidate for further clinical investigations.

Crystalline Calcium Alendronate Obtained by Octacalcium Phosphate Digestion: A New Chance for Local Treatment of Bone Loss Diseases?

Octacalcium phosphate (OCP) interaction with alendronate (AL) solution results in the complete digestion of OCP: calcium ion is recruited by the bisphosphonate to yield quantitative precipitation of crystalline calcium alendronate monohydrate. This compound improves osteoblast differentiation and inhibits osteoclast proliferation and activity, both alone and, even more, in combination with OCP.

Pre-clinical employment of the heparin binding domain of PRELP in the treatment of bone loss diseases

Pre-clinical employment of the heparin binding domain of PRELP in the treatment of bone loss diseases

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Vacuolar H(+)-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC(50) of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.