Receptor Activator Of NF-κB Research Articles

Phlpp1 Expression in Osteoblasts Plays a Modest Role in Bone Homeostasis.

Prior work demonstrated that Phlpp1 deficiency alters limb length and bone mass, but the cell types involved and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within bone-forming osteoblasts, we crossed Phlpp1 floxed mice with mice harboring type 1 collagen (Col1a12.3kb)-Cre. Mineralization of bone marrow stromal cell cultures derived from Phlpp1 cKOCol1a1 was unchanged, but levels of inflammatory genes (eg, Ifng, Il6, Ccl8) and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratios were enhanced by either Phlpp1 ablation or chemical inhibition. Micro-computed tomography of the distal femur and L5 vertebral body of 12-week-old mice revealed no alteration in bone volume per total volume, but compromised femoral bone microarchitecture within Phlpp1 cKOCol1a1 conditional knockout females. Bone histomorphometry of the proximal tibia documented no changes in osteoblast or osteoclast number per bone surface but slight reductions in osteoclast surface per bone surface. Overall, our data show that deletion of Phlpp1 in type 1 collagen-expressing cells does not significantly alter attainment of peak bone mass of either males or females, but may enhance inflammatory gene expression and the ratio of RANKL/OPG. Future studies examining the role of Phlpp1 within models of advanced age, inflammation, or osteocytes, as well as functional redundancy with the related Phlpp2 isoform are warranted. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

The mechanism of metformin combined with total flavonoids of Rhizoma Drynariae on ovariectomy-induced osteoporotic rats

The present study evaluated the in vitro effect of metformin (Met) and total flavonoids of Rhizoma Drynariae (TFRD) on osteoclasts, osteocytes, and osteoblasts at different stages. We also assessed the effect and mechanism of treatment with Met combined with TFRD on ovariectomy (OVX)-induced osteoporosis in rats. The results showed that Met combined with TFRD significantly promoted the migration of osteoprogenitor cells and stimulated the differentiation and maturation of osteoblast precursor cells. Furthermore, Met combined with TFRD treatment significantly inhibited the osteogenic inhibitor sclerostin (SOST)/dickkopf 1 (DKK1) protein expression and the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) ratio in osteocytes. In the in vivo study, Met combined with TFRD effectively reduced bone resorption markers levels, including type-I collagen carboxy-terminal peptide (CTX-1) and tartrate-resistant acid phosphatase (TRAP), and remarkably increased the bone formation marker propeptide of type I procollagen (PINP) level in the serum of rats with osteoporosis. Met combined with TFRD treatment improved bone mineral density (BMD), trabecular microstructure, and mechanical properties of osteoporotic rats. Mechanistically, Met combined with TFRD downregulated SOST and DKK1 levels, and upregulated Wnt10b, β-catenin, runt-related transcription factor 2 (Runx2) et al. Meanwhile, Met combined with TFRD treatment reduced the RANKL/OPG ratio, and reduced the receptor activator of nuclear factor-κB (RANK), nuclear factor of activated T cells c1 (NFATC1), and TRAP levels. In conclusion, Met combined with TFRD ameliorated bone mass in osteoporotic rats through regulating Wnt/β-catenin signaling pathway and OPG/RANKL/RANK axis.

Co-targeting RANK pathway treats and prevents acquired resistance to CDK4/6 inhibitors in luminal breast cancer

The combination of endocrine therapy (ET) and cyclin-dependent kinase 4/6 (CDK4/6) inhibitors (CDK4/6i) was a hallmark in metastatic luminal breast cancer (BC). However, intrinsic and acquired resistance affects long-term efficacy. Here, we study the role of the receptor activator of nuclear factor-κB (RANK) pathway in CDK4/6i resistance. We find that RANK overexpression in luminal BC is associated with intrinsic resistance to CDK4/6i, both invitro and in mouse xenografts, and decreased proliferation rate and chronic interferon (IFN) γ response are highlighted as resistance drivers. Gene expression data from the NeoPalAna CDK4/6i clinical trial, and studies with palbociclib-resistant cell lines, show that RANK is upregulated after treatment with CDK4/6i, supporting a role in acquired resistance. Our study shows that RANK ligand (RANKL) inhibitors can restore sensitivity to CDK4/6i and prevent acquired resistance. On the basis of these findings, we conclude that pharmacological inhibition of the RANK pathway through RANKL blocking could represent an add-on to ET+ CDK4/6i, warranting further clinical studies.

Neuropsychiatric adverse reactions in patients treated with denosumab: two case reports and a review of data from the FDA Adverse Event Reporting System (FAERS).

Denosumab is a human monoclonal antibody indicated for patients with osteoporosis and a high risk of fractures. It targets RANKL, the receptor activator of NF-κB (RANK) ligand, blocking RANKL-RANK interaction and leading to rapid osteoclast-mediated bone resorption inhibition. But RANK is widely expressed in neurons, microglia, and astrocytes. RANKL/RANK/NF-κB system can play an important role in the neuroinflammatory response, depressive behavior, memory impairments, and neurotrophism. We present two well-documented case reports of recurrent neuropsychiatric manifestations in patients treated with denosumab and a descriptive review of similar cases reported to the Food and Drug Administration Adverse Event Reporting System (FAERS) database between 2012 and 2022. Only those reported by healthcare professionals, coding denosumab as the only suspected drug, were retained. An 81-year-old woman with pre-existing mild cognitive impairment suffered two acute confusional episodes and another 81-year-old woman with depression in remission suffered two depressive recurrences with anxiety and psychomotor inhibition, in both cases following sequential administrations of denosumab without underlying calcium/phosphate imbalance. Scores on Naranjo Adverse Drug Reaction Probability Scale were 6 and 7, respectively, suggesting a probable causal relationship. Of the 91,151 cases with denosumab exposure reported to FAERS, 5.7% were related to psychiatric/neurological disorders and 23.8% of these corresponded to cognitive impairment, depressive/mood disturbances, or psychomotor retardation. Denosumab may cause transient but severe neuropsychiatric symptoms by several mechanisms involving RANKL blockade and subsequent immuno-inflammatory changes, at least in subjects with pre-existing neurobiological vulnerability. We recommend caution and careful monitoring of these patients following denosumab administrations.

Fracture risks and their mechanisms in atopic dermatitis, focusing on receptor activator of nuclear factor kappa-Β ligand.

Recent multiple studies have shown that the long-term consequences of AD include an increased risk of osteoporosis and fracture, especially in hip, pelvic, spinal, and wrist fractures. AD is very common worldwide, and some kinds of fractures, such as hip fractures, are associated with increased mortality, which has a substantial socio-economic impact; however, the precise mechanisms for that remain unclear. Receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) and osteoprotegerin (OPG) are members of the tumor necrosis factor ligand and receptor family, members of which also are known as bone biomarkers. Alterations in the RANKL/RANK/OPG system and the balance among these factors (represented by the RANKL/OPG ratio) are central to the pathogenesis of bone loss from osteoporosis, and it is postulated that the potential association between the serum levels of RANKL and OPG, and bone density or fracture. Recently, we demonstrated that serum RANKL/OPG ratio positively correlated with AD severity and suggests fracture risk in older women with AD. This review summarizes and discusses the risk and mechanisms of osteoporotic fracture in AD. RANKL may be involved in the pathogenesis of AD, regarding not only bone abnormality but also inflammation. Although further investigation will be needed to verify the hypotheses, recent findings may provide new insights into AD's pathogenesis and therapeutic targets.

Role of Iron Accumulation in Osteoporosis and the Underlying Mechanisms.

Osteoporosis is prevalent in postmenopausal women. The underlying reason is mainly estrogen deficiency, but recent studies have indicated that osteoporosis is also associated with iron accumulation after menopause. It has been confirmed that some methods of decreasing iron accumulation can improve the abnormal bone metabolism associated with postmenopausal osteoporosis. However, the mechanism of iron accumulation-induced osteoporosis is still unclear. Iron accumulation may inhibit the canonical Wnt/β-catenin pathway via oxidative stress, leading to osteoporosis by decreasing bone formation and increasing bone resorption via the osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-B ligand (RANKL)/receptor activator of nuclear factor kappa-B (RANK) system. In addition to oxidative stress, iron accumulation also has been reported to inhibit either osteoblastogenesis or osteoblastic function as well as to stimulate either osteoclastogenesis or osteoclastic function directly. Furthermore, serum ferritin has been widely used for the prediction of bone status, and nontraumatic measurement of iron content by magnetic resonance imaging may be a promising early indicator of postmenopausal osteoporosis.

Vitamin D(3) and methylenebisphosphonic acid in the correction of mineral metabolism disorders and bone remodeling associated with glucocorticoid-induced osteoporosis

The study was aimed at evaluating therapeutic efficacy of vitamin D3 (VD3, 1000 IU/kg of b.w., 30 days) and sodium salt of methylenebisphosphonic acid (MBPA, 17 mg/kg of b.w., 30 days) monotherapies as well as their effect in combination in preventing mineral metabolism and bone remodeling disturbances associated with glucocorticoid(GC)-induced osteoporosis. Osteoporosis in rats was induced by long-term (30 days) administration of the synthetic glucocorticoid prednisolone (5 mg/kg of b.w.). Calcium and inorganic phosphate levels, activity of alkaline phosphatase (ALP) in serum, bone tissue and bone marrow were determined spectrophotometrically. The protein levels of VD3 receptor (VDR), receptor activator of nuclear factor kappa-B (RANK), its ligand (RANKL), and osteoprotegerin (OPG) in bone tissue were determined by Western blotting. Serum 25-hydroxyvitamin D3 (25OHD3) content was assayed by ELISA. It was shown that prednisolone caused the development of hypocalcemia and hypophosphatemia, increased the alkaline phosphatase activity in the blood serum, while downregulating its activity in bone tissue and bone marrow. GC-induced osteoporosis was accompanied by a profound deficiency of VD3 and a decrease in the content of VDR. Evaluation of the NF-κB-associated cytokine axis RANK/RANKL/OPG, which regulates the balance of osteoblasts/osteoclasts, showed a simultaneous decrease in the RANK content and OPG/RANKL ratio. Vitamin D3 restored mineral metabolism and 25OHD3 level that led to the normalization of VDR-mediated signaling­ and RANK/RANKL/OPG functions in bone tissue. It has been shown that the administration of MBPA had a corrective effect on the content of mineral components in the blood serum and bone tissue, as well as on the activity­ of alkaline phosphatase only in combination with vitamin D3, indicating a low efficiency of bisphosphonate monotherapy in GC-induced vitamin D3 deficiency and osteoporosis. Keywords: bone remode­ling, glucocorticoid-induced osteoporosis, methylenebisphosphonic acid, RANK/RANKL/OPG axis, vitamin D3

Citropten Ameliorates Osteoclastogenesis Related to MAPK and PLCγ/Ca2+ Signaling Pathways through the Regulation of Amyloid Beta.

Osteoporosis and Alzheimer's disease are typical types of dementia in seniors, which share common risk factors. Previous studies have shown that citizens with osteoporosis are more likely than healthy individuals to be at risk of Alzheimer's disease. Citropten, found in Citrus aurantifolia, has been reported to have several pharmacological activities; however, its antiosteoclastogenic activity remains unknown. Here, receptor activator nuclear factor κB ligand (RANKL)-induced osteoclast differentiation, formation, and function in the presence of amyloid beta (Aβ) were attenuated by citropten in the RAW 264.7 cell line. The expression of osteoclast specific genes and proteins indicated that citropten pretreatment lowers the MAPK and PLCγ/Ca2+ signaling pathways. Molecular docking simulations revealed that citropten interacts with the active sites of proteins in the calcium signaling pathway, which have negative binding affinities. These findings indicate that, through Aβ regulation, the RANKL-induced osteoclast can be suppressed by citropten, suggesting that citropten is a potential candidate for treating osteoclastogenesis-related diseases.

Histochemical assessment of osteoclast-like giant cells in Rankl−/− mice

ObjectivesWe examined mice with gene deletion of Receptor activator of nuclear factor-κB (Rank) ligand (Rankl) to histologically clarify whether they contained progenitor cells committed to osteoclastic differentiation up to the stage requiring RANK/RANKL signaling. MethodsThe tibiae and femora of ten-week-old male wild-type, c-fos−/−, and Rankl−/− mice were used for immunohistochemistry and transmission electron microscopy (TEM). ResultsIn Rankl−/− mice, we observed osteoclast-like giant cells, albeit in low numbers, with single or two nuclei, engulfing the mineralized extracellular matrix. TEM revealed that these giant cells contained large numbers of mitochondria, vesicles/vacuoles, and clear zone-like structures but no ruffled borders. They often engulfed fragmented bony/cartilaginous components of the extracellular matrix that had been degraded. Additionally, osteoclast-like giant cells exhibited immunoreactivity for vacuolar H+-ATPase, galectin-3, and siglec-15 but not for tartrate-resistant acid phosphatase, cathepsin K, or MMP-9, all of which are classical hallmarks of osteoclasts. Furthermore, osteoclast-like giant cells were ephrinB2-positive as they were near EphB4-positive osteoblasts that are also positive for alkaline phosphatase and Runx2 in Rankl−/− mice. Unlike Rankl−/− mice, c-fos−/− mice lacking osteoclast progenitors and mature osteoclasts had no ephrinB2-positive osteoclast-like cells or alkaline phosphatase-positive/Runx2-reactive osteoblasts. This suggests that similar to authentic osteoclasts, osteoclast-like giant cells might have the potential to activate osteoblasts in Rankl−/− mice. ConclusionsIt seems plausible that osteoclast-like giant cells may have acquired some osteoclastic traits and the ability to resorb mineralized matrices even when the absence of RANK/RANKL signaling halted the osteoclastic differentiation cascade.

Oncostatin M stimulates prostaglandin D2-induced osteoprotegerin and interleukin-6 synthesis in osteoblasts

Oncostatin M produced by osteal macrophages plays a significant role in fracture healing. Osteoprotegerin (OPG) secreted by osteoblasts, binds to the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) as a decoy receptor and prevents RANKL from binding to RANK, resulting in bone resorption suppression. Interleukin-6 (IL-6) is a pro-inflammatory cytokine and generally regulates bone resorption. However, accumulating evidence suggests that IL-6 plays pivotal roles in bone formation. We previously showed that prostaglandin D2 (PGD2) induces OPG synthesis by activating p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we demonstrated that PGD2 stimulates IL-6 synthesis by activating p38 MAP kinase and p44/p42 MAP kinase in MC3T3-E1 cells. In the present study, we investigated whether oncostatin M affects PGD2-stimulated OPG and IL-6 synthesis in MC3T3-E1 cells through MAP kinase activation. The osteoblast-like MC3T3-E1 cells and normal human osteoblasts were treated with oncostatin M and subsequently stimulated with PGD2. Consequently, oncostatin M significantly increased the PGD2-stimulated OPG and IL-6 release in both cells. Oncostatin M significantly enhanced mRNA expression levels of OPG and IL-6 induced by PGD2 similarly in both cells. Regarding the signaling mechanism, oncostatin M did not affect the phosphorylation of p38 MAP kinase, SAPK/JNK, and p44/p42 MAP kinase. Our results suggest that oncostatin M upregulates the PGD2-stimulated OPG and IL-6 synthesis in osteoblasts and therefore affects bone remodeling. However, OPG and IL-6 synthesis are not mediated through p38 MAP kinase, p44/p42 MAP kinase, or SAPK/JNK pathways.

Therapeutics of Charcot neuroarthropathy and pharmacological mechanisms: A bone metabolism perspective.

Charcot neuroarthropathy (CN) is a chronic, destructive, and painless damage of the skeletal system that affects the life quality of patients. CN, with an unclear mechanism, is characterized with invasive destruction of bones and a serious abnormality of bone metabolism. Unfortunately, development of an effective prevention and treatment strategy for CN is still a great challenge. Of note, recent studies providing an insight into the molecular mechanisms of bone metabolism and homeostasis have propelled development of novel CN therapeutic strategies. Therefore, this review aims to shed light on the pathogenesis, diagnosis, and treatment of CN. In particular, we highlight the eminent role of the osteoprotegerin (OPG)-receptor activator of nuclear factor-κB (RANK)-RANK ligand (RANKL) system in the development of CN. Furthermore, we summarize and discuss the diagnostic biomarkers of CN as well as the potential pharmacological mechanisms of current treatment regimens from the perspective of bone metabolism. We believe that this review will enhance the current state of knowledge on the diagnosis, prevention, and therapeutic efficacy of CN.

The mechanism underlying the remodeling effect of lactoferrin on midpalatal sutures during maxillary expansion and relapse in rats

The remodeling effects of intragastric administration and intramaxillary injection of lactoferrin (LF) on midpalatal sutures (MPS) during maxillary expansion and relapse in rats were studied to explore the underlying bone remodeling mechanism. Using a rat model of maxillary expansion and relapse, rats were treated with LF by intragastric administration (1 g·kg-1·d-1) or intramaxillary injection (5 mg·25 μl-1·d-1). The effects of LF on the osteogenic and osteoclast activities of MPS were observed by microcomputed tomography, histologic staining, and immunohistochemical staining, and the expressions of key factors in the extracellular regulated protein kinase 1/2 (ERK1/2) pathway and osteoprotegerin (OPG)-receptor activator of nuclear factor-KB ligand (RANKL)-receptor activator of nuclear factor-KB (RANK) axis were detected. Compared with the group with maxillary expansion alone, osteogenic activity was relatively enhanced, whereas osteoclast activity was relatively weakened in the groups administered LF, and the phosphorylated-ERK1/2: ERK1/2 and OPG: RANKL expression ratios increased significantly. The difference was more significant in the group administered LF intramaxillary. Administration of LF promoted osteogenic activity at MPS and inhibited osteoclast activity during maxillary expansion and relapse in rats, which may have occurred through regulation of the ERK1/2 pathway and the OPG-RANKL-RANK axis. The efficiency of intramaxillary LF injection was greater than that of intragastric LF administration.

Mechanism of artesunate on bone destruction in experimental rheumatoid arthritis based on transcriptomics and network pharmacology

The present study investigated the mechanism of artesunate in the treatment of bone destruction in experimental rheumatoid arthritis(RA) based on transcriptomics and network pharmacology. The transcriptome sequencing data of artesunate in the inhibition of osteoclast differentiation were analyzed to obtain differentially expressed genes(DEGs). GraphPad Prism 8 software was used to plot volcano maps and heat maps were plotted through the website of bioinformatics. GeneCards and OMIM were used to collect information on key targets of bone destruction in RA. The DEGs of artesunate in inhibiting osteoclast differentiation and key target genes of bone destruction in RA were intersected by the Venny 2.1.0 platform, and the intersection target genes were analyzed by Gene Ontology(GO)/Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment. Finally, the receptor activator of nuclear factor-κB(RANKL)-induced osteoclast differentiation model and collagen-induced arthritis(CIA) model were established. Quantitative real time polymerase chain reaction(q-PCR), immunofluorescence, and immunohistochemistry were used to verify the pharmacological effect and molecular mechanism of artesunate in the treatment of bone destruction in RA. In this study, the RANKL-induced osteoclast differentiation model in vitro was established and intervened with artesunate, and transcriptome sequencing data were analyzed to obtain 744 DEGs of artesunate in inhibiting osteoclast differentiation. A total of 1 291 major target genes of bone destruction in RA were obtained from GeneCards and OMIM. The target genes of artesunate in inhibiting osteoclast differentiation and the target genes of bone destruction in RA were intersected to obtain 61 target genes of artesunate against bone destruction in RA. The intersected target genes were analyzed by GO/KEGG enrichment. According to the results previously reported, the cytokine-cytokine receptor interaction signaling pathway was selected for experimental verification. Artesunate intervention in the RANKL-induced osteoclast differentiation model showed that artesunate inhibited CC chemokine receptor 3(CCR3), CC chemokine receptor 1(CCR1) and leukemia inhibitory factor(LIF) mRNA expression in osteoclasts in a dose-dependent manner compared with the RANKL-induced group. Meanwhile, the results of immunofluorescence and immunohistochemistry showed that artesunate could dose-dependently reduce the expression of CCR3 in osteoclasts and joint tissues of the CIA rat model in vitro. This study indicated that artesunate regulated the CCR3 in the cytokine-cytokine receptor interaction signaling pathway in the treatment of bone destruction in RA and provided a new target gene for the treatment of bone destruction in RA.

Inflammatory cytokine-enriched microenvironment plays key roles in the development of breast cancers.

As the incidence of breast cancer continues to increase, it is critical to develop prevention strategies for this disease. Inflammation underlies the onset of the disease, and NF-κB is a master transcription factor for inflammation. Nuclear factor-κB (NF-κB) is activated in a variety of cell types, including normal epithelial cells, cancer cells, cancer-associated fibroblasts (CAFs), and immune cells. Ductal carcinoma in situ (DCIS) is the earliest stage of breast cancer, and not all DCIS lesions develop into invasive breast cancers (IBC). Currently, most patients with DCIS undergo surgery with postoperative therapy, although there is a risk of overtreatment. In BRCA mutants, receptor activator of NF-κB (RANK)-positive progenitors serve as the cell of origin, and treatment using the RANK monoclonal antibody reduces the risk of IBC. There is still an unmet need to diagnose malignant DCIS, which has the potential to progress to IBC, and to establish appropriate prevention strategies. We recently demonstrated novel molecular mechanisms for NF-κB activation in premalignant mammary tissues, which include DCIS, and the resultant cytokine-enriched microenvironment is essential for breast cancer development. On the early endosomes in a few epithelial cells, the adaptor protein FRS2β, forming a complex with ErbB2, carries the IκB kinase (IKK) complex and leads to the activation of NF-κB, thereby inducing a variety of cytokines. Therefore, the FRS2β-NFκB axis in the inflammatory premalignant environment could be targetable to prevent IBC. Further analysis of the molecular mechanisms of inflammation in the premalignant microenvironment is necessary to prevent the risk of IBC.

Pulsed Electromagnetic Field (PEMF) Treatment Ameliorates Murine Model of Collagen-Induced Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease of the joint synovial membranes. RA is difficult to prevent or treat; however, blocking proinflammatory cytokines is a general therapeutic strategy. Pulsed electromagnetic field (PEMF) is reported to alleviate RA's inflammatory response and is being studied as a non-invasive physical therapy. In this current study, PEMF decreased paw inflammation in a collagen-induced arthritis (CIA) murine model. PEMF treatment at 10 Hz was more effective in ameliorating arthritis than at 75 Hz. In the PEMF-treated CIA group, the gross inflammation score and cartilage destruction were lower than in the untreated CIA group. The CIA group treated with PEMF also showed lower serum levels of IL-1β but not IL-6, IL-17, or TNF-α. Serum levels of total anti-type II collagen IgG and IgG subclasses (IgG1, IgG2a, and IgG2b) remained unchanged. In contrast, tissue protein levels of IL-1β, IL-6, TNF-α, receptor activator of nuclear factor kappa-Β (RANK), RANK ligand (RANKL), IL-6 receptor (IL-6R), and TNF-α receptor1 (TNFR1) were all lower in the ankle joints of the PEMF-treated CIA group compared with the CIA group. The results of this study suggest that PEMF treatment can preserve joint morphology cartilage and delay the occurrence of CIA. PEMF has potential as an effective adjuvant therapy that can suppress the progression of RA.

The Ethanol Extracts of Osmanthus fragrans Leaves Ameliorate the Bone Loss via the Inhibition of Osteoclastogenesis in Osteoporosis

The aim of this study was to evaluate the anti-osteoporosis effects of Osmanthus fragrans leaf ethanol extract (OFLEE) in bone marrow-derived macrophages (BMM) and animals with osteoporosis. OFLEE not only suppressed tartrate-resistant acid phosphatase (TRAP)-positive cells with multiple nuclei but also decreased TRAP activity in BMM treated with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). The formation of F-actin rings and the expression and activation of matrix metalloproteinases were decreased by OFLEE in BMM treated with M-CSF and RANKL. OFLEE suppressed M-CSF- and RANKL-induced osteoclastogenesis by inhibiting NF-κB phosphorylation, tumor necrosis factor receptor-associated factor 6, c-fos, the nuclear factor of activated T-cells, cytoplasmic 1, and cathepsin K in BMM. OFLEE downregulated reactive oxygen species, cyclooxygenase-2, inducible nitric oxide synthase, prostaglandin E2, tumor necrosis factor α, interleukin (IL)-1β, IL-6, IL-17, and RANKL in BMM treated with M-CSF and RANKL. Oral administration of OFLEE suppressed osteoporotic bone loss without hepatotoxicity in ovariectomy-induced osteoporosis animals. Our findings suggest that OFLEE, with anti-inflammatory effects, prevents osteoporotic bone loss through the suppression of osteoclastic differentiation in BMM and animals with osteoporosis.

RANKL inhibition: a new target of treating diabetes mellitus?

Accumulating evidence demonstrates the link between glucose and bone metabolism. The receptor activator of nuclear factor-kB ligand (RANKL)/the receptor activator of NF-κB (RANK)/osteoprotegerin (OPG) axis is an essential signaling axis maintaining the balance between bone resorption and bone formation. In recent years, it has been found that RANKL and RANK are distributed not only in bone but also in the liver, muscle, adipose tissue, pancreas, and other tissues that may influence glucose metabolism. Some scholars have suggested that the blockage of the RANKL signaling may protect islet β-cell function and prevent diabetes; simultaneously, there also exist different views that RANKL can improve insulin resistance through inducing the beige adipocyte differentiation and increase energy expenditure. Currently, the results of the regulatory effect on glucose metabolism of RANKL remain conflicting. Denosumab (Dmab), a fully human monoclonal antibody that can bind to RANKL and prevent osteoclast formation, is a commonly used antiosteoporosis drug. Recent basic studies have found that Dmab seems to regulate glucose homeostasis and β-cell function in humanized mice or in vitro human β-cell models. Besides, some clinical data have also reported the glucometabolic effects of Dmab, however, with limited and inconsistent results. This review mainly describes the impact of the RANKL signaling pathway on glucose metabolism and summarizes clinical evidence that links Dmab and DM to seek a new therapeutic strategy for diabetes.

Proinflammatory Activation of Osteoclasts Due to High Prolactin Level

High concentrations of prolactin (PRL) during the lactation period have an essential role in milk production by mammary glands stimulation. PRL may have an impact on calcium regulation and bone mineral density. We investigated if the PRL concentration during the lactation period could influence osteoclast (OC) activation and bone mineral density (BMD). In vivo, the Calcium Detection Assay, and ELISA were used to detect serum calcium, PRL, and inflammatory cytokines, respectively. BMD was evaluated by µ-CT in six months old female mice during lactation. The osteoclast (OC) activity was detected by Tartrate-resistant acid phosphatase (TRAP), Immunohistochemistry (IHC), and hematoxylin and eosin (H&E). In vitro, osteoclast differentiation, resorption and their activity markers TRAP, Matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), C-reactive protein (CRP), Receptor activator of nuclear factor kappa-Β (RANK) and inflammatory cytokines were measured in osteoclasts stimulated with recombinant prolactin protein (rPRL) or with an anti-prolactin blocker. We found that serum calcium, PRL, and inflammatory markers were increased. BMD was significantly reduced in lactating mice; TRAP activity was increased and tubercular was reduced in lactating mice compared to normal mice. In vitro, the osteoclast number, resorption, and activation markers TRAP, MMP-9, CTSK, CRP, and RANK were significantly increased after treatment with rPRL protein, but not in osteoclasts treated with anti-prolactin receptor antibody and rPRL. The gene expression of TNF-α, IL-6, and Monocyte chemoattractant protein-1 (MCP-1) but not IL-1b were significantly increased in osteoclasts with PRL treatment compared to the untreated osteoclasts. Taken together, the high level of PRL could activate osteoclasts and proinflammatory cytokines expression which reduce BMD in the lactation period.

Neutrophil extracellular traps induce the bone erosion of gout

ObjectiveTo investigate the relationships between monosodium urate (MSU) crystals -induced neutrophil extracellular traps (NETs) and bone erosion in gout.MethodsAnimal models were used to study the relationship between NETs induced by MSU crystals and bone erosion. Neutrophils were treated with MSU crystals to induce NETs. The osteoblasts-like cells (OB) were then treated with NETs, and the supernatant was co-incubated with osteoclasts-like cells (OC). The NETs were digested with DNase, and the neutrophil elastase (NE) was inhibited with sivelestat sodium. Cell viability, mRNA, and protein expression were also assessed.ResultsAfter treating OB with NETs, the cell viability decreased. Yet, after digesting the DNA and inhibiting NE, the viability was moderately improved. The expression level of osteoprotegerin (OPG) and alkaline phosphatase (ALP) was up-regulated, while the expression level of receptor activator of nuclear factor kappa-B ligand (RANKL) was down-regulated in the sivelestat sodium + MSU group compared with MSU group. The number of OC was significantly elevated. In contrast, the number of OB was not increased in the tibia after establishing the gout model. The supernatant obtained from OB was treated with NETs promoting OC differentiation. The expression level of receptor activator of nuclear factor kappa-B (RANK), tartrate-resistant acid phosphatase (TRAP), and cathepsin K (Cst K) was up-regulated in the MSU group compared with the normal control (NC) group.ConclusionNETs induced by MSU crystals could inhibit osteoblasts viability and enhance the activity of osteoclasts.

Effects of customized CAD/CAM abutments on cytokine levels in peri-implant crevicular fluid during early implant healing: a pilot study

ObjectivesThis study aimed to assess levels of biomarkers associated with inflammation and tissue destruction in peri-implant crevicular fluid (PICF) of implants provided with customized or standard healing abutments during early implant healing.Materials and methodsThirty implants were placed in 22 patients with partial posterior edentulism. Subsequently, test group implants (n=15) received one-piece titanium abutments that were fabricated using computer-aided design/computer-aided manufacturing (CAD/CAM). Control group implants (n=15) were provided with standard abutments. PICF collection and standardized periapical radiographs were carried out at suture removal one week later, following crown delivery after 3 months and at 6 months. Expression of C-reactive protein (CRP), interferon-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12A, IL-17A, macrophage inflammatory protein (MIP)-1α, matrix metalloproteinase (MMP)-13, osteopontin, osteoactivin, Receptor Activator of NF-κB (RANK), and TGF-β were analyzed using a multiplex ELISA kit.ResultsBoth groups showed a significant decrease in protein expression of CRP, IL-1β, IL-6, IL-8, MIP-1α, osteopontin, osteoactivin, and TGF-β, while MMP-13 levels increased during the observation period. A rise in OPG and RANK levels was detected among customized abutments. Expression of CRP was higher, whereas IL-1β, IL-1α, and MIP-1α were decreased in control compared to test group implants after 6 months. Marginal bone loss did not depend on abutment modality.ConclusionsBoth abutment types showed distinctive temporal expression of inflammatory biomarkers during 6 months following implant placement.Trial registrationISRCTN98477184, registration date 18/05/2022Clinical relevanceCustomized healing abutments exert similar effects on inflammation during early implant healing compared to standard healing abutments.