Increased Expression of Receptor Activator of Nuclear Factor-κB Ligand in Osteoblasts from Adolescent Idiopathic Scoliosis Patients with Low Bone Mineral Density

Persistent generalized low bone mineral density (BMD) has been reported in patients with adolescent idiopathic scoliosis (AIS).However,the exact mechanisms and causes of the low BMD in AIS patients are largely unknown.The purpose of this study was to examine the relationship between the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) levels in osteoblasts (OBs) from AIS patients with low BMD and with comparison made between the patients and controls.Twenty AIS patients and eight age-matched controls were included in the present study.The BMD of lumbar spine and proximal femur was measured in all subjects.OBs from the cancellous bone of each subject was harvested and primarily cultured.The mRNA and protein expression of RANKL and OPG in OBs was detected by RT-PCR and Western blotting.The results showed BMD was lower in AIS patients than in controls.A significantly higher mRNA and protein expression of RANKL was observed in OBs from AIS patients,while no significant difference was found in the expression of OPG between AIS patients and controls.As a result,RANKL/OPG ratio in patients with AIS was remarkably higher than controls.Our study preliminarily demonstrated expression of RANKL was higher in OBs from AIS patients with low BMD as compared with controls,suggesting the unbalanced RANKL/OPG ratio caused by an over-expression of RANKL in OBs may be responsible for the low BMD in AIS patients.

Effect of Triptolide on Expression of Receptor Activator of Nuclear Factor-κB Ligand in Rat Adjuvant Induced Arthritis

The effect of triptolide （TP） on the expression of receptor activator of nuclear factor-κB ligand （RANKL） and osteoprotegerin （OPG） was explored in rat adjuvant induced arthritis （AA）. AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate （MTX） at the onset （day 9） of arthritis. On the peak of arthritis （day 24）, the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells （PBMC） was detected. TNF-α and IL-1β levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group （.P〈0.01） ; The expression levels of RANKL in the synovium （P〈0.01） and bone （P〈0.05）, and OPG level in synovium （P〈0.05） were lower in TP group than in AA group （P〈0.05）. In TP group, the expression levels of RANKL mRNA and TNF-α, IL-1β in PBMC were lower than in AA group （all P〈0.01）. It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL.

Influence of baicalin on the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin in human periodontal ligament cells

Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.

Receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression in chronic apical periodontitis：possible association with inflammatory cells

Background Receptor activator of nuclear factor kappa B （NF-κB） ligand （RANKL） and osteoprotegerin （OPG） have been recently shown to play important roles in bone resorption. The aim of this study was to investigate the possible association between the expression of bone resorption regulators （RANKL and OPG） and inflammatory cell infiltration in chronic apical periodontitis.Methods The samples of chronic periapical lesions （n=40） and healthy periapical tissues （n=10） were examined for immunohistochemical analysis of RANKL and OPG. Lesion samples were further analyzed for the inflammatory infiltration condition. The inflammatory cell infiltration was scored in relation to immunohistochemical reactivity for CD3, CD20 and CD68.Results The number of RANKL-positive cells and the ratio of RANKL/OPG in chronic apical periodontitis were significantly higher than those in healthy periapical tissues （P＜0.001）. The number of RANKL-positive cells was higher in lesions with severe inflammatory infiltration than in those with light inflammatory infiltration （P＜0.05）. Significantly increased RANKL expression was found with T lymphocytes （CD3＋）, macrophages （CD68＋） and B lymphocytes （CD20＋）infiltration （P＜0.05）. No association was found between the ratio of RANKL/OPG and inflammatory cell infiltration.Conclusions RANKL expression was increased with T, B lymphocytes and macrophages infiltration, respectively in chronic periapical lesions. RANKL appears to be closely related to periapical inflammatory infiltrates. The relative ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.

Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

Background The cannabinoid receptor-2 （CB2） is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist （AM630） on receptor activator of nuclear factor kappa B （RANK） ligand （RANKL）induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase （TRAP） staining using a commercial kit. Total ribonucleic acid （RNA）was isolated and real-time reverse transcriptase-polymerase chain reaction （RT-PCR） was done to examine the expression of RANK, cathepsin K （CPK） and nuclear factor kappa B （NF-κB）. The extracellular signal-regulated kinase （ERK）,phosphorylation of ERK （P-ERK） and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide （MTT） assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of 〉100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB.Conclusion AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

Effect of Wenhua Juanbi Recipe(温化蠲痹方) on Expression of Receptor Activator of Nuclear Factor Kappa B Ligand,Osteoprotegerin,and Tumor Necrosis Factor Receptor Superfamily Member 14 in Rats with Collagen-Induced Arthritis

Objective： To study the effect of Wenhua Juanbi Recipe（温化蠲痹方, WJR） on expression of receptor activator of nuclear factor kappa B ligand（RANKL）, osteoprotegerin（OPG）, and tumor necrosis factor receptor superfamily member 14（TNFRSF14, also known as LIGHT） in rats with collagen-induced arthritis（CIA）. Methods： CIA rats were generated by subcutaneous injection of bovine collagen type-Ⅱ at the tail base. Sixty CIA rats were randomly assigned（10 animals/group） to： model, methotrexate（MTX）-treated（0.78 mg/kg body weight）, and WJR-treated（22.9 g/kg） groups. Healthy normal rats（n=10） were used as the normal control. Treatments or saline were administered once daily by oral gavage. Rats were sacrificed at day 28 post-treatment and knee synovium and peripheral blood serum were collected. Toe swelling degree and expression of RANKL, OPG, and LIGHT were determined by Western blot and immunohistochemistry. Results： Compared with the normal group, toe swelling degree was significantly increased in the model group（P〈0.01）. After treatment, toe swelling degree decreased significantly in the WJR and MTX groups compared with the model group（P〈0.01）. Compared with the normal group, expression of RANKL and LIGHT were significantly increased and OPG significantly decreased in peripheral blood and synovium of the model group（P〈0.01）. Conversely, RANKL and LIGHT expression were significantly reduced and OPG increased in the WJR and MTX groups compared with the model group（P〈0.01）. No statistically significant difference existed between WJR and MTX groups. Conclusion： WJR likely acts by reducing RANKL expression and increasing OPG expression, thus inhibiting RANKL/RANK interaction and reducing LIGHT expression, thereby inhibiting osteoclast formation/activation to block bone erosion.

Interleukin-1βinduces human cementoblasts to support osteoclastogenesis

Injury of the periodontium followed by inflammatory response often leads to mot resorption. Resorption is accomplished by osteoclasts and their generation may depend on an interaction with the cells in direct contact with the root, the cementoblasts. Our study aimed to investigate the role of human cementoblasts in the formation of osteoclasts and the effect of interleukin （IL）- 1β hereupon. Extracted teeth from healthy volunteers were subjected to sequential digestion by type I collagenase and trypsin. The effect of enzymatic digestion on the presence of cells on the root surface was analyzed by histology. Gene expression of primary human cementoblasts （pHCB） was compared with a human cementoblast cell line （HCEM）. The pHCBs were analyzed for their expression of IL-1 receptors as well as of receptor activator of nuclear factor kappa-B ligand （RANKL） and osteoprotegerin （OPG）. In a co-culture system consisting of osteoclast precursors （blood monocytes） and pHCBs, the formation of osteoclasts and their resorptive activity was assessed by osteo-assay and ivory slices. The cells obtained after a 120 min enzyme digestion expressed the highest level of bone sialoprotein, similar to that of HCEM. This fraction of isolated cells also shared a similar expression pattern of IL-1 receptors （ILl-R1 and ILl-R2）. Treatment with IL-11~ potently upregulated RANKL expression but not of OPG. pHCBs were shown to induce the formation of functional osteoclasts. This capacity was significantly stimulated by pretreating the pHCBs with IL-1β prior to their co-culture with human blood monocytes. Our study demonstrated that cementoblasts have the capacity to induce osteoclastogenesis, a capacity strongly promoted by IL-1β. These results may explain why osteoclasts can be formed next to the root of teeth.

Influences of IL-6R Antibody on PMMA Bone Cement-mediated Expression of OPG and RANKL in Synovial Fibroblasts

Effect of interleukin-6 receptor （IL-6R） antibody on polymethyl methacrylate （PMMA） bone cement-mediated expression of osteoprotegerin （OPG） and ：receptor activator of nuclear fac- tor-kappaB ligand （RANKL） in synovial fibroblasts was investigated. Synovial tissue obtained from to- tal knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry （SABC method） staining was used to identify synovial fibro- blasts. This experiment was divided into three groups according to different culture media： PMMA group （75μg/mL PMMA bone cement particles）, IL-6R antibody group （10 ng/mL IL-6R antibody＋75 μg/mL PMMA bone cement particles）, and control group （no IL-6R antibody or PMMA bone cement particles）. Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was meas- ured by cell counting kit-8 （CCK-8）. ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR （FQ-PCR） was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance （A） value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group （P〈0.01）, but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group （P〉0.05）. Re- suits of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group （P〈0.01）. The expression of RANKL was inhibited （P〈0.05）, and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group （P〉0.05）, but the expression of RANKL was higher in PMMA group than in control group （P〈0.05）, and there was a significant difference in the ratio of OPG/RANKL be- tween them （P〈0.05）. Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited （P〈0.01） and the expression of OPG mRNA was significantly increased （P〈0.01） in IL-6R an- tibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group （P〈0.05）, but the expression of OPG mRNA had no sig- nificant difference between them （P〉0.05）. IL-6R antibody could significantly increase the expression of OPC~ but inhibit the expression of RANKL, which might provide a theoretical basis of molecular bi- ology for the prevention and treatment of aseptic loosening of prosthesis.

Crosstalk between bone and other organs

Bone has long been considered as a silent organ that provides a reservoir of calcium and phosphorus,traditionally.Recently,further study of bone has revealed additional functions as an endocrine organ connecting systemic organs of the whole body.Communication between bone and other organs participates in most physiological and pathological events and is responsible for the maintenance of homeostasis.Here,we present an overview of the crosstalk between bone and other organs.Furthermore,we describe the factors mediating the cross-talk and review the mechanisms in the development of potential associated diseases.These connections shed new light on the pathogenesis of systemic diseases and provide novel potential targets for the treatment of systemic diseases.