Nuclear factor-κB Ligand Research Articles

Histone Deacetylase 6 Inhibitor CKD-WID Suppressed Monosodium Urate-Induced Osteoclast Formation by Blocking Calcineurin-NFAT Pathway in RAW 264.7 Cells

Histone deacetylase (HDAC) has been found to play a crucial role in the regulation of osteoclast differentiation and formation. This study was designed to identify the effect of the HDAC6 inhibitor CKD-WID on the receptor for the activation of nuclear factor-κB ligand (RANKL)-mediated osteoclast formation in the presence of monosodium urate (MSU) in RAW 264.7 murine macrophage cells. The expression of osteoclast-specific target genes, calcineurin, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was evaluated in RAW 264.7 murine macrophages treated with MSU, RANKL, or CKD-WID by real-time quantitative polymerase chain reaction and Western blot assay. The effect of CKD-WID on osteoclast formation was measured by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring formation staining, and assays for bone resorption activity. RANKL in the presence of MSU significantly induced HDAC6 gene and protein expression in RAW 264.7 cells. CKD-WID markedly suppressed the expression of osteoclast-related markers such as c-Fos, TRAP, cathepsin K, and carbonic anhydrase II induced by co-stimulation with RANKL and MSU in RAW 264.7 cells. Transcription factor NFATc1 mRNA expression and nuclear NFATc1 protein expression induced by co-stimulation with RANKL and MSU were significantly inhibited by CKD-WID treatment. CKD-WID also decreased the number of TRAP-positive multinuclear cells and F-actin ring-positive cells and attenuated bone resorption activity. Co-stimulation with RANKL and MSU increased calcineurin gene and protein expression, which was significantly blocked by CKD-WID treatment. The HDAC6 inhibitor CKD-WID suppressed MSU-induced osteoclast formation through blocking the calcineurin-NFAT pathway in RAW 264.7 cells. This suggests that HDAC6 is considered a therapeutic target in uric acid-mediated osteoclastogenesis.

The STING inhibitor C-176 attenuates osteoclast-related osteolytic diseases by inhibiting osteoclast differentiation.

Inflammatory osteolysis occurs primarily in the context of osteoarthritis, aseptic inflammation, prosthesis loosening, and other conditions. An excessive immune inflammatory response causes excessive activation of osteoclasts, leading to bone loss and bone destruction. The signaling protein stimulator of interferon gene (STING) can regulate the immune response of osteoclasts. C-176 is a furan derivative that can inhibit activation of the STING pathway and exert anti-inflammatory effects. The effect of C-176 on osteoclast differentiation is not yet clear. In this study, we found that C-176 could inhibit STING activation in osteoclast precursor cells and inhibit osteoclast activation induced by nuclear factor κB ligand receptor activator in a dose-dependent manner. After treatment with C-176, the expression of the osteoclast differentiation marker genes nuclear factor of activated T-cells c1(NFATc1), cathepsin K, calcitonin receptor, and V-ATPase a3 decreased. In addition, C-176 reduced actin loop formation and bone resorption capacity. The WB results showed that C-176 downregulated the expression of the osteoclast marker protein NFATc1 and inhibited activation of the STING-mediated NF-κB pathway. We also found that C-176 could inhibit the phosphorylation of mitogen-activated protein kinase signaling pathway factors induced by RANKL. Moreover, we verified that C-176 could reduce LPS-induced bone absorption in mice, reduce joint destruction in knee arthritis induced by meniscal instability, and protect against cartilage matrix loss in ankle arthritis induced by collagen immunity. In summary, our findings demonstrated that C-176 could inhibit the formation and activation of osteoclasts and could be used as a potential therapeutic agent for inflammatory osteolytic diseases.

Diosmin mitigates dexamethasone-induced osteoporosis in vivo: Role of Runx2, RANKL/OPG, and oxidative stress

Secondary osteoporosis is commonly caused by long-term intake of glucocorticoids (GCs), such as dexamethasone (DEX). Diosmin, a natural substance with potent antioxidant and anti-inflammatory properties, is clinically used for treating some vascular disorders. The current work targeted exploring the protective properties of diosmin to counteract DEX-induced osteoporosis in vivo. Rats were administered DEX (7 mg/kg) once weekly for 5 weeks, and in the second week, vehicle or diosmin (50 or 100 mg/kg/day) for the next four weeks. Femur bone tissues were collected and processed for histological and biochemical examinations. The study findings showed that diosmin alleviated the histological bone impairments caused by DEX. In addition, diosmin upregulated the expression of Runt-related transcription factor 2 (Runx2) and phosphorylated protein kinase B (p-AKT) and the mRNA transcripts of Wingless (Wnt) and osteocalcin. Furthermore, diosmin counteracted the rise in the mRNA levels of receptor activator of nuclear factor-kB ligand (RANKL) and the reduction in osteoprotegerin (OPG), both were induced by DEX. Diosmin restored the oxidant/antioxidant equilibrium and exerted significant antiapoptotic activity. The aforementioned effects were more pronounced at the dose level of 100 mg/kg. Collectively, diosmin has proven to protect rats against DEX-induced osteoporosis by augmenting osteoblast and bone development while hindering osteoclast and bone resorption. Our findings could be used as a stand for recommending supplementation of diosmin for patients chronically using GCs.

Insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis

Pulmonary alveolar microlithiasis is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a pulmonary alveolar microlithiasis lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes and other proteins suggested a role for osteoclast-like cells in the host response to microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activation in a receptor activator of nuclear factor-κB ligand and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.

Abstract PD9-03: Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment

Abstract Background: Estrogen receptor positive (ER+)/HER-2 negative breast cancer (BC) is considered to be an immunologically cold tumor compared to triple negative breast cancer. Therefore, the tumor microenvironment (TME) of ER+/HER-2 negative BC is understudied. The receptor activator of nuclear factor-kB ligand (RANKL)-RANK pathway was first identified, as mediator of T and dendritic cells interaction, but it is mostly known for its role as key regulator of bone remodeling and pathophysiology of bone metastases. RANK is a member of the tumor necrosis factor receptor (TNFR) superfamily that is activated upon RANKL binding, promoting cell proliferation, survival and differentiation. The RANKL-RANK pathway also emerged as a major mediator of hormone-driven breast carcinogenesis. The aim of this study is to investigate the TME and the immune response during neoadjuvant endocrine therapy (NET) and to correlate this with the treatment response in a real life setting. Methods: Expressions of immune checkpoint receptors and immune cells were examined immunohistochemically in pre- and post- NET on a cohort of 44 ER+/HER-2 negative BC patients. They were treated with tamoxifen (N=8), an aromatase inhibitor (N=36) or a combination of an aromatase inhibitor with a PI3K inhibitor (N= 7) for a median duration of 6 months (range 1-32) months. Monoclonal antibodies for PDL-1, PD-1, TIM-3, LAG-3, CTLA-4, CD4, CD68, FOXP3, RANK and RANKL were used. All staining were done according to validated protocols and scoring was done by a pathologist specialized in breast cancer. Positivity was defined as staining > 1% on TILs. Response to NET was evaluated according to tumor size change on imaging and Ki-67 change. Results: The median age was 62.5 (44–90.3) years. Diameter of tumor size decreased with a mean of 7.818 mm (p < 0.0001) during NET and the value of Ki-67 value decreased significantly after NET (value, p< 0.0004). An increase in PD-L1 expression after NET showed a trend towards significant (p= 0.088) and RANK expression on TILs significantly decreased with a median of 30% (range= -70 to 85) (p= 0.0007) after NET. A good response to NET defined as a decrease in tumor size and/or decrease of Ki-67 was found to be associated with a longer duration of NET, a change of CD4+ T-cells, a change of RANK expression on TILs and a higher number of CD68+ tumor-associated macrophages before the start of NET and also RANK expression on TILs before the start of NET. Conclusion: The immune micro-environment plays an important role in ER+/HER-2 negative BC. NET influences the composition and/or functional state of the infiltrating immune cells. Furthermore, changes in the immune micro-environment are also associated with treatment response. Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment Comparison of continuous and categorical parameters before and after NET. Comparison of the continuous parameters was done using Wilcoxon signed rank test. Comparison of the categorical parameters was done using a Chi-square test. sTIL: stromal tumour infiltrating lymphocytes and NET: neoadjuvant endocrine therapy. Bold values denote statistical significance at the p < 0.05 level. Citation Format: Gizem Oner, Glenn Broeckx, Christophe Van Berckelaer, Sevilay Altintas, Zafer Canturk, Wiebren Tjalma, Karen Zwaenepoel, Zwi Berneman, Marc Peeters, Patrick Pauwels, Peter A van Dam. Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment Breast Cancer Microenvironment Change After Neoadjuvant Endocrine Treatment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-03.

GCF Levels of Osteoclastogenesis-Related Cytokines in Periodontitis in Relation to Smoking During Non-Surgical Periodontal Therapy

Objective: Interleukins (IL) -1β, -34, receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) play a crucial role in osteoclastogenesis and bone resorption through modulating inflammatory processes and osteoclastogenesis. Smoking is the major risk factor in the initiation and progression of the periodontitis, and adversely affects the outcomes of non-surgical periodontal therapy. To date, there is no study investigating both gingival crevicular fluid (GCF) IL-1β, IL-34, RANKL, and OPG levels before and after non-surgical periodontal therapy in smoking and non-smoking patients with periodontitis stage 3, grade B and C. The aim of current research was to examine the GCF levels of some osteoclastogenesis-related cytokines in periodontitis in relation to smoking before and after periodontal therapy. Methods: At baseline, full-mouth periodontal status together with GCF samples were collected from 116 individuals, including 60 periodontitis patients (30 smokers and 30 nonsmokers) and 56 periodontally healthy controls (28 smokers and 28 nonsmokers). Non-surgical periodontal therapy, consisting of instruction for daily plaque control and scaling and root planing (SRP), was performed. GCF sampling and full-mouth periodontal measurements were repeated 6 weeks after completion of SRP. The GCF levels of biomarkers were measured by enzyme-linked immunosorbent assay. Results: The periodontitis groups exhibited significant improvement in clinical parameters. At baseline, the GCF IL-1β levels in periodontitis groups were significantly higher than periodontally healthy controls (p<0.05) and it was significantly decreased in periodontitis groups after non-surgical periodontal therapy. At baseline, the GCF IL-34 levels in periodontitis groups were significantly higher than periodontal healthy controls(p<0.05) and the GCF IL-34 level was significantly decreased in non-smoking periodontitis patients. At baseline and after periodontal therapy, the GCF RANKL levels were similar in all groups. The GCF OPG level was significantly lowest in non-smoking periodontitis patients at baseline and the GCF OPG level was significantly increased in smoking and non-smoking periodontitis patients after non-surgical periodontal therapy. Conclusion: In the periodontal inflammation process, GCF IL-34 level followed a similar pathway to GCF IL-1β, suggesting that IL-34 may be a marker in the pathogenesis of periodontal disease. The significant decrease in GCF IL-34 and a significant increase in GCF OPG level in the non-smoker periodontitis group after periodontal therapy suggest the negative effect of smoking on the response to periodontal therapy. More comprehensive studies are needed by increasing the number of samples included in the study groups in order to better understand the pathogenesis of periodontitis.

Pulsed frequency modulated ultrasound promotes therapeutic effects of osteoporosis induced by ovarian failure in mice

Low-intensity pulsed ultrasound (LIPUS) has been proved to be an effective technique for the treatment of osteoporosis. To better activate the bone formation-related markers, promote the different stages of osteogenesis, and further enhance the therapeutic effects of ultrasound, this study employed pulsed frequency modulated ultrasound (pFMUS) to treat mice with osteoporosis, which was caused by ovarian failure due to 4-vinylcyclohexene dioxide (VCD) injection. Healthy 8-week-old female C57BL/6J mice were randomly divided into four groups: Sham (S), VCD-control (V), VCD + LIPUS (VU), and VCD + pFMUS (VFU). VU and VFU groups were treated by LIPUS and pFMUS, respectively. Serum analysis, micro-computed tomography (micro-CT), mechanical testing and hematoxylin and eosin (HE) staining were performed to evaluate the therapeutic effects of ultrasound. Quantitative reverse-transcription PCR (qRT-PCR) and western blot analysis were used to explore the mechanism of ultrasound on osteoporosis. Results showed that pFMUS might have better therapeutic effects than traditional LIPUS in terms of bone microstructure and bone strength. In addition, pFMUS could promote bone formation by activating phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) pathway, and slow down bone resorption by increasing osteoprotegerin/receptor activator of nuclear factor κB ligand (OPG/RANKL) ratio. This study is of positive prognostic significance when understanding the mechanism of ultrasound regulation on osteoporosis and establishing novel treatment plan of osteoporosis by multi-frequency ultrasound.

Difference in apical resorption activity during rat molar root formation in response to mechanical force.

To investigate whether there is a difference in apical resorption activity during the development of roots in response to mechanical force in vivo. Maxillary first molars (M1) from postnatal day (PN) 21 and PN35 male rats were selected as representatives of the root-developing and root-completing groups, respectively. A mechanical force of 3 cN was applied to M1 on PN21 and PN35, and the maxilla was collected on PN28 and PN42. Odontoclastogenesis and root morphology were investigated using micro-focus X-ray computed tomography, followed by immunohistochemistry and quantitative real-time polymerase chain reaction to clarify root resorption activity. Development of the mesiobuccal root (MBR) preceded the mesial root (MR). In the PN28 force application (FA) group, the dentine was bent, but the histology, including Hertwig's epithelial root sheath (HERS), was intact. No odontoclasts and resorption lacunae were found in the apical area of the MRs, and only lateral root resorption was observed. External apical root resorption (EARR) was observed in the MR of PN42 (FA) and in the MBR of both PN28 (FA) and PN42 (FA). The expression of osteopontin changed accordingly. No significant change occurred in osteoprotegerin or receptor activator of nuclear factor-κB ligand expression in the MRs of the PN28 (FA) group. Our animal model did not adequately simulate the clinical process of tooth movement in humans. Force application delayed HERS dissociation on the compression side of the developing roots, leading to inhibitory effects on cementogenesis, which resulted in decreased odontoclast differentiation and prevention of EARR.

In Vitro Effect of Photobiomodulation Therapy with 980 nm Diode Laser on Gene Expression of Key Regulators of Bone Remodeling by Human Periodontal Ligament Cells under Mild Orthodontic Forces.

This study investigated the effect of photobiomodulation (PBM) with 980 nm diode laser as monotherapy and in combination with compressive and tensile orthodontic forces on expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), sclerostin (SOST) and periostin (POSTN), by human periodontal ligament cells. Isolated cells were cultured and subjected to either tensile (10% elongation) or compressive forces (25 g cm-2 ) for 24 and 48 h. Subsequently, the cells received PBM (100 mW power, 3 or 6 J cm-2 energy density) immediately after load cycle. RT-PCR was applied to assess the genes expression. Data were analyzed by one-way ANOVA, followed by post hoc Tukey test (P≤ 0.05). We found that PBM in combination with orthodontic forces led to upregulation of bone resorption genes (RANKL and SOST) at the pressure side and their downregulation at the tension side. The expression of osteogenic genes (OPG and POSTN) increased at the tension side and decreased at the pressure side. PBM alone did not affect gene expression. In conclusion, these findings suggest that this PBM protocol may be effective in enhancement of the gene expression in favor of bone remodeling acceleration that should be confirmed in future animal and human studies.

Chlorhexidine gel topical application ameliorates inflammatory bone loss in experimental periodontitis

This study aimed to evaluate the impact of chlorhexidine (CHX) gel on inflammation-induced periodontal tissue destruction, osteoclastogenesis, subgingival microbiota, and on the modulation of the RANKL/OPG as well as inflammatory mediators during bone remodeling invivo. Ligation- and LPS injection-induced experimental periodontitis were created to investigate the effect of topical application of CHX gel invivo. Alveolar bone loss, osteoclast number and gingival inflammation was evaluated by micro-CT, histological, immunohistochemistry and biochemical analysis. The composition of the subgingival microbiota was characterized by 16S rRNA gene sequencing. Data shows significant decreases in the alveolar bone destruction in rats from ligation-plus-CHX gel group compared to ligation group. In addition, significant decreases in the number of osteoclasts on bone surface and the protein level of receptor activator of nuclear factor κB ligand (RANKL) in gingival tissue were observed in rats from ligation-plus-CHX gel group. Moreover, data shows significantly decreased inflammatory cell infiltration and decreased expression of cyclooxygenase (COX-2) and inducible NO synthase (iNOS) in gingival tissue from ligation-plus-CHX gel group versus ligation group. Assessment of the subgingival microbiota revealed changes in rats with CHX gel application treatment. HX gel presents protective effect on gingival tissue inflammation, osteoclastogenesis, RANKL/OPG expression, inflammatory mediators, and alveolar bone loss invivo, which may have a translational impact on the adjunctive use in the management of inflammation-induced alveolar bone loss.

A dual functional Ti-Ga alloy: inhibiting biofilm formation and osteoclastogenesis differentiation via disturbing iron metabolism

BackgroundAlthough biomedical implants have been widely used in orthopedic treatments, two major clinical challenges remain to be solved, one is the bacterial infection resulting in biofilm formation, and the other is aseptic loosening during implantation due to over-activated osteoclastogenesis. These factors can cause many clinical issues and even lead to implant failure. Thus, it is necessary to endow implants with antibiofilm and aseptic loosening-prevention properties, to facilitate the integration between implants and bone tissues for successful implantation. To achieve this goal, this study aimed to develop a biocompatible titanium alloy with antibiofilm and anti-aseptic loosening dual function by utilizing gallium (Ga) as a component.MethodsA series of Ti-Ga alloys were prepared. We examined the Ga content, Ga distribution, hardness, tensile strength, biocompatibility, and anti-biofilm performance in vitro and in vivo. We also explored how Ga3+ ions inhibited the biofilm formation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and osteoclast differentiation.ResultsThe alloy exhibited outstanding antibiofilm properties against both S. aureus and E. coli in vitro and decent antibiofilm performance against S. aureus in vivo. The proteomics results demonstrated that Ga3+ ions could disturb the bacterial Fe metabolism of both S. aureus and E. coli, inhibiting bacterial biofilm formation. In addition, Ti-Ga alloys could inhibit receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteoclast differentiation and function by targeting iron metabolism, then suppressing the activation of the NF-κB signaling pathway, thus, showing their potential to prevent aseptic loosening.ConclusionThis study provides an advanced Ti-Ga alloy that can be used as a promising orthopedic implant raw material for various clinical scenarios. This work also revealed that iron metabolism is the common target of Ga3+ ions to inhibit biofilm formation and osteoclast differentiation.

Rheumatic disease Risk factors, pathophysiology and management

Rheumatic diseases, because of their systemic inflammatory component, are associated with several shared comorbidities. The high affinity of shared epitope for citrullinated peptides could accelerate the amount of human leukocyte antigen peptide complexes on the surface of antigen-presenting cells, thus leading to a (possible joint-specific) T- cell response. Molecules such as receptor activator of nuclear factor κB ligand, prostaglandins, and matrix metalloproteinases are induced by pro-inflammatory cytokines, involving tumor necrosis factor and interleukin 6, and mediate signs and symptoms of the disease, involving pain and swelling, and degradation of cartilage and bone. Steroids at dosages equivalent to less than 10 mg of prednisone daily are highly effective for relieving symptoms of rheumatoid arthritis and can slow joint injury.

BML-111 inhibits osteoclast differentiation by suppressing the MAPK and NF-κB pathways, alleviating deterioration of the knee joints in a CIA rat model.

Irreversible destruction of joints is the hallmark of rheumatoid arthritis (RA). Osteoclasts are the only bone-resorbing cells and play an important role in joint rebuilding. BML-111 (5(S),6(R),7-trihydroxyheptanoic acid methyl ester, C8 H16 O5 ) is a synthetic lipoxin A4 agonist with antioxidant and anti-inflammatory properties. The present study aimed to investigate the effect of BML-111 on osteoclasts in vivo and in vitro, to investigate its therapeutic effect on joint destruction in RA. Cell Counting Kit-8assay and flow cytometry were used to exclude cytotoxic effects of BML-111 to bone marrow-derived macrophages(BMMs). Then, osteoclasts were differentiated in vitro from BMMs by used macrophage colony-stimulating factorand receptor activator of nuclear factor-κB ligand,and osteoclasts were observed following tartrate-resistant acid phosphatase staining with or without BML-111 treatment. Meanwhile, absorption pit assay and immunofluorescence staining of the fibrous actin ring were used to observe osteoclast function. Moreover, we examined mitogen-activated protein kinase(MAPK) and nuclear factor-κB (NF-κB) activation. We established collagen-induced arthritisin a rat model and, after treatment with BML-111, joint swelling was measured and the knee joints were processed for histology. We also examined serum and tissue for osteoclastogenesis-related markers. BML-111 inhibited osteoclast formation and differentiation in a time- and concentration-dependent manner, and downregulated the expression levels of MAPK and NF-κB in vitro. Meanwhile, BML-111 effectively alleviated joint structural damage and inhibited osteoclast formation in vivo. BML-111 inhibited osteoclast formation and differentiation in vitro and in vivo, and delayed the progression of joint destruction.

Isosinensetin alleviates estrogen deficiency-induced osteoporosis via suppressing ROS-mediated NF-κB/MAPK signaling pathways

The formation of osteoclasts and their hyperactive bone resorption are related to the aggregation of intracellular reactive oxygen species (ROS). Flavonoids, derived from plant active ingredients, can alleviate the symptoms of osteoporosis (OP). Isosinensetin (Iss) is a flavonoid with antioxidant effects obtained mainly from citrus fruits, and its effect on osteoclastogenesis has not been reported. In this study, we investigated the antioxidant activity of Iss on osteoclast differentiation and function, as well as the therapeutic impact of Iss on OP. We found that Iss inhibited osteoclastogenesis and suppressed the bone resorption function of osteoclasts. Additionally, Iss reduced receptor activator of nuclear factor-κB ligand (RANKL)-induced intracellular ROS. Using quantitative real-time polymerase chain reaction and western blot, we further found that Iss inhibited osteoclast-specific genes and related proteins, while promoting the expression of antioxidant enzyme-related genes and proteins. Mechanistically, Iss reduces intracellular ROS by activating nuclear factor-erythroid 2-related factor 2 (Nrf2) and its related antioxidant enzymes and inhibits the downstream nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways of ROS, which in turn inhibits nuclear factor of activated T cells 1 (NFATc1), and ultimately inhibits osteoclastogenesis. In vivo, by micro-computed tomography (Micro-CT) assay and histological analyses, we found that Iss could reduce bone loss in ovariectomized (OVX) mice. Therefore, Iss has the potential as an OP preventative and therapeutic drug option.

α-Linolenic Acid Inhibits RANKL-Induced Osteoclastogenesis In Vitro and Prevents Inflammation In Vivo.

Inflammation is an important risk factor for bone-destroying diseases. Our preliminary research found that Zanthoxylum bungeanum seed oil (ZBSO) is abundant in unsaturated fatty acids and could inhibit osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells. However, the key constituents in ZBSO in the prevention of osteoclastogenesis and its possible mechanism related to inflammation are still unclear. Therefore, in this study, oleic acid (OA), linoleic acid (LA), palmitoleic acid (PLA), and alpha-linolenic acid (ALA) in ZBSO, havingthe strongest effect on RANKL-induced osteoclastogenesis, were selected by a tartrate-resistant acid phosphatase (TRAP) staining method. Furthermore, the effects of the selected fatty acids on anti-inflammation and anti-osteoclastogenesis in vitro and in vivo were assessed using RT-qPCR. Among the four major unsaturated fatty acids we tested, ALA displayed the strongest inhibitory effect on osteoclastogenesis. The increased expression of free fatty acid receptor 4 (FFAR4) and β-arrestin2 (βarr2), as well as the decreased expression of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), nuclear factor of activated T-cells c1 (NFATc1), and tartrate-resistant acid phosphatase (TRAP) in RAW264.7 cells after ALA treatment were observed. Moreover, in ovariectomized osteoporotic rats with ALA preventive intervention, we found that the expression of TNF-α, interleukin-6 (IL-6), interleukin-1β (IL-1β), NFATc1, and TRAP were decreased, while with the ALA therapeutic intervention, downregulated expression of NF-κB, NFATc1, TRAP, and transforming growth factor beta-activated kinase 1 (TAK1) were noticed. These results indicate that ALA, as the major unsaturated fatty acid in ZBSO, could inhibit RANKL-induced osteoclastogenesis via the FFAR4/βarr2 signaling pathway and could prevent inflammation, suggesting that ZBSO may be a promising potential natural product of unsaturated fatty acids and a dietary supplement for the prevention of osteoclastogenesis and inflammatory diseases.

Eldecalcitol effectively prevents alveolar bone loss by partially improving Th17/Treg cell balance in diabetes-associated periodontitis.

Background: Diabetes-associated periodontitis (DPD) is an inflammatory and destructive disease of periodontal tissues in the diabetic population. The disease is manifested as more severe periodontal destruction and is more difficult to treat when compared with periodontitis (PD). Eldecalcitol (ELD) is a novel active vitamin D3 analog; however, little clinical evidence is available on its role on improving PD and DPD, and its specific mechanisms remain unclear. In this study, we evaluated the preventative effects of ELD toward PD and DPD and explored its underlying molecular mechanisms. Methods: Experimental PD and DPD mouse models were established by ligation combined with lipopolysaccharide (LPS) from Porphyromonas gingivalis injection in C57BL/6J and C57BLKS/J Iar- + Leprdb/+Leprdb (db/db) mice, respectively. Simultaneously, ELD (0.25μg/kg) was orally administered to mice via an intragastric method. Micro-computed tomography (CT), hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), and tartrate-resistant acid phosphatase (TRAP) staining were used to evaluate alveolar bone alterations in vivo. Flow cytometry, immunofluorescence, and real-time polymerase chain reaction (qRT-PCR) were also used to examine gene expression and probe systemic and local changes in Treg and Th17 cell numbers. Additionally, western blotting and immunofluorescence staining were used to examine changes in STAT3/STAT5 signaling. Results: Micro-CT and HE staining showed that the DPD group had higher alveolar bone loss when compared with the PD group. After applying ELD, alveolar bone loss decreased significantly in both PD and DPD groups, and particularly evident in the DPD group. IHC and TRAP staining also showed that ELD promoted osteoblast activity while inhibiting the number of osteoclasts, and after ELD treatment, the receptor activator of nuclear factor-κB ligand (RANKL) to osteoprotegerin (OPG) ratio decreased. More importantly, this decreasing trend was more obvious in the DPD group. Flow cytometry and qRT-PCR also showed that the systemic Th17/Treg imbalance in PD and DPD groups was partially resolved when animals were supplemented with ELD, while immunofluorescence staining and qRT-PCR data showed the Th17/Treg imbalance was partially resolved in the alveolar bone of both ELD supplemented groups. Western blotting and immunofluorescence staining showed increased p-STAT5 and decreased p-STAT3 levels after ELD application. Conclusion: ELD exerted preventative effects toward PD and DPD by partially rectifying Th17/Treg cell imbalance via STAT3/STAT5 signaling. More importantly, given the severity of DPD, we found ELD was more advantageous in preventing DPD.

Lichen Planus After Denosumab

AbstractLichenoid reactions have been described as a side effect of several drugs. Here we describe the development of lichen planus (LP) of the mammilla following off-label use of denosumab in an adolescent patient with mono-ostotic fibrous dysplasia. Lichen planus is considered an autoimmune disorder. Since receptor activator of nuclear factor κΒ ligand (RANKL), the target of denosumab action, has regulatory function in the immune system, it is feasible, that LP may be caused as part of an immunological dysregulation.

Diagnostic potential of salivary interleukin-17, RANKL, and OPG to differentiate between periodontal health and disease and discriminate stable and unstable periodontitis: A case-control study.

Limitations of the conventional diagnostic techniques urged researchers to seek novel methods to predict, diagnose, and monitor periodontal disease. Use of the biomarkers available in oral fluids could be a revolutionary surrogate for the manual probing/diagnostic radiograph. Several salivary biomarkers have the potential to accurately discriminate periodontal health and disease. This study aimed to determine the diagnostic sensitivity and specificity of salivary interleukin (IL)-17, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), RANKL/OPG for differentiating (1) periodontal health from disease and (2) stable and unstable periodontitis. Participants with periodontitis (n = 50) and gingivitis (n = 25), both diseases represented the cases, and subjects with healthy periodontium (n = 15) as a control were recruited for this study. Periodontitis cases were further equally subdivided into stable and unstable. Whole unstimulated salivary sample were collected from all participants. Periodontal parameters including bleeding on probing, probing pocket depth, clinical attachment loss, and number of missing teeth were recorded. The protein levels of salivary IL-17, RANKL, and OPG were determined by using enzyme-linked immunosorbent assays technique. Salivary IL-17, OPG, RANKL, and RANKL/OPG showed high sensitivity and specificity to differentiate periodontal health from gingivitis and periodontitis. Similar pattern was observed in discriminating stable and unstable periodontitis. Salivary IL-17 and RANKL showed a good accuracy to differentiate gingivitis from periodontitis. However, OPG and RANKL/OPG did not exhibit enough sensitivity and specificity to differentiate the latter conditions. Salivary IL-17, RANKL, OPG, and RANKL/OPG system are potential candidates for differentiating periodontal health and disease and discriminate stable and unstable periodontitis.

Isoimperatorin alleviates lipopolysaccharide-induced periodontitis by downregulating ERK1/2 and NF-κB pathways.

Chronic periodontitis is an inflammatory disease characterized by inflammation of the soft tissues of the gums. To combat this disease, more effective drugs are still needed to identify and develop. Isoimperatorin is a kind of a natural compound, which has anti-inflammatory, analgesic, antitumor, antivirus, and other pharmacological effects. However, its possible effects on the progression of chronic periodontitis are still unclear. In this study, we used human periodontal membrane fibroblasts (hPDLCs), human bone marrow-derived macrophages, and found that isoimperatorin reduced hPDLCs viability. In addition, isoimperatorin alleviated the oxidative stress of periodontal membrane cells. Isoimperatorin reduced proinflammatory factor secretion and receptor activator for nuclear factor-κB ligand-induced osteoclast differentiation in periodontal membrane cells. Further, isoimperatorin inhibited the activation of ERK1/2 and nuclear factor-κB pathways. We, therefore, thought isoimperatorin could serve as a promising drug for the treatment of this disease.

Bardoxolone methyl ameliorates osteoarthritis by inhibiting osteoclastogenesis and protecting the extracellular matrix against degradation

Inflammation and oxidative damage are closely related to the development of osteoarthritis. Bardoxolone methyl (CDDO-Me), a semisynthetic oleanane triterpenoid, plays a strong anti-inflammatory and antioxidant role. The purpose of our research was to explore fundamental mechanisms of CDDO-Me in orthopaedics development. The results showed that CDDO-Me inhibited nuclear factor-κB ligand (RANKL)-induced osteoclast formation and extracellular matrix (ECM) degradation by activating the Nrf2/HO-1 signaling pathways and inhibiting NF-κB pathway activation and excess ROS production. In vivo, CDDO-Me significantly attenuated articular cartilage proteoglycan loss and the number of TRAP-positive osteoclasts in a destabilized medial meniscus (DMM) mouse model of OA. Taken together, these data demonstrate that CDDO-Me inhibits osteoclastogenesis and ECM degradation, underscoring its potential therapeutic value in treating OA.