Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r =  - 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer\'s patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants. KEY POINTS: • Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken. • The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression. • Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine.

Vascular calcification has a global health impact that is closely linked to bone loss. The Receptor Activator of Nuclear Factor Kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, fundamental for bone metabolism, also plays an important role in vascular calcification. The Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), a novel receptor for RANKL, regulates bone remodeling, and it appears to be involved in vascular calcification. Besides RANKL, LGR4 interacts with R-spondins (RSPOs), which are known for their roles in bone but are less understood in vascular calcification. Studies were conducted in rats with chronic renal failure fed normal or high phosphorus diets for 18 weeks, with and without control of circulating parathormone (PTH) levels, resulting in different degrees of aortic calcification. Additionally, vascular smooth muscle cells (VSMCs) were cultured under non-calcifying (1 mM phosphate) and calcifying (3 mM phosphate) media with different concentrations of PTH. To explore the role of RANKL in VSMC calcification, increasing concentrations of soluble RANKL were added to non-calcifying and calcifying media. The effects mediated by RANKL binding to its receptor LGR4 were investigated by silencing the LGR4 receptor in VSMCs. Furthermore, the gene expression of the RANK/RANKL/OPG system and the ligands of LGR4 was assessed in human epigastric arteries obtained from kidney transplant recipients with calcification scores (Kauppila Index). Increased aortic calcium in rats coincided with elevated systolic blood pressure, upregulated Lgr4 and Rankl gene expression, downregulated Opg gene expression, and higher serum RANKL/OPG ratio without changes in Rspos gene expression. Elevated phosphate in vitro increased calcium content and expression of Rankl and Lgr4 while reducing Opg. Elevated PTH in the presence of high phosphate exacerbated the increase in calcium content. No changes in Rspos were observed under the conditions employed. The addition of soluble RANKL to VSMCs induced genotypic differentiation and calcification, partly prevented by LGR4 silencing. In the epigastric arteries of individuals presenting vascular calcification, the gene expression of RANKL was higher. While RSPOs show minimal impact on VSMC calcification, RANKL, interacting with LGR4, drives osteogenic differentiation in VSMCs, unveiling a novel mechanism beyond RANKL-RANK binding.

The global prevalence of osteoporosis is being exacerbated by the increasing number of aging societies and longer life expectancies. In response, numerous drugs have been developed in recent years to mitigate bone resorption and enhance bone density. Nonetheless, the efficacy and safety of these pharmaceutical interventions remain constrained. Corylin (CL), a naturally occurring compound derived from the anti-osteoporosis plant Psoralea corylifolia L., has exhibited promising potential in impeding osteoclast differentiation. This study aims to evaluate the effect and molecular mechanisms of CL regulating osteoclast differentiation in vitro and its potential as a therapeutic agent for osteoporosis treatment in vivo. Our investigation revealed that CL effectively inhibits osteoclast formation and their bone resorption capacity by downregulating the transcription factors NFATc1 and c-fos, consequently resulting in the downregulation of genes associated with bone resorption. Furthermore, it has been observed that CL can effectively mitigate the migration and fusion of pre-osteoclast, while also attenuating the activation of mitochondrial mass and function. The results obtained from an in vivo study have demonstrated that CL is capable of attenuating the bone loss induced by ovariectomy (OVX). Based on these significant findings, it is proposed that CL exhibits considerable potential as a novel drug strategy for inhibiting osteoclast differentiation, thereby offering a promising approach for the treatment of osteoporosis.

Bone disease associated with multiple myeloma (MM) is characterized by osteolytic lesions and pathological fractures, which remain a therapeutic priority despite new drugs improving MM patient survival. Antiresorptive molecules represent the main option for the treatment of MM-associated bone disease (MMBD), whereas osteoanabolic molecules are under investigation. Among these latter, we here focused on the myokine irisin, which is able to enhance bone mass in healthy mice, prevent bone loss in osteoporotic mouse models, and accelerate fracture healing in mice. Therefore, we investigated irisin effect on MMBD in a mouse model of MM induced by intratibial injection of myeloma cells followed by weekly administration of 100 μg/kg of recombinant irisin for 5 wk. By micro-Ct analysis, we demonstrated that irisin improves MM-induced trabecular bone damage by partially preventing the reduction of femur Trabecular Bone Volume/Total Volume (P = .0028), Trabecular Number (P = .0076), Trabecular Fractal Dimension (P = .0044), and increasing Trabecular Separation (P = .0003) in MM mice. In cortical bone, irisin downregulates the expression of Sclerostin, a bone formation inhibitor, and RankL, a pro-osteoclastogenic molecule, while in BM it upregulates Opg, an anti-osteoclastogenic cytokine. We found that in the BM tibia of irisin-treated MM mice, the percentage of MM cells displays a reduction trend, while in the femur it decreases significantly. This is in line with the in vitro reduction of myeloma cell viability after 48 h of irisin stimulation at both 200 and 500 ng/mL and, after 72 h already at 100 ng/mL rec-irisin. These results could be due to irisin ability to downregulate the expression of Notch 3, which is important for cell-to-cell communication in the tumor niche, and Cyclin D1, supporting an inhibitory effect of irisin on MM cell proliferation. Overall, our findings suggest that irisin could be a new promising strategy to counteract MMBD and tumor burden in one shot.

The global incidence of Type 2 diabetes (T2D) is on the rise, fueled by factors such as obesity, sedentary lifestyles, socio-economic factors, and ethnic backgrounds. T2D is a multifaceted condition often associated with various health complications, including adverse effects on bone health. This study aims to assess key biomarkers linked to bone health and remodeling-Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL), and Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB)-among individuals with diabetes while exploring the impact of ethnicity on these biomarkers. A cross-sectional analysis was conducted on a cohort of 2083 individuals from diverse ethnic backgrounds residing in Kuwait. The results indicate significantly elevated levels of these markers in individuals with T2D compared to non-diabetic counterparts, with OPG at 826.47 (405.8) pg/mL, RANKL at 9.25 (17.3) pg/mL, and GPNMB at 21.44 (7) ng/mL versus 653.75 (231.7) pg/mL, 0.21 (9.94) pg/mL, and 18.65 (5) ng/mL in non-diabetic individuals, respectively. Notably, this elevation was consistent across Arab and Asian populations, except for lower levels of RANKL observed in Arabs with T2D. Furthermore, a positive and significant correlation between OPG and GPNMB was observed regardless of ethnicity or diabetes status, with the strongest correlation (r = 0.473, p < 0.001) found among Arab individuals with T2D. Similarly, a positive and significant correlation between GPNMB and RANKL was noted among Asian individuals with T2D (r = 0.401, p = 0.001). Interestingly, a significant inverse correlation was detected between OPG and RANKL in non-diabetic Arab individuals. These findings highlight dysregulation in bone remodeling markers among individuals with T2D and emphasize the importance of considering ethnic variations in T2D-related complications. The performance of further studies is warranted to understand the underlying mechanisms and develop interventions based on ethnicity for personalized treatment approaches.

BACKGROUND: Postmenopausal osteoporosis is a chronic metabolic bone disease caused by excessive osteoclast formation and function. Targeting osteoclast differentiation and activity can modulate bone resorption and alleviate osteoporosis. Cirsilineol, an active constituent of Vestita Wall, has shown numerous biological activities and has been used to treat many metabolic diseases. However, whether cirsilineol inhibits osteoclast activity and prevents postmenopausal osteoporosis still remain unknown.
METHODS: Primary bone marrow macrophages (BMMs) and RAW264.7 cells were used. Osteoclast activity was measured by TRAP staining, F-actin staining, and bone resorption assay after BMMs were treated with cirsilineol at concentrations of 0, 1, 2.5 and 5 µM. RT-PCR and western blotting were performed to evaluate the expression of osteoclast-related genes. In addition, female C57BL/6 mice underwent OVX surgery and were treated with cirsilineol (20 mg/kg) to demonstrate the effect of cirsilineol on osteoporosis.
RESULTS: Cirsilineol significantly inhibited receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation in a concentration- and time-dependent manner, respectively. Additionally, cirsilineol inhibited F-actin ring formation, thus reducing the activation of bone resorption ability. Cirsilineol suppressed the expression of osteoclast-related genes and proteins via blocking nuclear factor (NF)-κb, ERK, and p38 signaling cascades. More importantly, cirsilineol treatment in mice with osteoporosis alleviated osteoclasts hyperactivation and bone mass loss caused by estrogen depletion.
CONCLUSIONS: In this study, the protective effect of cirsilineol on osteoporosis has been investigated for the first time. In conclusion, our findings prove the inhibitory effect of cirsilineol on osteoclast activity via NF-κb/ERK/p38 signaling pathways and strongapplication of cirsilineol can be proposed as a potential therapeutic strategy.

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments.

UNASSIGNED: As the pivotal cellular mediators of bone resorption and pathological bone remodeling, osteoclasts have emerged as a prominent target for anti-resorptive interventions. Pinocembrin (PIN), a predominant flavonoid found in damiana, honey, fingerroot, and propolis, has been recognized for its potential therapeutic effects in osteolysis. The purpose of our project is to investigate the potential of PIN to prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms.
UNASSIGNED: The study commenced by employing protein-ligand molecular docking to ascertain the specific interaction between PIN and nuclear factor-κB (NF-κB) ligand (RANKL). Subsequently, PIN was introduced to bone marrow macrophages (BMMs) under the stimulation of RANKL. The impact of PIN on osteoclastic activity was assessed through the utilization of a positive TRAcP staining kit and a hydroxyapatite resorption assay. Furthermore, the study investigated the generation of reactive oxygen species (ROS) in osteoclasts induced by RANKL using H2DCFDA. To delve deeper into the underlying mechanisms, molecular cascades triggered by RANKL, including NF-κB, ROS, calcium oscillations, and NFATc1-mediated signaling pathways, were explored using Luciferase gene report, western blot analysis, and quantitative real-time polymerase chain reaction. Moreover, an estrogen-deficient osteoporosis murine model was established to evaluate the therapeutic effects of PIN in vivo.
UNASSIGNED: In this study, we elucidated the profound inhibitory effects of PIN on osteoclastogenesis and bone resorption, achieved through repression of NF-κB and NFATc1-mediated signaling pathways. Notably, PIN also exhibited potent anti-oxidative properties by mitigating RANKL-induced ROS generation and augmenting activities of ROS-scavenging enzymes, ultimately leading to a reduction in intracellular ROS levels. Moreover, PIN effectively abrogated the expression of osteoclast-specific marker genes (Acp5, Cathepsin K, Atp6v0d2, Nfatc1, c-fos, and Mmp9), further underscoring its inhibitory impact on osteoclast differentiation and function. Additionally, employing an in vivo mouse model, we demonstrated that PIN effectively prevented osteoclast-induced bone loss resultant from estrogen deficiency.
UNASSIGNED: Our findings highlight the potent inhibitory effects of PIN on osteoclastogenesis, bone resorption, and RANKL-induced signaling pathways, thereby establishing PIN as a promising therapeutic candidate for the prevention and management of osteolytic bone diseases.
UNASSIGNED: PIN serves as a promising therapeutic agent for the prevention and management of osteolytic bone diseases and holds promise for future clinical applications in addressing conditions characterized by excessive bone resorption. PIN is a natural compound found in various sources, including damiana, honey, fingerroot, and propolis. Its widespread availability and potential for therapeutic use make it an attractive candidate for further investigation and development as a clinical intervention.

Improper mechanical stress may induce side effects during orthodontic treatment. If the roots and alveolar bones are extensively resorbed following excess mechanical stress, unplanned tooth mobility and inflammation can occur. Although multiple factors are believed to contribute to the development of side effects, the cause is still unknown. Sonic hedgehog (Shh), one of the hedgehog signals significantly associated with cell growth and cancer development, promotes osteoclast formation in the jawbone. Shh may be associated with root and bone resorptions during orthodontic treatment. In this study, we investigated the relationships between Shh, RANKL, and IL-6 in human periodontal ligament (hPDL) cells exposed to improper mechanical force. Weights were placed on hPDL cells and human gingival fibroblasts (HGFs) for an optimal orthodontic force group (1.0 g/cm2) and a heavy orthodontic force group (4.0 g/cm2). A group with no orthodontic force was used as a control group. Real-time PCR, SDS-PAGE, and Western blotting were performed to examine the effects of orthodontic forces on the expression of Shh, RANKL, and IL-6 at 2, 4, 6, 8, 12, and 24 h after the addition of pressure. The protein expression of Shh was not clearly induced by orthodontic forces of 1.0 and 4.0 g/cm2 compared with the control in HGFs and hPDL cells. In contrast, RANKL and IL-6 gene and protein expression was significantly induced by 1.0 and 4.0 g/cm2 in hPDL cells for forces lasting 6~24 h. However, neither protein was expressed in HGFs. RANKL and IL-6 expressions in response to orthodontic forces and in the control were clearly inhibited by Shh inhibitor RU-SKI 43. Shh did not directly link to RANKL and IL-6 for root and bone resorptions by orthodontic force but was associated with cell activities to be finally guided by the production of cytokines in hPDL cells.

BACKGROUND: Lung fibrosis is a chronic lung disease with a high mortality rate with only two approved drugs (pirfenidone and nintedanib) to attenuate its progression. To date, there are no reliable biomarkers to assess fibrosis development and/or treatment effects for these two drugs. Osteoprotegerin (OPG) is used as a serum marker to diagnose liver fibrosis and we have previously shown it associates with lung fibrosis as well.
METHODS: Here we used murine and human precision-cut lung slices to investigate the regulation of OPG in lung tissue to elucidate whether it tracks with (early) fibrosis development and responds to antifibrotic treatment to assess its potential use as a biomarker.
RESULTS: OPG mRNA expression in murine lung slices was higher after treatment with profibrotic cytokines TGFβ1 or IL13, and closely correlated with Fn and PAI1 mRNA expression. More OPG protein was released from fibrotic human lung slices than from the control human slices and from TGFβ1 and IL13-stimulated murine lung slices compared to control murine slices. This OPG release was inhibited when murine slices were treated with pirfenidone or nintedanib. OPG release from human fibrotic lung slices was inhibited by pirfenidone treatment.
CONCLUSIONS: OPG can already be detected during the early stages of fibrosis development and responds, both in early- and late-stage fibrosis, to treatment with antifibrotic drugs currently on the market for lung fibrosis. Therefore, OPG should be further investigated as a potential biomarker for lung fibrosis and a potential surrogate marker for treatment effect.