Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-κB ligand–induced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of mitogen-activated protein kinase/activator protein 1 and Ca2+–nuclear factor of activated T-cells cytoplasmic 1 signaling pathways

Receptor activator of NF-κB ligand (RANKL) stimulation leads to the activation of mitogen-activated protein kinase (MAPK)/AP-1 and Ca2+–nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) signaling pathways in osteoclastogenesis. Targeting these pathways has been an encouraging strategy for bone-related diseases, such as postmenopausal osteoporosis. In this study, we examined the effects of caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) on osteoclastogenesis. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, CADPE suppressed RANKL-induced osteoclast differentiation and actin-ring formation in a dose-dependent manner within non–growth inhibitory concentrations at the early stage, while CADPE had no effect on macrophage colony-stimulating factor (M-CSF)-induced proliferation and differentiation. At the molecular level, CADPE inhibited RANKL-induced phosphorylation of MAPKs, including extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), without significantly affecting the NF-κB signaling pathway. CADPE abrogated RANKL-induced activator protein 1 (AP-1)/FBJ murine osteosarcoma viral oncogene homolog (c-Fos) nuclear translocation and activation. Overexpression of c-Fos prevented the inhibition by CADPE of osteoclast differentiation. Furthermore, CADPE suppressed RANKL-induced the tumor necrosis factor receptor associated factor 6 (TRAF6) interaction with c-src tyrosine kinase (c-Src), blocked RANKL-induced the phosphorylation of protein kinase B (AKT), and inhibited RANKL-induced Ca2+ oscillation. As a result, CADPE decreased osteoclastogenesis-related marker gene expression, including NFATc1, TRAP, cathepsin K, and c-Src. To test the effects of CADPE on osteoclast activity in vivo, we showed that CADPE prevented ovariectomy-induced bone loss by inhibiting osteoclast activity. Together, our data demonstrate that CADPE suppresses osteoclastogenesis and bone loss through inhibiting RANKL-induced MAPKs and Ca2+-NFATc1 signaling pathways. CADPE is a novel agent in the treatment of osteoclast-related diseases, such as osteoporosis.     

Leonurine hydrochloride inhibits osteoclastogenesis and prevents osteoporosis associated with estrogen deficiency by inhibiting the NF-κB and PI3K/Akt signaling pathways

Osteoclasts, the primary bone resorbing cells, are responsible for destructive bone diseases such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis. Many plant-derived traditional medicines that might suppress the formation and/or function of osteoclasts are promising treatments for osteoclast-related diseases. In this study, we investigated the effects of leonurine hydrochloride (LH) on receptor activator NF-κB ligand (RANKL)-induced osteoclastogenesis and ovariectomy-induced bone loss. LH is a synthetic chemical compound based on the structure of leonurine, which is found in motherwort and has been reported to exhibit phytoestrogenic activity. In RAW 264.7 cells and mouse bone marrow monocytes (BMMs), LH suppressed RANKL-induced osteoclastogenesis and actin ring formation in a dose-dependent manner. LH targeted RANKL-induced osteoclastogenesis and bone resorption at an early stage. Molecular analysis demonstrated that LH attenuated RANKL-induced NF-κB signaling by inhibiting the phosphorylation and degradation of IκBα and NF-κB p65 nuclear translocation. LH inhibited the RANK-TRAF6 association triggered by RANKL binding and the phosphatidylinositol 3-kinase (PI3K)/Akt axis, without significantly affecting the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and AP-1 signaling pathways. LH attenuated the RANKL-stimulated expression of osteoclast-related genes including NFATc1, tartrate resistant acid phosphatase (TRAP), cathepsin K, and osteoclast-associated receptor (OSCAR). Consistent with the in vitro results, LH administration attenuated osteoclast activity, thus preventing bone loss caused by estrogen deficiency in mice. In this study, LH suppressed RANKL-induced osteoclastogenesis via RANK-TRAF6, NF-κB, and PI3K/Akt signaling. These data provide the first evidence that LH might be a promising therapeutic compound to treat osteoclast-related diseases, such as osteoporosis.     

Inhibition of the classical NF-κB pathway prevents osteoclast bone-resorbing activity

The classical NF-κB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-κB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-κB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-κB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p < 0.001) and depths (p < 0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-κB pathway plays a pivotal role in osteoclast bone-resorbing activity.     

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-κB pathways

Summary  Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-κB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. Introduction  A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-κB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. Methods  The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-κB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-κB activity. Results  Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-κB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. Conclusions  Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-κB activation. R.-S. Yang, and W.-M. Fu contributed equally to this study.     

High-frequency near-infrared diode laser irradiation suppresses IL-1β-induced inflammatory cytokine expression and NF-κB signaling pathways in human primary chondrocytes

Lasers in Medical Science - Osteoarthritis (OA) and rheumatoid arthritis (RA) are common inflammation-associated cartilage degenerative diseases. Recent studies have shown that low-level diode...     

TSG-6 inhibits hypertrophic scar fibroblast proliferation by regulating IRE1α/TRAF2/NF-κB signalling

TNF-stimulated gene (TSG-6) was reported to suppress hypertrophic scar (HS) formation in a rabbit ear model, and the overexpression of TSG-6 in human HS fibroblasts (HSFs) was found to induce their apoptotic death. The molecular basis for these findings, however, remains to be clarified. HSFs were subjected to TSG-6 treatment. Treatment with TSG-6 significantly suppressed HSF proliferation and induced them to undergo apoptosis. Moreover, TSG-6 exposure led to reductions in collagen I, collagen III, and α-SMA mRNA and protein levels, with a corresponding drop in proliferating cell nuclear antigen (PCNA) expression indicative of impaired proliferative activity. Endoplasmic reticulum (ER) stress was also suppressed in these HSFs as demonstrated by decreases in Bip and p-IRE1α expression, downstream inositol requiring enzyme 1 alpha (IRE1α) -Tumor necrosis factor receptor associated factor 2 (TRAF2) pathway signalling was inhibited and treated cells failed to induce NF-κB, TNF-α, IL-1β, and IL-6 expression. Overall, ER stress was found to trigger inflammatory activity in HSFs via the IRE1α-TRAF2 axis, as confirmed with the specific inhibitor of IRE1α STF083010. Additionally, the effects of TSG-6 on apoptosis, collagen I, collagen III, α-SMA, and PCNA of HSFs were reversed by the IRE1α activator thapsigargin (TG). These data suggest that TSG-6 administration can effectively suppress the proliferation of HSFs in part via the inhibition of IRE1α-mediated ER stress-induced inflammation (IRE1α/TRAF2/NF-κB signalling).     

Angelicin regulates LPS-induced inflammation via inhibiting MAPK/NF-κB pathways

Background

Angelicin is a furocoumarin found in Psoralea corylifolia L. fruit. The purpose of this study was to investigate the protective ability of angelicin against inflammation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and LPS-induced in vivo acute lung injury model.

Materials and methods

The concentrations of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 in the culture supernatants of RAW 264.7 cells were determined 24 h after LPS administration. ALI was induced by intratracheal instillation of LPS. Six hours after LPS inhalation, bronchoalveolar lavage fluid and lung tissue samples were obtained for enzyme-linked immunosorbent assay, histologic, and Western blotting analyses.

Results

The results showed that pretreatment with angelicin markedly downregulated TNF-α and IL-6 levels in vitro and in vivo, and significantly decreased the amount of inflammatory cells, lung wet-to-dry weight ratio, and myeloperoxidase activity in LPS-induced ALI mice. Furthermore, Western blotting analysis results demonstrated that angelicin blocked the phosphorylation of IκBα, NF-κBp65, p38 MAPK, and JNK in LPS-induced ALI.

Conclusions

These results suggest that angelicin was potentially advantageous to prevent inflammatory diseases by inhibiting NF-κB and MAPK pathways. Our data indicated that angelicin might be a potential new agent for prevention of inflammatory reactions and diseases in the clinic.     

Simultaneous blockade of the CD40/CD40L and NF-κB pathways prolonged islet allograft survival

Activation of NF-κB pathway and co-stimulatory system CD40/CD40L promotes the inflammation, which plays a key role in the failure of islet graft. Therefore, the purpose of this study was to determine if simultaneous blockade of CD40/CD40L and IκB/NF-κB pathways could protect islet graft. Streptozocin-induced diabetic Wistar rats were transplanted intraportally with 2000 IEQ islets isolated from Sprague-Dawley rats. The rats were divided into five groups: nontreatment group, AdGFP-treated group, Ad-IκBα-treated group, Ad-sCD40LIg-treated group, and Ad-IκBα-IRES(2) -sCD40L-treated group. The islet graft mean survival time (MST), insulin expression of islet grafts, and the levels of cytokines in peripheral blood, were measured for the animals in each group. Our study confirmed that islet cells transfected with low doses of adenovirus could achieve high transfection efficiency, and would not affect the function of islet cells (P > 0.05). Splenocytes cultured with Ad-IκBα-IRES2-CD40L-transfected islets resulted in homospecific hyporesponsiveness. The islet graft MST (>100 d) in the Ad-IκBα-IRES2-sCD40L-treated group was dramatically prolonged compared with that in the nontreatment group (7.1 ± 1.16 d). In addition, TNF-α, IL-1β, and IFN-γ were diminished in the Ad-IκBα-IRES2-sCD40L-treated group, which was commensurate with the reduced cellular infiltration (P < 0.01). Simultaneous blockade of the CD40/CD40L and IκB/NF-κB pathways could effectively extend the survival of islet grafts.