Introduction: miR-146a is one of the first identified miRNAs expressed differentially in osteoarthritis (OA) cartilage. However, the role it plays in OA pathogenesis is not clear. The aim of this study is to identify a molecular target of miR-146a, thereby elucidating its function in chondrocytes during OA pathogenesis. Methods: Primary chondrocytes from Sprague-Dawley rats were treated with IL-1β before the expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by real-time PCR and/or western blotting. The effect of miR-146a on cellular response to transforming growth factor (TGF)-β1 was quantified by a luciferase reporter harboring TGF-β1 responsive elements and by extracellular signal-regulated kinase assay. The effect of miR-146a on apoptosis was quantified by the TUNEL assay. OA pathogenesis was surgically induced with joint instability in rats, evaluated by histopathological analysis with safranin O staining, and the expression levels of miR-146a, Smad4, and VEGF were quantified using real-time PCR and/or immunohistochemistry. Results: IL-1β treatment of chondrocytes increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in a time-dependent manner. miR-146a upregulated VEGF expression and downregulated Smad4 expression in chondrocytes, while a miR-146a inhibitor acted in a converse manner. Smad4, a common mediator of the TGF-β pathway, is identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3'-UTR region of its mRNA. Mutation of the binding sequence significantly relieved the inhibition of the Smad4 reporter activity by miR-146a. Furthermore, miR-146a upregulation of VEGF is mediated by Smad4. Expression of miR-146a led to a reduction of cellular responsiveness to TGF-β and an increase of apoptosis rate in chondrocytes. In vivo, cartilage from surgically induced OA rats displayed higher levels of miR-146a and VEGF compared with the sham group. In contrast, Smad4 expression level was lower in the OA group than the sham group. Conclusion: IL-1β responsive miR-146a is overexpressed in an experimentally induced OA model, accompanied by upregulation of VEGF and downregulation of Smad4 in vivo. miR-146a may contribute to OA pathogenesis by increasing VEGF levels and by impairing the TGF-β signaling pathway through targeted inhibition of Smad4 in cartilage.

Osteoarthritis (OA) is an inflammatory disease of load-bearing synovial joints that is currently treated with drugs that exhibit numerous side effects and are only temporarily effective in treating pain, the main symptom of the disease. Consequently, there is an acute need for novel, safe, and more effective chemotherapeutic agents for the treatment of osteoarthritis and related arthritic diseases. Curcumin, the principal curcuminoid and the most active component in turmeric, is a biologically active phytochemical. Evidence from several recent in vitro studies suggests that curcumin may exert a chondroprotective effect through actions such as anti-inflammatory, anti-oxidative stress, and anti-catabolic activity that are critical for mitigating OA disease pathogenesis and symptoms. In the present study, we investigated the protective mechanisms of curcumin on interleukin 1β (IL-1β)-stimulated primary chondrocytes in vitro. The treatment of interleukin (IL)-1β significantly reduces the cell viability of chondrocytes in dose and time dependent manners. Co-treatment of curcumin with IL-1β significantly decreased the growth inhibition. We observed that curcumin inhibited IL-1β-induced apoptosis and caspase-3 activation in chondrocytes. Curcumin can increase the expression of phosphorylated extracellular signal-regulated kinases 1/2 (ERK1/2), autophagy marker light chain 3 (LC3)-II, and Beclin-1 in chondrocytes. The expression of autophagy markers could be decreased when the chondrocytes were incubated with ERK1/2 inhibitor U0126. Our results suggest that curcumin suppresses apoptosis and inflammatory signaling through its actions on the ERK1/2-induced autophagy in chondrocytes. We propose that curcumin should be explored further for the prophylactic treatment of osteoarthritis in humans and companion animals.

Aseptic loosening caused by wear particles is a common complication after total hip arthroplasty. We investigated the effect of the quercetin on wear particle‐mediated macrophage polarization, inflammatory response and osteolysis. In vitro, we verified that Ti particles promoted the differentiation of RAW264.7 cells into M1 macrophages through p‐38α/β signalling pathway by using flow cytometry, immunofluorescence assay and small interfering p‐38α/β RNA. We used enzyme‐linked immunosorbent assays to confirm that the protein expression of M1 macrophages increased in the presence of Ti particles and that these pro‐inflammatory factors further regulated the imbalance of OPG/RANKL and promoted the differentiation of osteoclasts. However, this could be suppressed, and the protein expression of M2 macrophages was increased by the presence of the quercetin. In vivo, we revealed similar results in the mouse skull by μ‐CT, H&E staining, immunohistochemistry and immunofluorescence assay. We obtained samples from patients with osteolytic tissue. Immunofluorescence analysis indicated that most of the macrophages surrounding the wear particles were M1 macrophages and that pro‐inflammatory factors were released. Titanium particle‐mediated M1 macrophage polarization, which caused the release of pro‐inflammatory factors through the p‐38α/β signalling pathway, regulated OPG/RANKL balance. Macrophage polarization is expected to become a new clinical drug therapeutic target.

Wear particles that detach from the surface of prostheses induce excessive activation of osteoclast and immoderate release of inflammatory cytokines that lead to peri-implant osteolysis and aseptic loosening. In this work, we investigated whether magnoflorine, a quaternary aporphine alkaloid extracted from the Chinese herb Magnolia or Aristolochia, could effectively inhibit inflammatory calvarial osteolysis caused by titanium particles in mouse models, inflammatory response as well as osteoclastogenesis in vitro mediated via receptor activator of NF-κB ligand (RANKL). Micro-computed tomography and histological examination of mice treated with magnoflorine revealed fewer resorption pits, less osteoclasts formation and inflammatory cytokine expression. Moreover, in vitro differentiation of osteoclasts and bone resorption as well as titanium particle-induced inflammatory response were dose-dependently inhibited by magnoflorine. These were accompanied by reduced transcription of osteoclast-specific genes encoding tartrate-resistant acid phosphatase (TRAP), V-ATPase d2, c-Fos, cathepsin K, nuclear factor of activated T cells (NFAT) c1, and calcitonin receptor (CTR). Further research on mechanism showed that the inhibition of phosphorylation of TAK1 and subsequent activation of MAPK and NF-κB signaling pathways were found to mediate the suppressive effects of magnoflorine. Collectively, these results suggested that magnoflorine treatment could effectively prevent peri-implant osteolysis due to wear debris as well as other diseases caused by chronic inflammation and excessive osteoclast activation.

The central physiological role of the bone marrow renders bone marrow stromal cells (BMSCs) particularly sensitive to aging. With bone aging, BMSCs acquire a differentiation potential bias in favor of adipogenesis over osteogenesis, and the underlying molecular mechanisms remain unclear. Herein, we investigated the factors underlying age-related changes in the bone marrow and their roles in BMSCs’ differentiation. Antibody array revealed that CC chemokine ligand 3 (CCL3) accumulation occurred in the serum of naturally aged mice along with bone aging phenotypes, including bone loss, bone marrow adiposity, and imbalanced BMSC differentiation. In vivo Ccl3 deletion could rescue these phenotypes in aged mice. CCL3 improved the adipogenic differentiation potential of BMSCs, with a positive feedback loop between CCL3 and C/EBPα. CCL3 activated C/EBPα expression via STAT3, while C/EBPα activated CCL3 expression through direct promoter binding, facilitated by DNA hypomethylation. Moreover, CCL3 inhibited BMSCs’ osteogenic differentiation potential by blocking β-catenin activity mediated by ERK-activated Dickkopf-related protein 1 upregulation. Blocking CCL3 in vivo via neutralizing antibodies ameliorated trabecular bone loss and bone marrow adiposity in aged mice. This study provides insights regarding age-related bone loss and bone marrow adiposity pathogenesis and lays a foundation for the identification of new targets for senile osteoporosis treatment.