This study explored the mechanism of curcumin (CUR) suppressing osteoclastogenesis and evaluated its effects on osteoarthritis (OA) mouse. Bone marrow-derived macrophages were isolated as osteoclast precursors. In the presence or absence of CUR, cell proliferation was detected by CCK-8, osteoclastogenesis was detected by tartrate-resistant acid phosphatase (TRAP) staining, F-actin rings formation was detected by immunofluorescence, bone resorption was detected by bone slices, IκBα, nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were detected using western blot, osteoclastogenesis-related gens were measured using quantitative polymerase chain reaction. A knee OA mouse model was designed by destabilizing the medial meniscus (DMM). Thirty-six male mice were divided into sham+vehicle, OA+vehicle, and OA+CUR groups. Mice were administered with or without CUR at 25 mg/kg/d from the first post-operative day until sacrifice. After 4 and 8 weeks of OA induction, micro-computed tomography was performed to analyze microstructure changes in subchondral bone, hematoxylin and eosin staining was performed to calculate the thickness of the calcified and hyaline cartilage layers, toluidine blue O staining was performed to assess the degenerated cartilage, TRAP-stained osteoclasts were counted, and NF-κB, phosphorylated Jun N-terminal Kinases (p-JNK), and receptor activator of nuclear factor κB ligand (RANKL) were detected using immunohistochemistry. CUR suppressed osteoclastogenesis and bone resorption without cytotoxicity. CUR restrained RANKL-induced activation of NF-κB, p-JNK and up-regulation of osteoclastogenesis-related genes. CUR delayed cartilage degeneration by suppressing osteoclastogenesis and bone resorption in early OA. The mechanism of CUR inhibiting osteoclastogenesis might be associated with NF-κB/JNK signaling pathway, indicating a novel strategy for OA treatment.

BACKGROUND: Mechanical unloading-induced bone loss threatens prolonged spaceflight and human health. Recent studies have confirmed that osteoporosis is associated with a significant reduction in bone microvessels, but the relationship between them and the underlying mechanism under mechanical unloading are still unclear.
METHODS: We established a 2D clinostat and hindlimb-unloaded (HLU) mouse model to simulate unloading in vitro and in vivo. Micro-CT scanning was performed to assess changes in the bone microstructure and mass of the tibia. The levels of CD31, Endomucin (EMCN) and histone deacetylase 6 (HDAC6) in tibial microvessels were detected by immunofluorescence (IF) staining. In addition, we established a coculture system of microvascular endothelial cells (MVECs) and osteoblasts, and qRT‒PCR or western blotting was used to detect RNA and protein expression; cell proliferation was detected by CCK‒8 and EdU assays. ChIP was used to detect whether HDAC6 binds to the miRNA promoter region.
RESULTS: Bone mass and bone microvessels were simultaneously significantly reduced in HLU mice. Furthermore, MVECs effectively promoted the proliferation and differentiation of osteoblasts under coculture conditions in vitro. Mechanistically, we found that the HDAC6 content was significantly reduced in the bone microvessels of HLU mice and that HDAC6 inhibited the expression of miR-375-3p by reducing histone acetylation in the miR-375 promoter region in MVECs. miR-375-3p was upregulated under unloading and it could inhibit MVEC proliferation by directly targeting low-density lipoprotein-related receptor 5 (LRP5) expression. In addition, silencing HDAC6 promoted the miR-375-3p/LRP5 pathway to suppress MVEC proliferation under mechanical unloading, and regulation of HDAC6/miR-375-3p axis in MVECs could affect osteoblast proliferation under coculture conditions.
CONCLUSIONS: Our study revealed that disuse-induced bone loss may be closely related to a reduction in the number of bone microvessels and that the modulation of MVEC function could improve bone loss induced by unloading. Mechanistically, the HDAC6/miR-375-3p/LRP5 pathway in MVECs might be a promising strategy for the clinical treatment of unloading-induced bone loss.

BACKGROUND: Bone loss caused by microgravity exposure presents a serious threat to the health of astronauts, but existing treatment strategies have specific restrictions. This research aimed to investigate whether salidroside (SAL) can mitigate microgravity-induced bone loss and its underlying mechanism.
METHODS: In this research, we used hindlimb unloading (HLU) and the Rotary Cell Culture System (RCCS) to imitate microgravity in vivo and in vitro.
RESULTS: The results showed that salidroside primarily enhances bone density, microstructure, and biomechanical properties by stimulating bone formation and suppressing bone resorption, thereby preserving bone mass in HLU rats. In MC3T3-E1 cells cultured under simulated microgravity in rotary wall vessel bioreactors, the expression of osteogenic genes significantly increased after salidroside administration, indicating that salidroside can promote osteoblast differentiation under microgravity conditions. Furthermore, the Nrf2 inhibitor ML385 diminished the therapeutic impact of salidroside on microgravity-induced bone loss. Overall, this research provides the first evidence that salidroside can mitigate bone loss induced by microgravity exposure through stimulating the Nrf2/HO-1 pathway.
CONCLUSIONS: These findings indicate that salidroside has great potential for treating space-related bone loss in astronauts and suggest that Nrf2/HO-1 is a viable target for counteracting microgravity-induced bone damage.

BACKGROUND: The polarization of macrophages into an anti-inflammatory phenotype is crucial for resolving periodontal inflammation. It has been reported that B10 cells can regulate the immune response of macrophages during inflammation and are also able to regulate inflammation in periodontitis. However, whether B10 cells\' regulation function in periodontitis is related to macrophage polarization remains unclear. This study aims to investigate whether B10 cells can regulate macrophage polarization in periodontitis.
METHODS: Macrophages were cocultured with B10 cells in vitro for 5 days. After coculture, macrophages were obtained for analysis directly or followed by stimulation with Pg-LPS/IFN-γ or IL-4/IL-13. Flow cytometry and/or reverse transcriptase-polymerase chain reaction (RT-PCR) were employed to detect the expression of IL-1β, iNOS, TNF-α, CD206, and ARG-1 in macrophages. B10 cells were transferred on the 5th day after ligation in wild or macrophage-depletion mice. Toluidine blue and TRAP staining were used to evaluate alveolar bone resorption and osteoclast activation. Immunohistochemistry was employed to detect the expression of CD68, IL-1β, TNF-α, iNOS, ARG-1, and IL-10. Immunofluorescence was used to detect the expression of CD68+CD86+M1 macrophages and CD68+CD206+M2 macrophages.
RESULTS: In vitro, B10 cells inhibit the expression of IL-1β, iNOS, and TNF-α in macrophages while increasing the expression of CD206 and ARG-1. In experimental periodontitis, B10 cells inhibit the polarization of CD68+CD86+M1 macrophages and iNOS expression but enhance the polarization of CD68+CD206+M2 macrophages and ARG-1 expression. Importantly, the depletion of macrophages partially weakened the regulation function of B10 cells in periodontitis.
CONCLUSIONS: B10 cells promote M2 macrophage polarization, inhibit M1 macrophage polarization in periodontitis, and alleviate periodontitis partially by regulating macrophage polarization.

OBJECTIVE: The objective of this study was to investigate the potential of salidroside (SAL) (a major active compound in Rhodiola rosea L.) in regulating osteoclast differentiation and function by modulating the HIF-1α pathway and its downstream target genes.
METHODS: The expression of HIF-1α and its downstream target genes was examined at both mRNA and protein levels in osteoclasts treated with SAL. Immunofluorescence analysis was performed to assess the nuclear translocation and transcriptional activity of HIF-1α in response to SAL. MTT, flow cytometry, qPCR, TRAP staining and bone resorption assays were used to evaluate the potential effect of salidroside on osteoclasts.
RESULTS: SAL enhanced the expression of HIF-1α and its downstream target genes in osteoclasts. Immunofluorescence analysis confirmed the facilitation of HIF-1α nuclear translocation and transcriptional activity by SAL. In addition, SAL enhanced osteoclast viability, differentiation and bone resorption activity in an autocrine manner through HIF-1α/VEGF, IL-6 and ANGPTL4 pathways.
CONCLUSIONS: SAL promotes osteoclast proliferation, differentiation and bone resorption through HIF-1α/VEGF, IL-6 and ANGPTL4 pathways.

Osteoclast activity plays a crucial role in the pathological mechanisms of osteoporosis and bone remodeling. The treatment of these disorders involves the use of pharmacological medicines that work by inhibiting the activity of osteoclasts. Nevertheless, the prevalent and infrequent negative consequences of current antiresorptive and bone anabolic treatments pose significant drawbacks, hence restricting their prolonged administration in patients, particularly those who are elderly and/or suffer from many medical conditions. We are currently in the process of creating a new molecule called N-(4-methoxyphen) methyl caffeamide (MPMCA), which is a derivative of caffeic acid. This compound has shown potential in preventing the production of osteoclasts and causing existing osteoclasts to undergo cell apoptosis. Our investigation discovered that MPMCA hinders osteoclast function via suppressing the MAPK pathways. The expectation is that the findings of this study will stimulate the advancement of a novel approach to treating anti-resorption.

Bone aging and decreased autophagic activity are related but poorly explored in the jawbone. This study aimed to characterize the aging jawbones and jawbone-derived stromal cells (JBSCs) and determine the role of autophagy in jawbone mass decline. We observed that the jawbones of older individuals and mice exhibited similar age-related bone loss. Furthermore, leptin receptor (LepR)-lineage cells served as the primary source for in vitro cultured and expanded JBSCs, referred to as LepR-Cre+/JBSCs. RNA-sequencing data from the jawbones and LepR-Cre+/JBSCs showed the upregulated expression of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway during aging. Through single-cell transcriptomics, we identified a decrease in the proportion of osteogenic lineage cells and the activation of the PI3K/AKT pathway in LepR-lineage cells in aging bone tissues. Reduced basal autophagic activity, diminished autophagic flux, and decreased osteogenesis occurred in the jawbones and LepR-Cre+/JBSCs from older mice (O-mice; O-JBSCs). Pharmacologic and constitutive autophagy activation alleviated the impaired osteogenesis in O-JBSCs. In addition, the suppression of mTOR-induced autophagy improved the aging phenotype of O-JBSCs. The activation of autophagy in LepR-Cre+/JBSCs using chemical autophagic activators reduced the alveolar bone resorption in O-mice. Therefore, our study demonstrated that ATG molecules and pathways are crucial in jawbone aging, providing novel approaches to understanding age-related jawbone loss.

Treatments to reduce periodontal inflammation and rescue periodontitis bone resorption have been of interest to researchers. Bone tissue engineering materials have been gradually used in the treatment of bone defects, but periodontal bone tissue regeneration still faces challenges. Considering the biocompatibility factor, constructing bionic scaffolds with natural extracellular matrix properties is an ideal therapeutic pathway. Based on the pathological mechanism of periodontitis, in this study, short peptide and nanometer inorganic particles were comingled to construct NapKFF-nano CaF2 supramolecular composite hydrogels with different ratios. Material characterization experiments confirmed that the composite hydrogel had suitable mechanical properties and a three-dimensional structure that can function in the resorption region of the alveolar bone and provide spaces for cell proliferation and adhesion. The release of low concentrations of fluoride and calcium ions has been shown to have positive biological effects in both in vivo and in vitro experiments. Vitro experiments confirmed that the composite hydrogel had good biocompatibility and promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Microbiological experiments confirmed that the composite hydrogel inhibited the activity of periodontal pathogenic bacteria. In animal studies, composite hydrogel applied to periodontitis rats in vivo can effectively repair alveolar bone resorption. This composite hydrogel has a simple preparation method and is inexpensive to produce, yet it has antibacterial and osteogenesis-promoting incremental effects, which makes it well suited for the treatment of periodontitis bone resorption, providing a new strategy for periodontal bone tissue engineering.

OBJECTIVE: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma.
METHODS: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis.
RESULTS: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group.
CONCLUSIONS: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.
目的: 骨质进行性吸收破坏是中耳胆脂瘤最重要的临床特征之一，可导致一系列颅内外并发症，而目前中耳胆脂瘤骨破坏的机制尚未明确。本研究旨在探究甲状旁腺激素相关蛋白(parathyroid hormone-related protein，PTHrP)参与中耳胆脂瘤骨破坏的机制。方法: 收集后天性中耳胆脂瘤患者的25例胆脂瘤标本和13例外耳道正常皮肤组织标本。采用免疫组织化学染色方法检测PTHrP、核因子κB受体活化因子配体(receptor activator for nuclear factor-kappa B ligand，RANKL)和骨保护素(osteoprotegerin，OPG)在中耳胆脂瘤和外耳道正常皮肤组织中的表达，抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase，TRAP)染色法检测中耳胆脂瘤和外耳道正常皮肤组织中是否存在TRAP阳性多核巨噬细胞。选取小鼠单核巨噬细胞RAW264.7细胞进行干预，分为RANKL干预组和PTHrP+RANKL共同干预组，采用TRAP染色法检测2组破骨细胞的生成情况，实时聚合酶链反应(real-time polymerase chain reaction，real-time PCR)检测干预后2组破骨细胞相关基因TRAP、组织蛋白酶K(cathepsin K，CTSK)和活化T细胞核因子1(nuclear factor of activated T cell cytoplasmic 1，NFATc1)的mRNA表达水平，骨吸收陷窝实验检测2组破骨细胞的骨吸收功能。结果: 免疫组织化学染色结果显示，PTHrP和RANKL在中耳胆脂瘤组织中的表达均显著增高，OPG表达降低(均P<0.05)，且PTHrP的表达与RANKL、RANKL/OPG比值均呈显著正相关，与OPG表达呈显著负相关(分别r=0.385、r=0.417、r=-0.316，均P<0.05)。同时，PTHrP、RANKL的表达水平与中耳胆脂瘤的骨破坏程度均呈显著正相关(分别r=0.413、r=0.505，均P<0.05)。TRAP染色结果显示中耳胆脂瘤上皮周围基质中有大量TRAP阳性细胞，并存在细胞核数量为3个或3个以上的TRAP阳性破骨细胞。RANKL或PTHrP+RANKL联合干预5 d后，与RANKL干预组相比，PTHrP+RANKL联合干预组的破骨细胞数量显著增加(P<0.05)，且破骨细胞相关基因TRAP、CTSK和NFATc1的mRNA表达水平均升高(均P<0.05)。骨吸收陷窝扫描电镜结果显示RANKL干预组、PTHrP+RANKL联合干预组的骨片表面均形成骨吸收陷窝；与RANKL干预组相比，PTHrP+RANKL联合干预组的骨片表面骨吸收陷窝数量显著增加(P<0.05)，面积也更大。结论: PTHrP可能通过促进RANKL诱导胆脂瘤组织周围基质中的巨噬细胞分化为破骨细胞，参与中耳胆脂瘤骨破坏。.

Lysosomes serve as catabolic centers and signaling hubs in cells, regulating a multitude of cellular processes such as intracellular environment homeostasis, macromolecule degradation, intracellular vesicle trafficking and autophagy. Alterations in lysosomal level and function are crucial for cellular adaptation to external stimuli, with lysosome dysfunction being implicated in the pathogenesis of numerous diseases. Osteoclasts (OCs), as multinucleated cells responsible for bone resorption and maintaining bone homeostasis, have a complex relationship with lysosomes that is not fully understood. Dysregulated function of OCs can disrupt bone homeostasis leading to the development of various bone disorders. The regulation of OC differentiation and bone resorption for the treatment of bone disease have received considerable attention in recent years, yet the role and regulation of lysosomes in OCs, as well as the potential therapeutic implications of intervening in lysosomal biologic behavior for the treatment of bone diseases, remain relatively understudied. This review aims to elucidate the mechanisms involved in lysosomal biogenesis and to discuss the functions of lysosomes in OCs, specifically in relation to differentiation, bone resorption, and autophagy. Finally, we explore the potential therapeutic implication of targeting lysosomes in the treatment of bone metabolic disorders.