Expression Levels Of Receptor Activator Research Articles

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 Morinda officinalis Polysaccharides on the Proliferation and Viability of Osteoblasts in Rats with Osteoporosis

We aimed to evaluate the effects of Morinda officinalis polysaccharides on the proliferation and viability of osteoblasts in rats with osteoporosis. 24 non-parous Sprague-Dawley rats were assigned into normal, model, low-dose and high-dose Morinda officinalis polysaccharide groups. The model of osteoporosis was established by ovariectomy. After successful modeling, 50 mg/kg and 75 mg/kg Morinda officinalis polysaccharides were subcutaneously injected in low-dose and high-dose Morinda officinalis polysaccharide groups, respectively. After 3 w of intervention, osteoblasts were prepared using the skull and their proliferation index and alkaline phosphatase activity were measured. The histopathological changes in femoral tissues were observed. The bone mineral density and levels of serum osteocalcin, calcium and phosphorus, as well as the protein levels of receptor activator of nuclear factor kappa-B, receptor activator of nuclear factor kappa-B ligand and osteoprotegerin were determined. The proliferation index and alkaline phosphatase activity were highest in high-dose Morinda officinalis polysaccharide group (p<0.05). Bone tissues had decreased cell spaces and gradually reduced spaces between the medullary cavities in Morinda officinalis polysaccharide groups. Model group had the highest bone mineral density and levels of serum osteocalcin, calcium and phosphorus (p<0.05). The protein expression levels of receptor activator of nuclear factor kappa-B ligand and receptor activator of nuclear factor kappa-B showed a decreasing trend, while that of osteoprotegerin displayed a rising trend sequentially in model, low-dose, high-dose Morinda officinalis polysaccharide and normal groups (p<0.05). Morinda officinalis polysaccharides promotes the proliferation of osteoblasts, enhances their viability and maintains the balance between bone resorption and formation in rats with osteoporosis probably by modulating the protein expression of the receptor activator of nuclear factor kappa-B ligand/receptor activator of nuclear factor kappa-B/osteoprotegerin pathway.

Selective β2-Adrenoceptor Blockade Rescues Mandibular Growth Retardation in Adolescent Rats Exposed to Chronic Intermittent Hypoxia

Activation of the sympathoadrenal system is associated with sleep apnea-related symptoms and metabolic dysfunction induced by chronic intermittent hypoxia (IH). IH can induce hormonal imbalances and growth retardation of the craniofacial bones. However, the relationship between IH and β2-adrenergic receptor signaling in the context of skeletal growth regulation is unclear. This study aimed to investigate the role of β2-adrenergic receptors in IH-induced mandibular growth retardation and bone metabolic alterations. Male 7-week-old Sprague–Dawley rats were subjected to IH for 3 weeks. IH conditions were established using original customized hypoxic chambers; IH was induced at a rate of 20 cycles per hour (oxygen levels changed from 4 to 21% in one cycle) for 8 h per day during the 12 h “lights on” period. The rats received intraperitoneal administration of a β2-adrenergic antagonist (butoxamine) or saline. To exclude dietary effects on general growth, the normoxic rats with saline, normoxic rats with butoxamine, and IH rats with butoxamine were subjected to food restriction to match the body weight gains between IH and other three groups. Body weight, heart rate, blood pressure, and plasma concentrations of leptin, serotonin, and growth hormone were measured. Bone growth and metabolism were evaluated using radiography, microcomputed tomography, and immunohistochemical staining. Plasma leptin levels were significantly increased, whereas that of serotonin and growth hormone were significantly decreased following IH exposure. Leptin levels recovered following butoxamine administration. Butoxamine rescued IH-induced mandibular growth retardation, with alterations in bone mineral density at the condylar head of the mandible. Immunohistochemical analysis revealed significantly lower expression levels of receptor activator of nuclear factor-kappa B ligand (RANKL) in the condylar head of IH-exposed rats. Conversely, recovery of RANKL expression was observed in IH-exposed rats administered with butoxamine. Collectively, our findings suggest that the activation of β2-adrenergic receptors and leptin signaling during growth may be involved in IH-induced skeletal growth retardation of the mandible, which may be mediated by concomitant changes in RANKL expression at the growing condyle.

Diphlorethohydroxycamalol isolated from Ishige okamurae prevents H2O2-induced oxidative damage via BMP2/Runx2 signaling in osteoblastic MC3T3-E1 cells

Accumulating evidence has shown an association between osteoporosis and oxidative damage. In the present study, the protective effects of diphlorethohydroxycarmalol (DPHC) isolated from the brown algae Ishige okamurae against H2O2-induced oxidative damage via bone morphogenetic protein 2 (BMP2)/ runt-related transcription factor 2 (Runx2) signaling were investigated using MC3T3-E1 osteoblastic cells. DPHC counteracted the reduction in cell viability caused by H2O2 exposure and protected against H2O2-induced dysfunction, demonstrated by improved cellular alkaline phosphatase (ALP) activity and calcium deposition. In addition, treatment with 0.05–0.2 mM DPHC elevated the protein expression of osteoblast differentiation factors type 1 collagen, ALP, p-Smad1/5, Osterix, BMP2, and Runx2, in response to H2O2-induced oxidative damage. Importantly, DPHC decreased the expression levels of receptor activator of nuclear factor kappa-B ligand, which promotes bone resorption, and inhibited the H2O2-induced generation of reactive oxygen species. Taken together, the results suggest that DPHC counteracts the effects of oxidative stress in osteoblastic cells and has the potential to be effective in preventing and alleviating osteoporosis.

The Mechanisms of Action of Zuogui Pill on Osteoporosis in Ovariectomized Rats

Traditional Chinese medicine recommends kidney-tonifying treatment for osteoporosis. Zuogui pill is one such treatment composed of eight well characterized Chinese medicinal herbs (Radix rehmanniae preparata, Fructus lycii, Rhizoma dioscoreae, F. corni, Semen cuscutae, Colla cornus cervi, C. carapacis et Plastri testudinis, and R. achyranthis bidentatae). The aim of the study is to evaluate the effects of the Zuogui pill on osteoporosis in ovariectomized rats and to identify the signaling pathways that mediate its action on the bones. A total of 46 6-month-old female Sprague-Dawley rats were divided into four groups: sham-operated control, bilaterally ovariectomized, ovariectomized rats receiving diethylstilbestrol (the positive control group), and ovariectomized rats receiving the Zuogui pill. By the end of 8-week treatments, the following parameters were assessed: (a) bone mineral density and trabecular histomorphology, (b) the expression levels of receptor activator of nuclear factor kappa B ligand and osteoprotegerin in rat tibial osteoblasts and (c) bone marrow stromal cells. The Zuogui pill treatment elevated the trabecular bone area and trabecular thickness and reduced the trabecular spacing in rats with ovariectomy-induced osteoporosis. Compared to the ovariectomized group, the Zuogui pill treatment significantly increased the levels of bone mineral density and osteoprotegerin and suppressed the level of receptor activator of the nuclear factor kappa B ligand. These data led us to conclude that the Zuogui pill regulates bone metabolism and improves osteoporosis symptoms possibly through the osteoprotegerin/receptor activator of the nuclear factor kappa B ligand/receptor activator of the nuclear factor kappa B signaling pathway.

Effect of Psoralen on the Intestinal Barrier and Alveolar Bone Loss in Rats With Chronic Periodontitis.

To study the effects of psoralen on the intestinal barrier and alveolar bone loss (ABL) in rats with chronic periodontitis. Fifty-two 8-week-old specific pathogen-free (SPF) male Sprague-Dawley (SD) rats were randomly divided into the following four groups: Control group (Control), psoralen group of healthy rats (Pso), periodontitis model group (Model), and psoralen group of periodontitis rats (Peri+Pso). The alveolar bone resorption of maxillary molars was observed via haematoxylin-eosin staining and micro-computed tomography. The expression level of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in periodontal tissues was evaluated by immunofluorescence staining. The changes in serum tumour necrosis factor (TNF)-α, interleukin (IL)-10, IL-6, intestinal mucosal occludin, and claudin-5 were detected using enzyme-linked immunosorbent assay (ELISA). The level of intestinal mucosal NOD2 was detected using immunohistochemical methods. DNA was extracted from the intestinal contents and the 16s rRNA gene was sequenced using an Illumina MiSeq platform. The expression of NOD2 protein in the intestinal tract of periodontitis rats decreased after intragastric psoralen administration. Psoralen increased the intestinal microbiota diversity of rats. The level of serum pro-inflammatory factor TNF-α decreased and the level of anti-inflammatory factor IL-10 increased. ABL was observed to be significantly decreased in rats treated with psoralen. Psoralen decreased the RANKL/OPG ratio of periodontitis rats. Psoralen may affect the intestinal immune barrier and ecological barrier, mediate immune response, promote the secretion of anti-inflammatory factor IL-10, and reduce the secretion of the pro-inflammatory factor TNF-α, thus reducing ABL in experimental periodontitis in rats.

Total Flavonoids of Bidens pilosa Ameliorates Bone Destruction in Collagen-Induced Arthritis.

Rheumatoid arthritis is a chronic autoimmune disease characterized by the infiltration of synovial inflammatory cells and progressive joint destruction. Total flavonoids of Bidens pilosa have been used against inflammation in rheumatoid arthritis, but its role in bone destruction remains to be explored. The aim of this paper was to study whether total flavonoids of B. pilosa relieve the severity of collagen-induced arthritis in rats, particularly whether it regulates the production of proinflammatory cytokines and the receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin signaling pathway. In this research, a collagen-induced disease model was induced in adult rats by subcutaneous injection of collagen II. Total flavonoids of B. pilosa at different doses (40, 80, and 160 mg/kg/d) were administered intragastrically, while methotrexate (1 mg/kg/w) was injected intraperitoneally as a positive control. Paw swelling, arthritis score, and body weight were assessed and evaluated. The severity of joint damage was determined using X-ray and confirmed by histopathology. The expression levels of receptor activator of nuclear factor-κB ligand, osteoprotegerin, IL-1β, IL-17, and TNF in the serum and tissue were assayed using ELISA and immunohistochemistry. We found that total flavonoids of B. pilosa attenuated collagen-induced arthritis at the macroscopic level, and total flavonoids of B. pilosa-treated rats showed reduced paw swelling, arthritis scores, and X-ray appearance of collagen-induced arthritis in addition to improved histopathological results. These findings were consistent with reduced serum and tissue receptor activator of nuclear factor-κB ligand, TNF, IL-1β, and IL-17 levels but increased osteoprotegerin levels. Our data suggest that total flavonoids of B. pilosa attenuate collagen-induced arthritis by suppressing the receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factor-κB/osteoprotegerin pathway and the subsequent production of proinflammatory cytokines. In addition, total flavonoids of B. pilosa may be a promising therapeutic candidate for the management of rheumatoid arthritis.

10-Hydroxydecanoic Acid Potentially Elicits Antigen-Specific IgA Responses.

The effective antigen (Ag) uptake by microfold cells (M-cells) is important for the induction of an efficient mucosal immune responses. Here, we show that 10-hydroxydecanoic acid (10-HDAA) from royal jelly (RJ) potentially supports M-cell differentiation and induces effective antigen-specific mucosal immune responses in cynomolgus macaques. 10-HDAA increases the expression level of receptor activator of nuclear factor-kappaB (NF-κB) (RANK) in Caco-2 cells, which suggests that 10-HDAA potentially prompts the differentiation of Caco-2 cells into M-cells and increased transcytosis efficiency. This idea is supported by the following observations. Intranasal administration of 10-HDAA increased the number of M-cells in the epithelium overlying nasopharynx-associated lymphoid tissue (NALT) in macaques. Oral administration of 10-HDAA increased the number of M-cells in the follicle-associated epithelium (FAE) covering Peyer's patches (PPs) and significantly increased the antigen-specific immunoglobulin A (IgA) level in macaques. These findings suggest that the exogenous honeybee-derived medium-chain fatty acid 10-HDAA may effectively enhance antigen-specific immune responses.

Involvement of the G-Protein-Coupled Receptor 4 in the Increased Expression of RANK/RANKL/OPG System and Neurotrophins by Nucleus Pulposus Cells under the Degenerated Intervertebral Disc-Like Acidic Microenvironment.

Intervertebral disc (IVD) degeneration is associated with local inflammation and increased expression of neurotrophins. Acidic microenvironment is believed to cause the progression of IVD degeneration. However, there is a paucity of information regarding the relationship between acidic microenvironment and the inflammation and expression of neurotrophins in IVD. G-protein-coupled receptor 4 (GPR4) is a pH-sensing receptor, which can activate the inflammation and increase the expression levels of nerve growth factor in acidic microenvironment. In this study, culture media with pH 7.2 (representing the normal IVD-like acidic condition) and pH 6.5 (degenerated IVD-like acidic condition) were prepared. The gene and protein expression levels of GPR4 in SD rat nucleus pulposus cells were determined under the acidic conditions. And cyclic AMP (cAMP), the second messenger of GPR4, was assayed. Furthermore, the expression levels of receptor activator of nuclear factor κ B (RANK), RANKL ligand (RANKL), osteoprotegerin (OPG), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were also determined. To clarify the involvement of GPR4 in the upregulation of the expression of RANK/RANKL/OPG system and neurotrophins, gene knockdown and forced expression of GPR4 and inhibiting its downstream cAMP accumulation and Ca2+ mobilization were performed. The alternation of the expression levels of matrix metalloproteinase-3 (MMP-3), MMP-13, and aggrecanase-2 (ADAMTS-5) were evaluated by RT-PCR and western blot. The results showed that GPR4 was expressed in rat nucleus pulposus cells, and the expression was upregulated under the degenerated IVD-like acidic microenvironment. cAMP accumulation levels were increased under the degenerated IVD-like acidic culture conditions. The expression levels of RANK, RANKL, OPG, NGF, and BNDF were significantly upregulated under the degenerated IVD-like acidic microenvironment. GPR4 knockdown and reduction of cAMP by the inhibitor SQ22536 abolished the upregulation of the expression of RANK, RANKL, OPG, NGF, and BNDF under the degenerated IVD-like acidic microenvironment. On the opposite, acidosis-induced cAMP accumulation and upregulation of RANK, RANKL, OPG, NGF, and BNDF were further promoted by GPR4 overexpression. The expression levels of MMP-3, MMP-13, and ADAMTS-5 were upregulated under the degenerated IVD-like acidic condition, which can be promoted or attenuated by GPR4 overexpression or knockdown, respectively. We concluded that GPR4-mediated cAMP accumulation was involved in the increased expression of RANK/RANKL/OPG system and neurotrophins by nucleus pulposus cells under the degenerated IVD-like acidic microenvironment.

Suppressive effect of melatonin on osteoclast function via osteocyte calcitonin.

Many studies have investigated the actions of melatonin on osteoblasts and osteoclasts. However, the underlying mechanisms, especially regarding osteocyte function, remain largely unknown. Therefore, this study aimed to clarify the underlying mechanisms of melatonin action on bone tissue via osteocyte function. Chick calvariae were employed as a model. In ovo injection of melatonin (5, 50 and 500 µg) dose-dependently decreased the mRNA expression levels of cathepsin K and matrix metalloproteinase 9 (MMP9) in chick calvariae without affecting the expression levels of receptor activator of NF-κB ligand or osteoprotegerin. Surprisingly enough, the expression of calcitonin mRNA in chick calvariae was significantly raised. After 3 days of in vitro treatment of melatonin (10-7 and 10-5 M) on newly hatched chick calvariae, both calcitonin mRNA expression in calvariae and the concentration of calcitonin in cultured medium were augmented in a dose-dependent manner, coincident with the decreased mRNA expression levels of cathepsin K and MMP9. Immunohistochemical analyses revealed expression of melatonin receptors and calcitonin by osteocytes buried in bone matrix. Moreover, the mRNA expression levels of melatonin receptors, calcitonin and sclerostin (a marker of osteocyte), were strongly and positively correlated. In conclusion, we demonstrated the expression of melatonin receptors and calcitonin expression in osteocytes for the first time and suggest a new mechanism underlying the suppressive effect of melatonin on osteoclasts via upregulation of calcitonin secretion by osteocytes.

The effect of risedronate on orthodontic tooth movement in ovariectomized rats

ObjectiveThis study aimed to investigate the effect of risedronate on orthodontic tooth movement in ovariectomized rats. MethodsForty-five female Sprague-Dawley rats were divided into three groups. Bilateral ovariectomies were performed on the rats in the ovariectomy and ovariectomy + risedronate groups. Two weeks after ovariectomy, risedronate was administered intraperitoneally every three days in the ovariectomy + risedronate group, while rats in the ovariectomy and sham groups were administered saline until they were sacrificed. One month after ovariectomy, nickel-titanium alloy closed-coil springs were placed between the maxillary left first molar and the maxillary left incisor. On days 3, 7 and 14, five rats from each group were sacrificed. The distance of orthodontic tooth movement was measured by a digital caliper, and slides were obtained for histological analysis. ResultsThe distance of orthodontic tooth movement and the number of tartar-resistant acid phosphatase (TRAP)-positive cells were the highest in the ovariectomy group, followed by those in the ovariectomy + risedronate group, on days 3, 7 and 14. The positive expression levels of receptor activator of nuclear factor-kappa β (RANK) ligand and cathepsin K were the strongest while the positive expression of osteoprotegerin in the ovariectomy group was the weakest, followed by the corresponding expression levels in the ovariectomy + risedronate group, on days 3, 7 and 14. ConclusionsRisedronate can inhibit orthodontic tooth movement in ovariectomized rats and may function by regulating the RANK/RANK ligand/osteoprotegerin pathway.

Combined effect of recombinant human bone morphogenetic protein-2 and low level laser irradiation on bisphosphonate-treated osteoblasts.

ObjectivesThe purpose of this study was to evaluate the synergic effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-level laser therapy (LLLT) on bisphosphonate-treated osteoblasts.Materials and MethodsHuman fetal osteoblast cells (hFOB 1.19) were cultured with 100 µM alendronate. Low-level Ga-Al-As laser alone or with 100 ng/mL rhBMP-2 was then applied. Cell viability was measured with MTT assay. The expression levels of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) were analyzed for osteoblastic activity inducing osteoclastic activity. Collagen type and transforming growth factor beta-1 were also evaluated for bone matrix formation.ResultsThe results showed that rhBMP-2 and LLLT had a synergic effect on alendronate-treated osteoblasts for enhancing osteoblastic activity and bone matrix formation. Between rhBMP-2 and LLLT, rhBMP-2 exhibited a greater effect, but did not show a significant difference.ConclusionrhBMP-2 and LLLT have synergic effects on bisphosphonate-treated osteoblasts through enhancement of osteoblastic activity and bone formation activity.

Charcot Pathogenesis: A Study of In Vivo Gene Expression

Charcot neuroarthropathy is a rare but often difficult to manage disease in the neuropathic patient. Early signs such as unremarkable edema, marginal trauma, or minor infection can activate a cascade of bony destruction and lead to gross prominence or deformity, with dire consequences. The exact molecular mechanism is poorly understood. Current theory states that an inflammatory reaction leads to the activation of osteoclasts mediated by specific cytokines. Our study sought to test the genetic expression of certain biomarkers in diabetic patients with and without Charcot neuroarthropathy compared with patients with and without diabetes or neuropathy. A total of 30 patients participated in the study, 17 (57%) males and 13 (43%) females. Peripheral blood samples were drawn, and gene expression was measured using real-time polymerase chain reaction. The expression levels of receptor activator of nuclear factor kappa-B ligand and osteoprotegerin showed no significant increase in the Charcot neuroarthropathy group compared with the healthy control group. We determined that the levels of receptor activator of nuclear factor kappa-B ligand and osteoprotegerin were not significantly increased in Charcot neuroarthropathy patients compared with healthy control patients. These results demonstrate a need for further investigation into alternative molecular pathways to determine the exact mechanism of the disease process.

Increased level of cell-derived microparticles in the cyst fluids of odontogenic keratocysts.

The aim of this study was to examine the level and basic characteristics of cell-derived microparticles (MPs) in the cyst fluids of odontogenic keratocysts (OKCs). For this purpose, MPs from the cyst fluids (CFMPs) of OKCs were purified by a classic differential centrifugation method and characterized by a transmission electron microscope and fluorescence microscope. Flow cytometric analysis was used to determine the size, concentration and cellular origins of the CFMPs. Moreover, the expression level of receptor activator for nuclear factor-κB ligand in the OKCs was evaluated by immunohistochemical staining and then analyzed for its correlation with the concentration of CFMPs by Spearman's rank correlation test. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and tartaric-resistant acid phosphatase (TRAP) staining were performed to examine the osteoclastogenesis of mouse bone marrow-derived macrophages (BMMs) in response to CFMPs. The results revealed that the levels of total CFMPs were significantly elevated in OKCs compared with dentigerous cysts (DCs) and radicular cysts (RCs). In addition, in vitro experiments further revealed that CFMPs derived from the OKCs of patients could be taken up by BMMs, leading to a significant increase in the mRNA expression levels of nuclear factor of activated T-cells 1 (NFATc1) and TRAP. Moreover, TRAP-positive multinucleated osteoclasts were successfully cultured in the presence of macrophage colony-stimulating factor (M-CSF) and CFMPs with BMMs. On the whole, our findings indicate that patients with OKCs have higher levels of CFMPs compared with patients with DCs and RCs, which may be associated with the bone resorption of OKCs.

P38 MAPK and Its Upstream Activators MKK3 and MKK6 Play an Important Role on Lipopolysaccharide-Induced RANKL Expression by Periodontal Ligament Cells

Resident, non-immune cells express various pattern-recognition receptors and produce inflammatory cytokines in response to microbial antigens, during the innate immune response. Alveolar bone resorption is the hallmark of destructive periodontitis and it is caused by the host response to bacteria and their mediators present on the biofilm. The balance between the expression levels of receptor activator of nuclear factorkappa B ligand (RANKL) and osteoprotegerin (OPG) is pivotal for osteoclast differentiation and activity and has been implicated in the progression of bone loss in periodontitis. To assess the contribution of resident cells to the bone resorption mediated by innate immune signaling, we stimulated fibroblasts and osteoblastic cells with LPS from. Escherichia coli (TLR4 agonist), Porphyromonas gingivalis (TLR2 and -4 agonist), and interleukin-1 beta (as a control for cytokine signaling through Toll/IL-1receptor domain) in time-response experiments. Expression of RANKL and OPG mRNA was studied by RT-PCR, whereas the production of RANKL protein and the activation of p38 MAPK and NF-kB signaling pathways were analyzed by western blot. We used biochemical inhibitors to assess the relative contribution of p38 MAPK and NF-kB signaling to the expression of RANKL and OPG induced by TLR2, -4 and IL1β in these cells. Both p38 MAPK and NFkB pathways were activated by these stimuli in fibroblasts and osteoblasts, but the kinetics of this activation varied in each cell type and with the nature of the stimulation. E. coli LPS was a stronger inducer of RANKL mRNA in fibroblasts, whereas LPS from P. gingivalis downregulated RANKL mRNA in periodontal ligament cells but increased its expression in osteoblasts. IL-1β induced RANKL in both cell types and without a marked effect on OPG expression. p38 MAPK was more relevant than NF-kB for the expression of RANKL and OPG in these cell types.

Multipotent mesenchymal stromal cell sheet therapy for bisphosphonate-related osteonecrosis of the jaw in a rat model

Bisphosphonates (BPs) inhibit bone resorption and are frequently used to treat osteoporosis, bone metastasis, and other conditions that result in bone fragility. However, numerous studies have reported that BPs are closely related to the development of osteonecrosis of the jaw (BRONJ), which is an intractable disease. Recent studies have demonstrated that intravenous infusion of multipotent mesenchymal stromal cells (MSCs) is effective for the treatment of BRONJ-like disease models. However, the stability of injected MSCs is relatively low. In this study, the protein level of vascular endothelial growth factor in BP-treated MSCs was significantly lower than untreated-MSCs. The mRNA expression levels of receptor activator of nuclear factor κ-B ligand and osteoprotegerin were significantly decreased in BP-treated MSCs. We developed a tissue-engineered cell sheet of allogeneic enhanced green fluorescent protein (EGFP)-labeled MSCs and investigated the effect of MSC sheet transplantation in a BRONJ-like rat model. The MSC sheet group showed wound healing in most cases compared with the control group and MSC intravenous injection group (occurrence of bone exposure: 12.5% compared with 80% and 100%, respectively). Immunofluorescence staining revealed that EGFP-positive cells were localized around newly formed blood vessels in the transplanted sub-mucosa at 2weeks after transplantation. Blood vessels were significantly observed in the MSC sheet group compared to in the control group and MSC intravenous injection group (106±9.6 compared with 40±5.3 and 62±10.2 vessels/mm2, respectively). These results suggest that allogeneic MSC sheet transplantation is a promising alternative approach for treating BRONJ. Statement of SignificanceBisphosphonates are frequently used to treat osteoporosis, bone metastasis of various cancers, and other diseases. However, bisphosphonate related-osteonecrosis of the jaw (BRONJ) is an intractable disease because it often recurs after surgery or is exacerbated following conservative treatment. Therefore, an alternative approach for treating BRONJ is needed.In this study, we developed a bone marrow-derived multipotent mesenchymal stromal cell (MSC) sheet to treat BRONJ and investigated the effect of MSC sheet transplantation in a rat model of BRONJ-like disease. The MSC sheet transplantation group showed wound healing in most cases, while only minimal healing was observed in the control group and MSC intravenous injection group. Our results suggest that the MSC sheet is a promising alternative approach for the treatment of BRONJ.

Effects of a GSK-3β inhibitor on the renal expression levels of RANK, RANKL and NF-κB in a rat model of diabetic nephropathy.

The present study aimed to investigate the effects of glycogen synthase kinase-3β (GSK-3β) on the expression levels of receptor activator of nuclear factor (NF)-κB (RANK), RANK ligand (RANKL) and NF-κB in the renal tissues of rats modeling diabetic nephropathy (DN). The rats were allocated at random into three groups, as follows: Normal control group (NC), the DN model group (DNM group) and the DN model lithium chloride (LiCl) intervention group (DNI group). Urinary proteins were examined by staining with the Coomassie Brilliant Blue dye for 24 h. Histochemical analyses of kidney tissue sections were conducted using hematoxylin and eosin staining, after which the kidney pathology of the rats was observed. In addition, the mRNA and protein expression levels of GSK-3β, RANK, RANKL and NF-κB in the renal tissues were detected using reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively. As compared with the NC group, the level of urinary protein was significantly increased in the DNM group (P<0.05); however, as compared with the DNM Group, the level of urinary protein at 12 weeks was significantly decreased in the DNI group (P<0.05). As compared with the NC group, marked pathological changes were detected, and the mRNA and protein expression levels of GSK-3β, RANK, RANKL and NF-κB were significantly increased, in the renal tissues of the DNM group. Conversely, pathological alterations in the renal tissues were attenuated, and the mRNA and protein expression levels of GSK-3β, RANK, RANKL and NF-κB were significantly decreased (P<0.05), in the DNI group, as compared with the DNM group. The results of the present study suggested that GSK-3β, RANK, RANKL and NF-κB may be crucially involved in the development of DN, and that LiCl may effectively attenuate DN by reducing the expression levels of GSK-3β, RANK, RANKL and NF-κB.

Icariin Prevents Cartilage and Bone Degradation in Experimental Models of Arthritis.

Background. Icariin (ICA) is an active compound extracted from Epimedium brevicornum Maxim. Previous reports have shown that icariin has a clinically significant therapeutic effect on rheumatoid arthritis. However, little is known about the mechanism by which icariin inhibits cartilage and bone degradation. Methods. New Zealand rabbits were immunized with antigen-induced arthritis (AIA) and treated with icariin. Joint tissues from rabbits were studied by histological analysis, transmission electron microscopy (TEM), and micro-CT. The expression levels of receptor activator of nuclear factor-B ligand (RANKL) and osteoprotegerin (OPG) in joint tissues were determined using immunohistochemistry and real-time PCR analysis. Results. Histological analysis and TEM sections of cartilage in the ICA treated group showed a low level of chondrocyte destruction. Micro-CT analysis showed that the bone mineral density value and bone structural level in ICA treated rabbits were significantly higher compared with those in the AIA group. Immunohistochemistry and real-time PCR analysis showed that icariin treatment reduced RANKL expression and enhanced OPG expression levels, as compared to the AIA group. Conclusion. These data indicate that ICA suppresses articular bone loss and prevents joint destruction. This study also determined that ICA regulated articular bone loss in part by regulating RANKL and OPG expression.

The effects of icariine concentration on osteoclasts bone resorption induced by titanium particles invitro.

In artificial joint replacement, osteoclast bone resorption induced by wear debris of the implant is a main reason for aseptic loosening. To extend the life of the prosthesis, detailed mechanisms of aseptic loosening and the ways to prevent it should be explored. The aim of this study was to investigate the in vitro effect of icariine on the bone resorption of osteoclasts induced by titanium particles. Macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL) were used to generate osteoclasts from RAW264.7 precursors. The proliferation of RAW264.7 precursors in the presence of different doses of icariine was evaluated by MTT assay. The cells were treated with titanium particles, titanium particles with icariine and culture medium only (control), respectively. At 48 h after treatment, the expression level of receptor activator of NF-kB (RANK) was detected by ELISA, and messenger RNA (mRNA) levels of tartrate-resistant acid phosphatase (TRAP), matrix metalloproteinase 9 (MMP-9), carbonic anhydrase II (CAII) and Cathepsin K (CtsK) were determined by real-time polymerase chain reaction. Western blot was applied to analyze the expression levels of TRAP, RANK and CtsK. In addition, bone chips were cultured in the above conditions, and Toluidine blue staining was then employed to calculate the number and area of resorption pits in the bone chips. After treatment with icariine, expression level of RANK was significantly decreased in the RAW264.7 cell that induced by titanium particle and its cultural medium, mRNA and protein levels of TRAP, CAII, MMP-9 and CtsK were reduced as well. In addition, the numbers of bone resorption pits and areas on bone slices were both reduced by icariine challenging. Icariine could inhibit bone resorption of osteoclast induced by titanium particle, and it might be used as a promising drug for treating of aseptic loosening.

Expression levels of receptor activator of nuclear factor-κB ligand and osteoprotegerin are associated with middle ear cholesteatoma risk

Conclusion: Overexpression of receptor activator of nuclear factor-κB ligand (RANKL) and low expression of osteoprotegerin (OPG) are typical features in middle ear cholesteatoma patients. The altered RANKL/OPG protein ratio suggests that alterations in the RANKL-OPG pathway may be major factors in the pathogenesis of middle ear cholesteatoma. Objective: Our meta-analysis explored the contribution of one important cytokine pathway, the RANKL and OPG pathway, in the development of middle ear cholesteatoma. Methods: We screened Embase, the Cochrane Library, CISCOM, CINAHL, Google Scholar, China BioMedicine, China National Knowledge Infrastructure, PubMed, and Web of Science for relevant articles. RANKL expression and ratio of RANKL/OPG were analyzed using Comprehensive Meta-Analysis Version 2 software. Results: The electronic literature search identified five studies that contained information on the correlation of RANKL and OPG expression with middle ear cholesteatoma. Increased RANKL expression positively correlated with middle ear cholesteatoma, while OPG expression showed an inverse association (p < 0.05). The ratio of RANKL/OPG in middle ear cholesteatoma cases was higher than in healthy controls, indicating that our observations are applicable to each individual case. Subgroup analysis based on country of study revealed that OPG levels decreased in China and Korea, and high RANKL expression was found in Poland, China, and Korea (all p < 0.05).