Receptor Activator Of NF-kappaB Ligand Expression Research Articles

Berberine inhibits RA-FLS cell proliferation and adhesion by regulating RAS/MAPK/FOXO/HIF-1 signal pathway in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology. Berberine inhibited proliferation and adhesion of RA-FLS cells, and significantly reduced the expression of MMP-1, MMP-3, RANKL, and TNF-α. Transcriptional results suggested that berberine intervention mainly regulated forkhead box O (FOXO) signal pathway, prolactin signal pathway, neurotrophic factor signal pathway, and hypoxia-inducible factor 1 (HIF-1) signal pathway. The effect of berberine on RA was related to the regulation of RAS/mitogen-activated protein kinase/FOXO/HIF-1 signal pathway in RA-FLS cells.Cite this article: Bone Joint Res2023;12(2):91-102.

Impact of PIEZO1-channel on inflammation and osteoclastogenesis mediated via periodontal ligament fibroblasts during mechanical loading.

The identification of mechanosensitive ion channels and their importance in innate immunity provides new starting points to elucidate the molecular mechanisms of orthodontic tooth movement. The mechanosensitive electron channel PIEZO1 (Piezo Type Mechanosensitive Ion Channel Component 1) may play a crucial role in orthodontic tooth movement. To investigate the role of the PIEZO1 channel, periodontal ligament fibroblasts (PDLF) were subsequently treated with a PIEZO1 inhibitor (GsMTx) with simultaneous pressure application or with an activator (JEDI2) without mechanical strain. The expression of genes and proteins involved in orthodontic tooth movement was examined by RT-qPCR, Western blot and ELISA. In addition, the effect on PDLF-mediated osteoclastogenesis was investigated in a coculture model using human monocytes. Inhibition of PIEZO1 under pressure application caused a reduction in RANKL (receptor activator of NF-kB ligand) expression, resulting in decreased osteoclastogenesis. On the other hand, activation of PIEZO1 without mechanical strain downregulated OPG (osteoprotegerin), resulting in increased osteoclastogenesis. PIEZO1 appears to play a role in the induction of inflammatory genes. It was also shown to influence osteoclastogenesis.

Human umbilical cord mesenchymal stem cells induction in peri-implantitis Rattus norvegicus accelerates and enhances osteogenesis activity and implant osseointegration

BackgroundPeri-implantitis additional treatment generally aims to repair damaged tissue through a regenerative approach. Human umbilical cord mesenchymal stem cells (hUCMSCs) produce a high osteogenic effect and are capable of modulating the immune system by suppressing inflammatory response, modulating bone resorption, and inducing endogenous osteogenesis. Aim: This study was intended to discover the effect of hUCMSCs on an implant osseointegration process in peri-implantitis rat subjects as assessed by several markers including interleukin-10 (IL-10), transforming growth factor-β (TGF-β), receptor activator of nuclear factor kappa- β ligand (RANKL), bone morphogenic protein (BMP-2), osterix (Osx), and osteoprotegerin (OPG). Material and methods: The research design implemented during this study represented a true experimental design incorporating the use of Rattus norvegicus (Wistar strain) as subjects. Results: Data analysed by means of a Brown Forsythe test indicated differences between the increase in BMP-2 expression (p < 0.000) and Osx expression (p < 0.001) and between RANKL expression (p < 0.001, Tukey HSD) and OPG expression (p < 0.000, Games Howell). Conclusion: According to the findings of this research, hUCMSCs induction is successful in accelerating and enhancing osteogenic activity and implant osseointegration in peri-implantitis rat subjects.

Effects of Co-exposure to Fluoride and Arsenic on TRAF-6 Signaling and NF-κB Pathway ofBoneMetabolism.

Little is known about the combined effect of fluoride (F)and arsenic (As) on bone metabolism. This study aims to explore the effect of co-exposure to F and As on the expressions of TNF receptor-associated factor 6 (TRAF-6), nuclear factor-kappa B (NF-κB), and the related factors in cell and animal experiments. With the rats exposed to different doses of F, As, and combined F-As, we found that F exposure doses were positively correlated with the protein expression of receptor activator of nuclear factor-kappa B ligand (RANKL), receptor activator of nuclear factor-kappa B (RANK), TRAF-6, NF-κB, and nuclear factor of activated T cells (NFAT-c1) (P < 0.001). As exposure doses were negatively correlated with RANK, TRAF-6, NF-κB, and NFAT-c1 (P < 0.001). The effect of F and As interaction on the protein expression of RANKL, TRAF-6, NF-κB, and NFAT-c1 was significant in bone tissue (P < 0.05). In the cellular experiment, F could promote the mRNA expression of RANK, TRAF-6, and NFAT-c1. A higher concentration of As could inhibit the mRNA expression of Tartrate-resistant acid phosphatase (TRAP), RANK, TRAF-6, and NFAT-c1. The effect of F and As interaction on the mRNA expression of TRAP, RANK, TRAF-6, and NFATc1 in osteoclasts was significant (P < 0.001). In conclusion, the expression of TRAF-6 and NF-κB pathway was affected by F and As co-exposure in osteogenic differentiation, and As could antagonize the promoting effect of F on the expression of TRAF-6, TRAP, RANKL, RANK, NF-κB, and NFAT-c1 in these exposure levels. These results could provide a scientific basis for understanding the interaction of F and As in bone formation.

Evaluation of Receptor Activator of Nuclear Factor Kappa B Ligand, Osteoprotegerin, Osteopontin, and Tumor Necrosis Factor Alpha on Chronic Apical Periodontitis in Smokers

IntroductionSmoking can be considered a risk factor for chronic apical periodontitis (CAP). This study compared the immunoexpression of biomarkers receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), osteopontin (OPN), and tumor necrosis factor alpha (TNF-α) in CAP in smokers and nonsmokers. MethodsTwelve smokers and 12 nonsmokers diagnosed with CAP and indicated for tooth extraction were selected. Exclusion factors were teeth with a diagnosis of root fracture, previous endodontic treatment, or endoperiodontal injury, in addition to individuals with systemic diseases, under 18 years of age, users of anti-inflammatory and/or antibiotics in the last 3 months, and drug users. Specimens were processed for histopathologic and immunohistochemical analysis. ResultsQualitative analysis of RANKL expression showed 66.66% weak/moderate and 33.33% strong in smokers and 100% weak/moderate in nonsmokers. OPG and OPN expressions were 100% negative to focal in the smoker group and 50% negative to focal and 50% weak/moderate in the nonsmoker group. TNF-α was 25% negative to focal and 75% weak/moderate in the smoker group and 33.33% negative to focal and 66.66% weak/moderate in the nonsmoker group. Quantitative analysis of the data using the Mann-Whitney U test showed that there was a significant difference in the immunoexpression of RANKL (P < .05), OPG (P < .05), and OPN (P < .05), but there was no statistical difference in the immunoexpression of TNF-α (P > .05) between the 2 groups. ConclusionsThese findings suggest that smoking is capable of altering the inflammatory response, influencing the evolution of CAP.

Salivary Gene Expression of RANK, RANKL, and OPG in Type 1 Diabetes Mellitus and Periodontal Disease Patients.

The relationship between type 1 diabetes mellitus (T1DM) and periodontal disease may exhibit by the alteration of bone metabolism. However, evidence for this relationship is scarce and inconclusive. Thus, the aims of the present study were to investigate salivary receptor activator of nuclear factor kappa-β (RANK), receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG) gene expression and the RANKL:OPG ratio in T1DM and non-T1DM. Secondary objective was to determine the relationships of RANK, RANKL and OPG gene expression to clinical parameters of T1DM and periodontal disease. Twenty patients with T1DM and twenty age-matched non-T1DM were recruited. Clinical periodontal parameters were measured. Total RNA was isolated from non-stimulated saliva, and the relative gene expressions of RANK, RANKL, OPG and RANKL:OPG ratio were determined by quantitative real-time polymerase chain reaction. The T1DM group had significantly higher mean periodontal parameters than the non-T1DM group, while the mean plaque scores of both groups were not significantly different. There was a trend of higher relative gene expression of RANK, RANKL, and the RANKL:OPG ratio and lower expression of OPG in T1DM group but no statistic significant different when compared to non-T1DM. In the T1DM group, RANKL:OPG correlated with the percentage of bleeding sites, whereas RANK, RANKL, and HbA1c levels correlated with pocket depth. Bone metabolisms demonstrating by decreased OPG gene expression and upregulated of RANK, RANKL, RANKL:OPG with higher pocket depth and bleeding in T1DM may play an important role in periodontal destruction in T1DM.

ODP564 Hypercalcemia in the Setting of HTLV-1 Infection and Normal PTHrP Levels

Abstract A 53 year old male with history of HTLV-1 infection associated myelopathy but with unknown mode of transmission, presented with acute on chronic bilateral lower extremity weakness and numbness with no other deficits. Examination revealed decreased strength (grade 1/5) in lower extremities with normal bulk, tone and sensations. Initial blood work revealed hypercalcemia with corrected Calcium of 16.2mg/dl(8.5-11.5), Phosphorus of 2.7mg/dl (2.5- 4.5) and Alkaline Phosphatase of 126mg/dl(20-140). Workup for hypercalcemia revealed Parathyroid hormone (PTH) level of 14pg/ml (10-65), 25 hydroxy vitamin D at 19.6 ng/ml(30-100), 1,25 dihydroxy vitamin D at 6.7pg/ml(19.9-79.3) and TSH of 1.265 microIU/ml(0.5-5). The Parathyroid hormone related protein(PTHrP) level was low at &amp;lt; 2pmol/L while Lactate Dehydrogenase (LDH) was 433U/L(100-220). Urine calcium creatinine ratio was 0.388. Reverse Transcriptase PCR was positive for HTLV-1 but negative for HTLV-2. Smudge cells and atypical lymphocytes were noted on peripheral smear. Imaging revealed generalized lymphadenopathy with splenomegaly but no spinal cord compression. Flow cytometry showed 7.8% atypical helper T cells, positive for CD3, CD4, CD5 and CD45. Clonal T cell receptor gene rearrangements were found, suggesting an underlying T cell neoplasm. Lymph node biopsy confirmed ATLL. He received treatment with fluids, Calcitonin and Denosumab after which serum Calcium levels fell (nadir 7.7mg/dl) and then normalized. He developed another episode of hypercalcemia 5 weeks later which was treated similarly. Discussion Human T cell Lymphotropic Virus-1 (HTLV-1) is a retrovirus causing asymptomatic infections, symptomatic systemic disorders and adult T cell Leukemia and lymphoma(ATLL). HTLV-1 infections, whether in asymptomatic or symptomatic patients, and ATLL are rare but important causes of hypercalcemia and hypercalcemic crises. Individuals with ATLL have the highest incidence of hypercalcemia(50-70%) among all hematologic malignancies. Hypercalcemia is a poor prognostic factor and predicts lower overall survival. It is caused by increased bone resorption, mediated by various factors with the largest role being played by Receptor Activator of Nuclear Factor Kappa B ligand (RANKL), followed by PTHrP, Interleukins, Macrophage Inflammatory Protein 1 alpha and gp 46. RANKL expression is increased upon transactivation by the viral TAX gene product and mediates conversion of osteoclast precursors to mature osteoclasts engaging in bone resorption. PTHrP expression is variable among patients, relating to levels of the TAX gene product that transactivates the PTHrP gene promoter. Thus, a low level, like in our patient, does not rule out HTLV infection as the cause of hypercalcemia. Management of hypercalcemia is in keeping with guidelines for management of other causes of hypercalcemia but may be better responsive to monoclonal antibodies against RANKL than bisphosphonates given the role of the ligand in mediating hypercalcemia in these cases. It can also be used in the setting of poor renal function, which is commonly encountered in hypercalcemic patients. Presentation: No date and time listed

Sinomenine Inhibits Orthodontic Tooth Movement and Root Resorption in Rats and Enhances Osteogenic Differentiation of PDLSCs.

PurposeTo investigate the effects of sinomenine on orthodontic tooth movement and root resorption in rats, as well as the effect of sinomenine on the osteogenesis of periodontal ligament stem cells (PDLSCs).MethodsFifty-four male Wistar rats were randomly divided into 3 groups: control group, 20 mg/kg sinomenine group and 40 mg/kg sinomenine group. Fifty-gram orthodontic force was applied to all groups. Each group was injected intraperitoneally with corresponding concentration of sinomenine every day. After 14 days, all rats were sacrificed. Micro-computed tomography (micro-CT) scan was used to analyze tooth movement, root resorption and alveolar bone changes. The effect on periodontal tissue was analyzed by Masson, tartrate-resistant acid phosphatase (TRAP) and immunohistochemical staining. In vitro, PDLSCs were extracted and identified. The effect of sinomenine on proliferation was determined by cell-counting kit-8. The effect of sinomenine on osteogenesis was investigated by alkaline phosphatase (ALP) activity and alizarin red staining. qPCR and Western blotting were performed to explore the effects of sinomenine on the expression levels of ALP, runt-related transcription factor 2 (RUNX2), receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG).ResultsThe tooth movement and root resorption of sinomenine groups were reduced. Sinomenine decreased trabecular spacing on compression side and increased alveolar bone volume and trabecular thickness on tension side. TRAP-positive cells in sinomenine groups decreased significantly. The expressions of TNF-α and RANKL were decreased, while the expressions of OPG, RUNX2 and osteocalcin were up-regulated. In vitro, 0.1 M and 0.5 M sinomenine enhanced ALP activity, mineral deposition and the expression of ALP, RUNX2 and OPG, and reduced the expression of RANKL.ConclusionSinomenine could inhibit tooth movement, reduce root resorption, and exert a positive effect on bone formation in rats. Moreover, sinomenine promoted the osteogenesis of PDLSCs.

Wnt5a-Ror2 signaling mediates root resorption

This study aimed to investigate the role of wingless-type MMTV integration site family member 5a (Wnt5a)-receptor tyrosine kinase-like orphan receptor 2 (Ror2) signaling in root resorption. The messenger RNA (mRNA) expression of Wnt5a, Ror2, and RANKL in periodontal ligament cells (PDLCs) under compression force (CF) with or without Ror2 small interfering RNA (siRNA) were measured by quantitative reverse transcription-polymerase chain reaction, and these proteins released into culture supernatants were measured using enzyme-linked immunosorbent assay. Then these PDLC-conditioned media under CF with or without Ror2 siRNA were used to culture osteoclast precursors to detect osteoclastogenesis effects via tartrate-resistant acid phosphatase staining. In invivo studies, the odontoclast number and the root resorption volume under excessive CF with or without Ror2 siRNA were investigated by tartrate-resistant acid phosphatase immunohistochemical staining and microcomputed tomography. The protein levels for Wnt5a, Ror2, and receptor activator of nuclear factor-kappa B ligand (RANKL) in the periodontal ligament tissues were also detected using immunohistochemical staining. Finally, the odontoclast number, root resorption volume, and the mRNA and protein expressions were compared between immature and mature teeth. The mRNA production and protein release level of Wnt5a, Ror2, and RANKL increased after CF, whereas they were significantly downregulated with Ror2 siRNA. The osteoclast number increased treating with culture medium from PDLC applying CF, but the increase was inhibited after adding Ror2 siRNA. In the animal model, the odontoclast number and root resorption volume significantly increased in the CF group but decreased in the CF with the Ror2 siRNA group. The protein levels of Wnt5a, Ror2, and RANKL in periodontal ligament were upregulated under excessive CF, and the pathway was inhibited with Ror2 siRNA. In the immature tooth group, the odontoclast number, root resorption volume, and the mRNA and protein expressions of Wnt5a-Ror2 signaling were reduced. Wnt5a-Ror2 signaling in PDLCs enhanced by excessive CF could promote RANKL release and induce precursor differentiation, partly leading to increased odontoclast activity and ultimate root resorption. The less resorption of the immature tooth may be due to odontoclastogenesis inhibition by decreased expression of Wnt5a-Ror2 signaling.

Human mast cells induce osteoclastogenesis through cell surface RANKL.

We employed the co-culture of CD34+ stem cell-derived human mast cells (HMC) and human monocyte-derived osteoclast precursors to evaluate if mast cells contribute to the pathogenesis of osteoporosis through regulation of osteoclast proliferation and activation. Mature HMC and osteoclast precursors were cultured from monocytes isolated from human buffy coat. The osteoclast precursors were incubated with HMC or receptor activator of nuclear factor kappa-B ligand (RANKL) for a week prior to determination of osteoclast maturation through characterization by their morphology and tartrate resistant acid phosphatase (TRAP) expression. The bone absorption activity was determined by pit formation on osteo-assay plate. Mature osteoclasts were identified following co-culture of osteoclast precursors with HMC for one week in the absence of RANKL and they were capable of bone resorption. These actions of HMC on osteoclasts were not affected by mast cell activators such anti-IgE or substance P but could be reversed by osteoprotegerin (OPG) in the co-culture system suggesting the involvement of RANKL. The expression of RANKL on the cell surface of HMC was confirmed by flow cytometry and the density was not affected by activation of HMC. Our study provided direct evidence confirming the initiation of osteoclast proliferation and activation by mast cells through cell surface RANKL suggesting that mast cells may contribute to bone destruction in pathological conditions such as osteoporosis.

The use of mucoadhesive oral patches containing epigallocatechin-3-gallate to treat periodontitis: an in vivo study

ObjectivesThe application of topical drugs such as mucoadhesive oral patches (MOPs) do not irritate the mucosa and are able to increase the permeability of drugs to oral tissue. Epigallocatechin-3-gallate (EGCG) is an active ingredient that exhibits significant antibacterial and anti-inflammatory effects. The purpose of this study was to analyze the therapeutic potential of a mucoadhesive oral patch containing EGCG (MOP-EGCG) in a model of periodontitis and investigate its effects on the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-kappa Β ligand (RANKL) and receptor activator of nuclear factor-κB (RANK). MethodsA model of periodontitis was induced in Rattus novergicus used Porphyromonas gingivalis by applying 0.03 ml of bacteria locally with 1 × 1010 colony-forming units (CFU) seven times at 2-day intervals in the central lower incisors. Periodontitis was then treated with MOP (control), a mucoadhesive oral patch containing doxycycline (MOP-doxy) or MOP-EGCG for 1 h/day for 21 days. On days 3, 5, 7, 14 and 21 after treatment, the central lower incisor was biopsied and analyzed by immunohistochemistry for RANK/RANKL and OPG expression in the gingiva tissue. ResultsMOP-EGCG extract significantly reduced the expression of RANKL and increased the expression of OPG and RANK (p < 0.05) when compared to the MOP-doxy and MOP groups. ConclusionMOP-EGCG extract reduced the expression of RANKL and increased the expression of OPG and RANK, thus suggesting that MOP-EGCG can inhibit the loss of alveolar bone in periodontitis.

Effects of Super-Activated Platelet Lysate on Early Healing of Tooth Extraction Sockets in Rats

PurposeTo evaluate the effect of super-activated platelet lysate (sPL) on wound healing of tooth extraction sockets in rats.MethodsRat models of the tooth extraction socket were established. Thirty-six rats were divided into control and sPL groups and sacrificed on days 7, 14, and 28 after tooth extraction. Bone formation in tooth extraction sockets were observed by microscopic computed tomography (micro-CT) and hematoxylin and eosin (HE) staining; osteoprotegerin (OPG), receptor activator of nuclear factor kappa-Β ligand (RANKL), interleukin 6(IL-6), and tumor necrosis factor-alpha (TNF-α) proteins were detected by immunohistochemistry; and chemokine and osteogenic gene expressions were detected by polymerase chain reaction (PCR).ResultssPL accelerated soft tissue wound healing in the extraction socket of rats. Micro-CT showed that the amount of bone formation and bone volume fraction were higher in the sPL group than the control 14 days after extraction. HE staining showed promotion of the formation of bony trabeculae by sPL in the apical third of the extraction socket 7 days after extraction and more mature and organized bony trabeculae in the sPL group than the control 14 days after extraction; mature bony trabeculae filling most of the fossa with lesser bone porosity in the socket in the sPL group than the control 28 days after extraction. Immunohistochemistry showed that sPL induced OPG expressions 7 and 14 days after tooth extraction but did not affect the RANKL expression while transiently promoting the IL-6 expression 7 days after extraction. PCR showed that sPL promoted chemokine expressions 7 and 14 days after extraction. The expressions of osteogenesis-related factors were higher in the sPL group than the control 7 and 28 days after extraction, while the opposite trend was observed 14 days after extraction.ConclusionsPL has a transient pro-inflammatory effect and promotes soft tissue healing and bone formation during early wound healing of extraction sockets in rats.

Piezo1-mediated fluid shear stress promotes OPG and inhibits RANKL via NOTCH3 in MLO-Y4 osteocytes

ABSTRACT Piezo1, a mechanosensitive ion channel, participates in a variety of biological processes in maintaining bone homeostasis. As the most abundant cells in bones of the mammals, osteocytes play an essential role in bone formation, remodeling, and bone mass maintenance. Here, by exposing MLO-Y4 osteocytes to the fluid shear stress (FSS) microenvironment, we explored the effect of Piezo1-mediated FSS on the expression of the molecules critical to the process of bone formation and resorption, Receptor Activator of Nuclear Factor-Kappa-B Ligand (RANKL) and Osteoprotegerin (OPG). It was found that 9 dyne/cm2 loading for 30 minutes showed an upregulation trend on Piezo1 when MLO-Y4 osteocytes were exposed to an FSS microenvironment. FSS promotes the expression of OPG and inhibits the expression of RANKL. The blocker of Piezo1, GsMTx4, downregulates the effect of FSS on the expression of these two molecules. In addition, NOTCH3 was involved in this process. Thus, the results demonstrated that Piezo1-mediated FSS promotes the expression of OPG and inhibits the expression of RANKL via NOTCH3 in MLO-Y4 osteocytes.

Melatonin inhibits osteoclastogenesis via RANKL/OPG suppression mediated by Rev‐Erbα in osteoblasts

Diabetic osteoporosis is secondary osteoporosis and a serious complication of diabetes with a high incidence rate and poor prognosis. The specific mechanism of diabetic osteoporosis is unclear, and prevention and treatment options are limited. Recently, melatonin has been found to prevent and treat diabetic osteoporosis. Herein, we investigated the mechanism whereby melatonin inhibits osteoclastogenesis and identified a new target for osteoporosis treatment. We established an in vitro osteoblast–osteoclast co‐culture system as a diabetic osteoporosis model. Osteoclastogenesis was determined using tartrate‐resistant acid phosphatase staining and cathepsin K expression. Real‐time PCR was used to ascertain expression of microRNA mir‐882, targeting Rev‐Erbα. Western blotting was performed to detect the expression of Rev‐Erbα, receptor activator of NF‐kB ligand (RANKL), and osteoprotegerin (OPG), and ELISA was utilized to analyse the secreted form of RANKL. High glucose promoted osteoclastogenesis and elevated the RANKL/OPG ratio in osteoblasts, while melatonin reversed these effects. High glucose inhibited Rev‐Erbα expression, while melatonin promoted its expression. Conversely, high glucose promoted mir‐882 expression, while melatonin inhibited it. We infer that melatonin inhibits RANKL expression in osteoblasts via the mir‐882/Rev‐Erbα axis, thus inhibiting osteoclastogenesis. Our findings provide insights into diabetic osteoporosis and identify a new therapeutic target for osteoporosis.

Alpha-7 Nicotinic Receptor Dampens Murine Osteoblastic Response to Inflammation and Age-Related Osteoarthritis

IntroductionOsteoarthritis (OA) is a whole-joint disease characterized by a low-grade inflammation that is involved in both cartilage degradation and subchondral bone remodeling. Since subchondral bone has a cholinergic innervation and that acetylcholine (Ach) might have an anti-inflammatory effect through the α7 nicotinic Ach receptor (α7nAchR), we aimed (i) to determine the expression of non-neuronal cholinergic system and nicotinic receptor subunits by murine and human osteoblasts, (ii) to address the role of α7nAchR in osteoblastic response to inflammation, and (iii) to study the role of α7nAchR in a spontaneous aging OA model.MethodsPrimary cultures of WT and α7nAchR knock-out mice (Chrna7-/-) murine osteoblasts and of subchondral bone human OA osteoblasts were performed. The expressions of the non-neuronal cholinergic system and of the nAchR subunits were assessed by PCR. In vitro, IL1β-stimulated WT, Chrna7-/-, and human osteoblasts were pretreated with nicotine. At 24 h, expressions of interleukin-6 (IL6) and metalloproteinase-3 and -13 (MMP), RANK-ligand (RANKL), and osteoprotegerin (OPG) were quantified by qPCR and ELISA. Spontaneous aging OA was evaluated and compared between male WT and Chrna7-/- mice of 9 and 12 months.ResultsMurine WT osteoblasts express the main components of the cholinergic system and α7 subunit composing α7nAchR. Nicotine partially prevented the IL1β-induced expression and production of IL6, MMP3, and RANKL in WT osteoblasts. The effect for IL6 and MMP was mediated by α7nAchR since nicotine had no effect on Chrna7-/- osteoblasts while the RANKL decrease persisted. Chrna7-/- mice displayed significantly higher cartilage lesions than their WT counterparts at 9 and 12 months, without difference in subchondral bone remodeling. Human OA osteoblasts also expressed the non-neuronal cholinergic system and α7 subunit as well as CHRFAM7A, the dominant negative duplicate of Chrna7. Nicotine pretreatment did not significantly reduce IL6 and MMP3 production in IL-1β-stimulated human osteoarthritic osteoblasts (n = 4), possibly due to CHRFAM7A.ConclusionCholinergic system counteracts murine osteoblastic response to IL-1β through α7nAchR. Since α7nAchR deletion may limit cartilage degradation during murine age-related OA, enhancing cholinergic system could be a new therapeutic target in OA but may depend on CHRFAM7A expression.

Nano-emulsion of mangosteen rind extract in a mucoadhesive patch for periodontitis regenerative treatment: An in vivo study

ObjectiveTo investigate the therapeutic potential of nano-emulsion of mangosteen rind extract in a mucoadhesive gingival patch on periodontitis, and its effect on tumor necrosis factor alpha (TNF-α), receptor activator of nuclear factor kappa Β ligand (RANKL), and interleukin 10 (IL-10) expression. MethodsSixty Wistar rats were divided into four groups: positive control group (mucoadhesive patch with doxycycline), negative control group (mucoadhesive patch), treatment group I (mucoadhesive patch with mangosteen rind extract), and treatment group II (mucoadhesive patch with nano-emulsion of mangosteen rind extract). An experimental model of Porphyromonas gingivalis-induced periodontitis was established in rats by treatment with 0.03 mL bacteria locally (1 × 1010 colony-forming units) seven times at 2-day intervals in the gingival sulcus of mandibular anterior teeth. Treatment was 1 h/day for 3 days. On days 3, 5, and 7, five rats from each group were killed. TNF-α, IL-10, and RANKL expression was determined by dissecting the lower jaw for immunohistochemistry. ResultsThe mucoadhesive patch with nano-emulsion mangosteen rind extract significantly decreased TNF-α and RANKL expression and increased IL-10 expression (p < 0.05) compared to the treatment I, positive and negative control groups. ConclusionA mucoadhesive gingival patch with nano-emulsion of mangosteen rind extract has the potential to treat periodontitis by decreasing TNF-α, RANKL, and increasing IL-10 expression.

Osteoblast Jmjd3 regulates osteoclastogenesis via EphB4 and RANKL signalling.

Osteoblasts suppress osteoclastogenesis during the reversal phase of bone remodelling and the mechanism needs to be further investigated. Here, we investigated the role of histone demethylase Jumonji domain-containing 3 (Jmjd3) in osteoblasts on regulating osteoclastogenesis. Jmjd3 expression was silenced in osteoblasts. Osteoblasts and osteoclasts were co-cultured in direct or indirect contact ways, and osteoclastogenesis was determined by tartrate-resistant acid phosphatase (TRAP) staining and Western blotting. Additionally, Ephrin receptor B4 (EphB4) and receptor activator of nuclear factor-kappa Β ligand (RANKL) expression were quantified in osteoblasts via real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. Subsequently, EphB4 was overexpressed in osteoblasts and RANKL expression and osteoclastogenesis was quantified. Osteoclastogenesis and marker protein expression levels was promoted when osteoclasts were co-cultured with Jmjd3-silenced osteoblasts. Silencing of Jmjd3 expression in osteoblasts decreased EphB4 expression, owing to suppression of demethylation of H3K27me3 on the promoter region of EphB4. Whereas RANKL expression was upregulated in Jmjd3-silenced osteoblasts. Overexpression of EphB4 in osteoblasts inhibited osteoclastogenesis and RANKL expression. Jmjd3 in osteoblasts is a crucial regulator of osteoblast-to-osteoclast communication through EphB4-EphrinB2, RANKL-RANK and EphB4-RANKL signalling axes, suggesting the pivotal role of Jmjd3 in bone remodelling process in bone destruction disease such as chronic apical periodontitis.

Evaluation of the Effects of Favipiravir Combined with Vitamin C on Alveolar Bone in Rats.

Introduction: Favipiravir and Vitamin C (Vit C) were used together in the treatment of the COVID-19 pandemic. However, the effects of favipiravir on the periodontium are still unknown. Therefore, the aim of this study was to investigate the effects of Favipiravir and Vit C treatment on alveolar bone metabolism. Experimental: Fifty healthy adult male Sprague-Dawley rats (2–3 months old) were randomly divided into five equal groups (n = 10): Control, Favi 20, Favi 100, Favi 20+Vit C, Favi 100+Vit C. Favipiravir (20 mg/kg and 100 mg/kg, i.m.) and Vit C (150 mg/kg/day, oral) were administered to the rats for 14 days. Alveolar bone loss (ABL) and histopathological changes were examined using a light microscope. Immunohistochemistry was used to determine levels of receptor activator of nuclear factor kappa-B ligand (RANKL), caspase-3, bone morphogenic protein 2 (BMP-2) and alkaline phosphatase (ALP) in the bone tissues. Results: Favipiravir increased the levels of RANKL and caspase-3 expression but decreased BMP-2 and ALP levels in a dose-dependent manner. Favi 20+Vit C and Favi 100 +Vit C groups showed decreased RANKL and caspase-3 levels in addition to increased BMP-2 and ALP levels. Conclusion: Favipiravir can cause histopathological damage to the periodontium, but administration of favipiravir combined with Vit C can provide a protective effect against this damage.

Effect of Cold Atmospheric Plasma (CAP) on Osteogenic Differentiation Potential of Human Osteoblasts.

Bone regeneration after oral and maxillofacial surgery is a long-term process, which involves various mechanisms. Recently, cold atmospheric plasma (CAP) has become known to accelerate wound healing and have an antimicrobial effect. Since the use of CAP in dentistry is not yet established, the aim of the present study was to investigate the effect of CAP on human calvaria osteoblasts (HCO). HCO were treated with CAP for different durations of time and distances to the cells. Cell proliferation was determined by MTT assay and cell toxicity by LDH assay. Additionally, RT-qPCR was used to investigate effects on osteogenic markers, such as alkaline phosphatase (ALP), bone morphogenic protein (BMP)2, collagen (COL)1A1, osteonectin (SPARC), osteoprotegerin (OPG), osterix (OSX), receptor activator of NF-κB (RANK), RANK Ligand (RANKL), and Runt-related transcription factor (RUNX)2. There were small differences in cell proliferation and LDH release regarding treatment duration and distance to the cells. However, an increase in the expression of RANK and RANKL was observed at longer treatment times. Additionally, CAP caused a significant increase in mRNA expression of genes relevant to osteogenesis. In conclusion, CAP has a stimulating effect on osteoblasts and may thus represent a potential therapeutic approach in the regeneration of hard tissue defects.

Mechanisms involved in suppression of osteoclast supportive activity by transforming growth factor-β1 via the ubiquitin-proteasome system.

Orthodontic treatment requires the regulation of bone remodeling in both compression and tension sides. Transforming growth factor-β1 (TGF-β1) is an important coupling factor for bone remodeling. However, the mechanism underlying the TGF-β1-mediated regulation of the osteoclast-supporting activity of osteoblasts and stromal cells remain unclear. The current study investigated the effect of TGF-β1 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in stromal cells induced by 1α,25(OH)2D3 (D3) and dexamethasone (Dex). TGF-β1 downregulated the expression of RANKL induced by D3 and Dex in mouse bone marrow stromal lineage, ST2 cells. Co-culture system revealed that TGF-β1 suppressed osteoclast differentiation from bone marrow cell induced by D3 and Dex-activated ST2 cells. The inhibitory effect of TGF-β1 on RANKL expression was recovered by inhibiting the interaction between TGF-β1 and the TGF-β type I/activin receptor or by downregulating of smad2/3 expression. Interestingly, TGF-β1 degraded the retinoid X receptor (RXR)-α protein which forms a complex with vitamin D receptor (VDR) and regulates transcriptional activity of RANKL without affecting nuclear translocation of VDR and phosphorylation of signal transducer and activator of transcription3 (STAT3). The degradation of RXR-α protein by TGF-β1 was recovered by a ubiquitin-proteasome inhibitor. We also observed that poly-ubiquitination of RXR-α protein was induced by TGF-β1 treatment. These results indicated that TGF-β1 downregulates RANKL expression and the osteoclast-supporting activity of osteoblasts/stromal cells induced by D3 and Dex through the degradation of the RXR-α protein mediated by ubiquitin-proteasome system.