Inhibits Osteoclast Differentiation Research Articles

Attenuative effects of collagen peptide from milkfish (Chanoschanos) scales on ovariectomy-induced osteoporosis.

Osteoporosis is characterized by low bone mass, bone microarchitecture disruption, and collagen loss, leading to increased fracture risk. In the current study, collagen peptides were extracted from milkfish scales (MS) to develop potential therapeutic candidates for osteoporosis. MS was used to synthesize a crude extract of fish scales (FS), collagen liquid (COL), and hydroxyapatite powder (HA). COL samples were further categorized according to the peptide size of total COL (0.1 mg/mL), COL < 1 kDa (0.1 mg/mL), COL: 1-10 kDa (0.1 mg/mL), and COL > 10 kDa (0.1 mg/mL) to determine it. Semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and immunofluorescence labeling were used to assess the expression levels of specific mRNA and proteins in vitro. For in vivo studies, mice ovariectomy (OVX)-induced postmenopausal osteoporosis were developed, while the sham surgery (Sham) group was treated as a control. Collagen peptides (CP) from MS inhibited osteoclast differentiation in RAW264.7 cells following an insult with nuclear factor kappa-B ligand (RANKL). CP also enhanced osteoblast proliferation in MG-63 cells, possibly through downregulating NFATc1 and TRAP mRNA expression and upregulating ALP and OPG mRNA levels. Furthermore, COL1 kDa also inhibited bone density loss in osteoporotic mice. Taken together, CP may reduce RANKL-induced osteoclast activity while promoting osteoblast synthesis, and therefore may act as a potential therapeutic agent for the prevention and control of osteoporosis.

Icaritin ameliorates RANKL-mediated osteoclastogenesis and ovariectomy-induced osteoporosis.

A rapidly aging society and longer life expectancy are causing osteoporosis to become a global epidemic. Over the last five decades, a number of drugs aimed at reducing bone resorption or restoring bone mass have been developed, but their efficacy and safety are limited. Icaritin (ICT) is a natural compound extracted from anti-osteoporosis herb Epimedium spp. and has been shown to inhibit osteoclast differentiation. However, the molecular mechanism by which ICT weaken RANKL-induced osteoclast differentiation has not been completely investigated. Here, we evaluated the anti-osteoclastogenic effect of ICT in vitro and the potential drug candidate for treating osteoporosis in vivo. In vitro study, ICT was found to inhibit osteoclast formation and bone resorption function via downregulating transcription factors activated T cell cytoplasm 1 (NFATc1) and c-fos, which further downregulate osteoclastogenesis-specific gene. In addition, the enhanced mitochondrial mass and function required for osteoclast differentiation was mitigated by ICT. The histomorphological results from an in vivo study showed that ICT attenuated the bone loss associated with ovariectomy (OVX). Based on these results, we propose ICT as a promising new drug strategy for osteoporosis that inhibits osteoclast differentiation.

Cabozantinib and IL-27 combinatorial therapy for bone-metastatic prostate cancer.

Introduction: Prostate cancer is the second leading cause of cancer-related death among American men. Prostate tumor cells exhibit significant tropism for the bone and once metastasis occurs, survival rates fall significantly. Current treatment options are not curative and focus on symptom management. Immunotherapies are rapidly emerging as a possible therapeutic option for a variety of cancers including prostate cancer, however, variable patient response remains a concern. Chemotherapies, like cabozantinib, can have immune-priming effects which sensitize tumors to immunotherapies. Additionally, lower doses of chemotherapy can be used in this context which can reduce patient side effects. We hypothesized that a combination of chemotherapy (cabozantinib) and immunotherapy [Interleukin-27 (IL-27)] could be used to treat bone-metastatic prostate cancer and exert pro-osteogenic effects. IL-27 is a multi-functional cytokine, which promotes immune cell recruitment to tumors, while also promoting bone repair. Methods: To test this hypothesis, in vivo experiments were performed where syngeneic C57BL/6J mice were implanted intratibially with TRAMP-C2ras-Luc cells that are able to form tumors in bone. Immunotherapy was administered in the form of intramuscular gene therapy, delivering plasmid DNA encoding a reporter gene (Lucia), and/or a therapeutic gene (IL-27). Sonoporation was used to aid gene delivery. Following immunotherapy, the animals received either cabozantinib or a vehicle control by oral gavage. Bioluminescence imaging was used to monitor tumor size over time. Results: Combinatorial therapy inhibited tumor growth and improved survival. Further, RNA sequencing was used to investigate the mechanisms involved. Microcomputed tomography and differentiation assays indicated that the combination therapy improved bone quality by enhancing osteoblast differentiation and inhibiting osteoclast differentiation. Discussion: Our conclusion is that a chemo-immunotherapy approach such as the one examined in this work has potential to emerge as a novel therapeutic strategy for treating bone-metastatic prostate cancer. This approach will enable a significant reduction in chemotherapy-associated toxicity, enhance sensitivity to immunotherapy, and improve bone quality.

Water Extract of Angelica dahurica Inhibits Osteoclast Differentiation and Bone Loss.

Angelica dahurica radix has a long history of traditional use in China and Korea for treating headaches, cold-damp pain and skin diseases. Despite various pharmacological studies on A. dahurica, its impact on bones remains unclear. Hence, this study investigated the inhibitory effect of A. dahurica's radix water extract (WEAD) on osteoclast differentiation. In vitro experiments showed that WEAD effectively suppresses osteoclast differentiation. Treatment of an osteoclast precursor with WEAD significantly suppressed the expression of nuclear factor of activated T-cells 1 (NFATc1), essential transcription factor for osteoclastogenesis, while increasing the expression of negative regulators, interferon regulatory factor 8 (Irf8) and v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB). Consistent with the in vitro findings, the oral administration of WEAD (100 and 300 mg/kg/day) to mice subjected to surgical ovariectomy for a duration of six weeks alleviated bone loss, while also mitigating weight gain and liver fat accumulation. In addition, we also identified phytochemicals present in WEAD, known to regulate osteoclastogenesis and/or bone loss. These results suggest the potential use of WEAD for treating various bone disorders caused by excessive bone resorption.

Lactate dehydrogenase A inhibition prevents RANKL-induced osteoclastogenesis by reducing enhanced glycolysis

Osteoclasts are multinucleated, specializes bone-resorbing cells that are derived from the monocyte/macrophage lineage. Excessive resorbing activities of osteoclasts are involved in destructive bone diseases. The detailed mechanism of acidification at the bone adhesion surface during the bone resorption process of osteoclasts remains to be defined. During glycolysis, pyruvate proceeds to the tricarboxylic cycle under aerobic conditions and pyruvate is converted to lactate via lactate dehydrogenase A (LDHA) under anaerobic conditions. However, tumor cells produce ATP during aerobic glycolysis and large amounts of pyruvate are converted to lactate and H+ by LDHA. Lactate and H+ are excreted outside the cell, whereby they are involved in invasion of tumor cells due to the pH drop around the cell. In this study, we focused on aerobic glycolysis and investigated the production of lactate by LDHA in osteoclasts. Expression of LDHA and monocarboxylate transporter 4 (MCT4) was upregulated during osteoclast differentiation. Intracellular and extracellular lactate levels increased with upregulation of LDHA and MCT4, respectively. FX11 (an LDHA inhibitor) inhibited osteoclast differentiation and suppressed the bone-resorbing activity of osteoclasts. We propose that inhibition of LDHA may represent a novel therapeutic strategy for controlling excessive bone resorption in osteoporosis and rheumatoid arthritis.

Inhibition of KIF11 ameliorates osteoclastogenesis via regulating mTORC1-mediated NF-κB signaling

Osteoporosis, characterized by over-production and activation of osteoclasts, has become a major health problem especially in elderly women. In our study, we first tested the effect of Caudatin (Cau) in osteoclastogenesis, which is separated from Cynanchum auriculatum as a species of C-21 steroidal glyosides. The results indicated that Cau suppressed osteoclastogenesis in a time- and dose-dependent manner in vitro. Mechanistically, Cau was identified to inhibit NF-κB signaling pathway via modulation of KIF11-mediated mTORC1 activity. In vivo, by establishing an ovariectomized (OVX) mouse model to mimic osteoporosis, we confirmed that Cau treatment prevented OVX-induced bone loss in mice. In conclusion, we demonstrated that Cau inhibited NF-κB signaling pathway via modulation of KIF11-mediated mTORC1 activity to suppress osteoclast differentiation in vitro as well as OVX-induced bone loss in vivo. This provides the possibility of a novel prospective drug for osteoporosis remedies.

Anti-resorption role of low-intensity pulsed ultrasound (LIPUS) during large-scale bone reconstruction using porous titanium alloy scaffolds through inhibiting osteoclast differentiation

BackgroundTi6Al4V biomaterials combine with low-intensity pulsed ultrasound (LIPUS) has been reported with great bone regeneration capacity. It is important to better understand how LIPUS benefits bone microenvironment to seek for target of therapeutic medicine. Osteoclast differentiation plays a crucial role in bone resorption. Recent advances in molecular biology have revealed that N6-methyladenosine (m6A) RNA modifications can modulate biological processes, but their role in bone biology, particularly in osteoclast differentiation, remains unclear. We aim to understand how LIPUS regulates bone microenvironment especially osteoclast formation during bone regeneration to provide new therapeutic options for preventing and delaying bone resorption, thus with better bone regeneration efficiency. Results1. LIPUS promoted bone ingrowth and bone maturity while inhibiting osteoclast formation within Ti6Al4V scaffolds in large-scale bone defect model. 2. LIPUS was found to inhibit osteoclast differentiation by decreasing the overall expression of osteoclast markers in vitro. 3. LIPUS decreases RNA m6A-modification level through upregulating FTO expression during osteoclast differentiation during. 4. Inhibiting FTO expression and function leads to less inhibition during osteoclast differentiation. ConclusionLIPUS suppresses osteoclast differentiation during bone regeneration through reducing m6A modification of osteoclastic RNAs by up regulating FTO expression.

Surface proteins of Propionibacterium freudenreichii MJ2 inhibit RANKL-induced osteoclast differentiation by lipocalin-2 upregulation and lipocalin-2-mediated NFATc1 inhibition

Osteoclasts degrade bone and osteoclast differentiation has been implicated in bone destruction in rheumatoid arthritis. The dairy bacterium Propionibacterium freudenreichii MJ2 (MJ2) isolated from raw milk inhibits osteoclast differentiation and ameliorates collagen-induced arthritis. This study aimed to investigate the inhibitory effect of the surface proteins of MJ2 on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and explain the underlying mechanism. The murine macrophage cell line RAW 264.7 was used to study the inhibition of osteoclast differentiation. The surface proteins significantly inhibited RANKL-induced osteoclast differentiation in a protein concentration-dependent manner by inhibiting the expression of genes and proteins related to osteoclast differentiation. RNA microarray analysis showed that the surface proteins significantly upregulated lipocalin-2 (lcn2) expression. In addition, they downregulated c-fos and NFATc1 and inhibited the expression of NFATc1-downstream genes Atp6v0d2, Calcr, and Ctsk. siRNA silencing of lcn2 decreased the extent of surface protein inhibition on osteoclast differentiation, suggesting that lcn2 plays an important role in the inhibition of RANKL-induced osteoclast differentiation. In conclusion, surface proteins of MJ2 show inhibitory effects on RANKL-induced osteoclast differentiation by upregulating lcn2 expression, in turn downregulating NFATc1, leading to the inhibition of NFATc1-downstream osteoclastogenesis-related gene expression.

A newly developed PLD1 inhibitor ameliorates rheumatoid arthritis by regulating pathogenic T and B cells and inhibiting osteoclast differentiation

Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays multiple roles in inflammation. We investigated the therapeutic effects of the newly developed PLD1 inhibitors A2998, A3000, and A3773 in vitro and in vivo rheumatoid arthritis (RA) model. A3373 reduced the levels of LPS-induced TNF-α, IL-6, and IgG in murine splenocytes in vitro. A3373 also decreased the levels of IFN-γ and IL-17 and the frequencies of Th1, Th17 cells and germinal-center B cells, in splenocytes in vitro. A3373 ameliorated the severity of collagen-induced arthritis (CIA) and suppressed infiltration of inflammatory cells into the joint tissues of mice with CIA compared with vehicle-treated mice. Moreover, A3373 prevented systemic bone demineralization in mice with CIA and suppressed osteoclast differentiation and the mRNA levels of osteoclastogenesis markers in vitro. These results suggest that A3373 has therapeutic potential for RA.

Euonymus alatus (Thunb.) Siebold Prevents Osteoclast Differentiation and Osteoporosis.

Euonymus alatus (Thunb.) Siebold, a traditional medicinal plant, has been used in China and several other Asian countries to address a variety of health concerns. The extensive research conducted on E. alatus is driven by its diverse pharmacological applications. However, its biological effects on osteoclastogenesis and osteoporosis have not been previously studied. In this research, we investigated the impact of an ethanolic extract of E. alatus (EEEA) on osteoclast differentiation and function as well as estrogen deficiency-induced bone loss. We found that EEEA inhibits osteoclast differentiation by downregulating the expression of the receptor activator of nuclear factor-κB ligand (RANKL) in osteoclast-supporting cells and by directly impeding RANKL-mediated signaling pathways for osteoclastogenesis in precursor cells. In addition, EEEA inhibited the bone-resorptive function of mature osteoclasts in vitro. Furthermore, oral administration of EEEA significantly alleviated bone loss in an ovariectomy-induced osteoporosis mouse model. Additionally, we identified phytochemicals in EEEA that have suppressive effects on osteoclast differentiation and bone loss. Collectively, these results suggest that EEEA holds potential as a biotherapeutic candidate for anti-postmenopausal osteoporosis.

Anemoside B4 attenuates RANKL-induced osteoclastogenesis by upregulating Nrf2 and dampens ovariectomy-induced bone loss

Increased numbers and functional overactivity of osteoclasts are the pathological basis for bone loss diseases such as osteoporosis, which are characterized by cortical bone thinning, decreased trabecular bone quantity, and reduced bone mineral density. Effective inhibition of osteoclast formation and bone resorption are important means of treating such skeletal diseases. Anemoside B4 (AB4), the main active component of Pulsatilla chinensis, possesses a wide range of anti-inflammatory and immunoregulatory effects. However, its effect and mechanism in osteoclast differentiation remain unclear. In this study, we found through tartrate-resistant acidic phosphatase (TRAcP) staining and immunofluorescence staining that AB4 inhibited the differentiation, fusion, and bone-resorption functions of osteoclasts induced by receptor activator of nuclear factor κB ligand (RANKL) in vitro. Additionally, real time PCR (RT-qPCR) and western blot analysis showed AB4 downregulated the expression of osteoclast marker genes, including Nfatc1, Fos, and Ctsk, while upregulating Nrf2 expression. AB4 (5 mg/kg) alleviated bone loss in ovariectomized mice by inhibiting osteoclast formation. Furthermore, the knockout of Nrf2 weakened the inhibitory effects of AB4 on osteoclast formation and related gene expression. In summary, the results suggest AB4 can inhibit osteoclast differentiation and function by activating Nrf2 and indicate AB4 may be a candidate drug for osteoporosis.

The role of prostate-specific antigen in the osteoblastic bone metastasis of prostate cancer: a literature review.

Prostate cancer is the only human malignancy that generates predominantly osteoblastic bone metastases, and osteoblastic bone metastases account for more than 90% of osseous metastases of prostate cancer. Prostate-specific antigen (PSA) plays an important role in the osteoblastic bone metastasis of prostate cancer, which can promote osteomimicry of prostate cancer cells, suppress osteoclast differentiation, and facilitate osteoblast proliferation and activation at metastatic sites. In the meantime, it can activate osteogenic factors, including insulin-like growth factor, transforming growth factor β2 and urokinase-type plasminogen activator, and meanwhile suppress osteolytic factors such as parathyroid hormone-related protein. To recapitulate, PSA plays a significant role in the osteoblastic predominance of prostate cancer bone metastasis and bone remodeling by regulating multiple cells and factors involved in osseous metastasis.

P7C3 Ameliorates Bone Loss by Inhibiting Osteoclast Differentiation and Promoting Osteogenesis.

Bone homeostasis, the equilibrium between bone resorption and formation, is essential for maintaining healthy bone tissue in adult humans. Disruptions of this process can lead to pathological conditions such as osteoporosis. Dual-targeted agents, capable of inhibiting excessive bone resorption and stimulating bone formation, are being explored as a promising strategy for developing new treatments to address osteoporosis. In this study, we investigated the effects of P7C3 on bone remodeling and its potential therapeutic role in osteoporosis treatment in mice. Specifically, P7C3 can remarkably suppress receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation in bone marrow macrophages via the Akt-NF-κB-NFATc1 signaling pathway. Additionally, RNA sequencing (RNAseq) analysis revealed that P7C3 promoted osteoblast differentiation and function through the Wnt/β-catenin signaling pathway, thereby enhancing bone formation. Furthermore, μCT analysis and histological examination of bone tissues from P7C3-treated mice showed attenuation of both Ti-induced bone erosion and ovariectomy (OVX)-induced bone loss. These findings suggest that P7C3 may have a novel function in bone remodeling and may be a promising therapeutic agent for the treatment of osteoporosis. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Metformin activates AMPK and mTOR to Inhibit RANKL-stimulated osteoclast formation.

Metformin is a medication used to treat type 2 diabetes by inhibiting hepatic glucose production through adenosine monophosphate-activated protein kinase (AMPK) activation. Autophagy is closely related to the homeostasis and stress mechanisms of the body. In recent years, much research has arisen on therapeutic methods utilizing autophagy mechanisms to treat diagnoses such as metabolic diseases, tumors, and Alzheimer's disease. This study thus aimed to investigate the effects of metformin treatment on the differentiation of osteoclasts and changes in AMPK mechanisms, which play an important role in regulating energy homeostasis, and mTOR, a highly conserved kinase that regulates autophagy. Experimentation, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, tartrate-resistant acid phosphate (TRAP) staining, pit formation assay, immunofluorescence, western blotting, and real-time polymerase chain reaction (PCR) was performed to investigate the effects of metformin on osteoclast differentiation. Additionally, to investigate its association with AMPK and pathways, the AMPK inhibitor compound C and mammalian targets of rapamycin (mTOR) activator leucine were used to examine the expression of osteoclast- or autophagy-related proteins, genes, and TRAP-positive cells. Metformin showed no cytotoxic effects on mouse osteoblastic cell lines (MC3T3-E1) and murine macrophage cell lines (RAW264.7) but did inhibit osteoclast differentiation. Furthermore, metformin was found to inhibit osteoclast differentiation through AMPK activation and mTOR inhibition. In turn, AMPK inhibition using compound C promoted osteoclast differentiation, and mTOR activation using leucine inhibited autophagy and osteoclast differentiation. Metformin activates the AMPK pathway while functioning as an activator of mTOR, thereby leading to the inhibition of autophagy and osteoclast differentiation.

Iris Koreana NAKAI Inhibits Osteoclast Formation via p38-Mediated Nuclear Factor of Activated T Cells 1 Signaling Pathway.

Iris Koreana NAKAI (IKN) is a flowering perennial plant that belongs to the Iridaceae family. In this study, we aimed to demonstrate the effects of IKN on osteoclast differentiation in vitro and in vivo. We also sought to verify the molecular mechanisms underlying its anti-osteoclastogenic effects. Osteoclasts were formed by culturing mouse bone marrow macrophage (BMM) cells with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Bone resorption assays were performed on dentin slices. mRNA expression levels were analyzed by quantitative polymerase chain reaction. Western blotting was performed to detect protein expression or activation. Lipopolysaccharide (LPS)-induced osteoclast formation was performed using a mouse calvarial model. In BMM cultures, an ethanol extract of the root part of IKN suppressed RANKL-induced osteoclast formation and bone resorptive activity. In contrast, an ethanol extract of the aerial parts of IKN had a minor effect on RANKL-induced osteoclast formation. Mechanistically, the root part of IKN suppressed RANKL-induced p38 mitogen-activated protein kinase (MAPK) activation, effectively abrogating the induction of c-Fos and nuclear factor of activated T cells 1 (NFATc1) expression. IKN administration decreased LPS-induced osteoclast formation in a calvarial osteolysis model in vivo. Our study suggested that the ethanol extract of the root part of IKN suppressed osteoclast differentiation and function partly by downregulating the p38 MAPK/c-Fos/NFATc1 signaling pathways. Thus, the root part.

Lipoteichoic acid inhibits osteoclast differentiation and bone resorption via interruption of gelsolin-actin dissociation.

Bone resorption can be caused by excessive differentiation and/or activation of bone-resorbing osteoclasts. While microbe-associated molecular patterns can influence the differentiation and activation of bone cells, little is known about the role of lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, in the regulation of bone metabolism. In this study, we investigated the effect of LTA on bone metabolism using wild-type Staphylococcus aureus and the LTA-deficient mutant strain. LTA-deficient S. aureus induced higher bone loss and osteoclast differentiation than wild-type S. aureus. LTA isolated from S. aureus (SaLTA) inhibited osteoclast differentiation from committed osteoclast precursors in the presence of various osteoclastogenic factors by downregulating the expression of NFATc1. Remarkably, SaLTA attenuated the osteoclast differentiation from committed osteoclast precursors of TLR2-/- or MyD88-/- mice and from the committed osteoclast precursors transfected with paired immunoglobulin-like receptor B-targeting siRNA. SaLTA directly interacted with gelsolin, interrupting the gelsolin-actin dissociation which is a critical process for osteoclastogenesis. Moreover, SaLTA suppressed the mRNA expression of dendritic cell-specific transmembrane protein, ATPase H+ transporting V0 subunit D2, and Integrin, which encode proteins involved in cell-cell fusion of osteoclasts. Notably, LTAs purified from probiotics, including Bacillus subtilis, Enterococcus faecalis, and Lactobacillus species, also suppressed Pam2CSK4- or RANKL-induced osteoclast differentiation. Taken together, these results suggest that LTAs have anti-resorptive activity through the inhibition of osteoclastogenesis by interfering with the gelsolin-actin dissociation and may be used as effective therapeutic agents for the prevention or treatment of inflammatory bone diseases.

Boeravinone B, a natural rotenoid, inhibits osteoclast differentiation through modulating NF-κB, MAPK and PI3K/Akt signaling pathways

Boeravinone B, a natural rotenoid, inhibits osteoclast differentiation through modulating NF-κB, MAPK and PI3K/Akt signaling pathways

CGK733 alleviates ovariectomy-induced bone loss through blocking RANKL-mediated Ca2+ oscillations and NF-κB/MAPK signaling pathways

Osteoporosis is a prevalent systemic metabolic disease in modern society, in which patients often suffer from bone loss due to over-activation of osteoclasts. Currently, amelioration of bone loss through modulation of osteoclast activity is a major therapeutic strategy. Ataxia telangiectasia mutated (ATM) inhibitor CGK733 (CG) was reported to have a sensitizing impact in treating malignancies. However, its effect on osteoporosis remains unclear. In this study, we investigated the effects of CG on osteoclast differentiation and function, as well as the therapeutic effects of CG on osteoporosis. Our study found that CG inhibits osteoclast differentiation and function. We further found that CG inhibits the activation of NFATc1 and ultimately osteoclast formation by inhibiting RANKL-mediated Ca2+ oscillation and the NF-κB/MAPK signaling pathway. Next, we constructed an ovariectomized mouse model and demonstrated that CG improved bone loss in ovariectomized mice. Therefore, CG may be a potential drug for the prevention and treatment of osteoporosis.

Synthesis and evaluation of 2-NMPA derivatives as potential agents for prevention of osteoporosis in vitro and in vivo

Abnormal osteoclast differentiation causes various bone disorders such as osteoporosis. Targeting the formation and activation of osteoclasts has been recognized as an effective approach for preventing osteoporosis. Herein, we synthesized eleven 2-NMPA derivatives which are (2-(2-chlorophenoxy)-N-(4-alkoxy-2-morpholinophenyl) acetamides, and evaluated their suppression effects on osteoclastogenesis in vitro by using TRAP-staining assay. Among the synthesized eleven novel 2-NMPAs, 4-(2-(2-chlorophenoxy)acetamido)-3-morpholinophenyl trifluoromethanesulfonate (11b), 4-(2-(2-chlorophenoxy) acetamido)-3-morpholinophenyl-3-(N-(2-oxo-2-((2-(phenylthio) phenyl) amino) ethyl)methylsulfonamido)benzoate (11d), and 4-(2-(2-chlorophenoxy) acetamido)-3-morpholinophenyl 4-acetamidobenzenesulfonate (11h) displayed highly inhibitory bioactivity on the differentiation of primary osteoclasts. 11h was selected for further investigation of the inhibitory effects and potential mechanism involved in the suppression of osteoclastogenesis. In vitro analysis suggested that 11h inhibited osteoclastogenesis with an IC50 of 358.29 nM, decreased the formation of F-action belts and bone resorption, without interfering cell viability and osteoblast differentiation. Furthermore, the mRNA expressions of osteoclast-specific genes such as Acp5, Nfatc1, Dc-stamp, Atp6v0d2, Mmp9, and Ctsk significantly decreased following 11h treatment. RANKL-induced osteoclast-specific proteins analysis demonstrated that 11h suppressed osteoclast differentiation by downregulating of RANKL-mediated TRAF6 expression, followed by inactivation of PI3K/AKT and IκBα/NF-κB signaling pathways. Finally, 11h inhibited ovariectomy-induced bone loss in vivo. Therefore, the current work highlighted the therapeutic potential of 11h as an anti-osteoporosis lead compound.

CX3CL1 promotes M1 macrophage polarization and osteoclast differentiation through NF-κB signaling pathway in ankylosing spondylitis in vitro.

Ankylosing spondylitis (AS) is an autoimmune disease with a genetic correlation and is characterized by inflammation in the axial skeleton and sacroiliac joints. Many AS patients also have inflammatory bowel diseases (IBD), but the underlying causes of intestinal inflammation and osteoporosis in AS are not well understood. CX3CL1, a protein involved in inflammation, has been found to be up-regulated in AS patients and AS-model mice. The authors investigated the effects of CX3CL1 on AS by studying its impact on macrophage polarization, inflammation factors, and osteoclast differentiation. Furthermore, the effects of inhibiting the NF-κB pathway and blocking CX3CL1 were assessed using BAY-117082 and anti-CX3CL1 mAb, respectively. AS model mice were used to evaluate the effects of anti-CX3CL1 mAb on limb thickness, spine rupture, and intestinal tissue damage. The authors found that CX3CL1 increased the expression of M1-type macrophage markers and inflammation factors, and promoted osteoclast differentiation. This effect was mediated through the NF-κB signaling pathway. Inhibition of the NF-κB pathway prevented M1-type macrophage polarization, reduced inflammation levels, and inhibited osteoclast differentiation. Injection of anti-CX3CL1 mAb alleviated limb thickness, spine rupture, and intestinal tissue damage in AS model mice by inhibiting M1-type macrophage polarization and reducing intestinal tissue inflammation. The study demonstrated that up-regulated CX3CL1 promotes M1-type macrophage polarization and osteoclast differentiation through the NF-κB signaling pathway. Inhibition of this pathway and blocking CX3CL1 can alleviate inflammation and bone destruction in AS. These findings contribute to a better understanding of the pathogenesis of AS and provide a basis for clinical diagnosis and treatment.