THE THREE KEY PLAYERS IN OSTEOIMMUNOLOGY: RANK, RANKL AND OSTEOPROTEGERIN.

The RANK, RANKL and OPG interaction plays a major role in bone resorption and remodelling. The history dates back to mid 1990s when the RANK/ RANKL interaction was found to mediate osteoblastic stromal cells to stimulate osteoclastic bone resorption. This interaction was found to induce several cytokines including the TNF superfamily, thereby activating the pathways of bone remodelling. The Osteoprotegerin (OPG) prevents the binding of RANKL to RANK, thereby preventing the excessive bone resorption. When there is an imbalance in the levels of RANK/RANKL/OPG, the metabolic activity of the bone cells gets altered and thus there is loss of balance between bone formation and resorption. Thus, studying the inter – relationship between RANK, RANKL and OPG becomes critical for assessing the osteoblastic and osteoclastic activity

Potential effect of platelet rich fibrin prepared under differen centrifugation protocols on stem cells from the apical papila / O potencial efeito da fibrina rica em plaquetas preparada sob diferentes protocolos de centrifugação nas células tronco da papila apical

Objetivo: Este estudo objetivou avaliar o potencial proliferativo e de diferenciação das células tronco da papila cultivadas conjuntamente com fibrina rica em plaquetas (PRF) preparados sob dois protocolos de centrifugação distintos. Material e Métodos: Protocolos padrão e avançado de PRF foram utilizados. As células foram divididas em 4 grupos: controle negativo, controle positivo, padrão (L-PRF) e avançado (A-PRF). A contagem de células e ensaio de viabilidade foram realizados para verificar a capacidade proliferativa. Coloração vermelho de alizarina S, atividade de fosfatase alcalina e imunofluorescência para o receptor ativador do fator nuclear kappa-B (RANKL) foram utilizados para avaliar o potencial osteogênico e de diferenciação celular. Resultados: Ambos os tipos de PRF aumentaram o número de células, viabilidade celular sem toxicidade o que refletiu no aumento da proliferação e diferenciação de acordo com os testes realizados. Conclusão: O grupo A-PRF aumentou significativamente a proliferação e diferenciação comparado com o grupo L-PRF.(AU)

Objectives: The present work was designed to evaluate the proliferation and differentiation potential of stem cells from the apical papilla (SCAP) seeded along with platelet rich fibrin (PRF) scaffolds prepared under two different centrifugation protocols. Materials and Methods: Standard and advanced PRF protocols were used. Cells were divided into 4 groups: negative control, positive control, standard (L-PRF) and advanced (A-PRF) groups. Cell count and cell viability assays were carried out to assess the proliferation capacity. Alizarin red S (ARS) stain, Alkaline phosphatase (ALP) activity and Receptor activator of nuclear factor-kappa B ligand (RANKL) immunofluorescence staining were used to evaluate the osteogenic potential in the differentiated cells. Results:Both types of platelet rich fibrin increased the cell count, cell viability with no cytotoxicity that was reflected on increased proliferation and differentiation in terms of the performed tests. Conclusion: A-PRF group showed significant increase in proliferation and differentiation potentials compared to L-PRF group

Study of resveratrol against bone loss by using in-silico and in-vitro methods / Estudo do resveratrol contra a perda óssea usando métodos in silico e in vitro

Abstract By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis ​​pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Study of resveratrol against bone loss by using in-silico and in-vitro methods / Estudo do resveratrol contra a perda óssea usando métodos in silico e in vitro

By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa-Β ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of −6.9, −7.6, −7.1, −6.9, −6.7, and −7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 μg / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 μg / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa-Β [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de −6,9, −7,6, −7,1, −6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 μg / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 μg / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Study of resveratrol against bone loss by using in-silico and in-vitro methods

Abstract By applying the in-silico method, resveratrol was docked on those proteins which are responsible for bone loss. The Molecular docking data between the resveratrol and Receptor activator of nuclear factor-kappa- ligand [RANKL] receptors proved that resveratrol binds tightly to the receptors, showed the highest binding affinities of 6.9, 7.6, 7.1, 6.9, 6.7, and 7.1 kcal/mol. According to in-vitro data, Resveratrol reduced the osteoclasts after treating Marrow-Derived Macrophages [BMM] with Macrophage colony-stimulating factor [MCSF] 20ng / ml and RANKL 50ng / ml, with different concentrations of resveratrol (2.5, 10 g / ml) For 7 days, the cells were treated with MCSF (20 ng / ml) and RANKL (40 ng / ml) together with concentrated trimethyl ether and resveratrol (2.5, 10 g / ml) within 12 hours. Which, not affect cell survival. After fixing osteoclast cells with formaldehyde fixative on glass coverslip followed by incubation with 0.1% Triton X-100 in PBS for 5 min and after that stain with rhodamine phalloidin staining for actin and Hoechst for nuclei. Fluorescence microscopy was performed to see the distribution of filaments actin [F.actin]. Finally, resveratrol reduced the actin ring formation. Resveratrol is the best bioactive compound for drug preparation against bone loss.

Resumo Com a aplicação do método in-silico, o resveratrol foi ancorado nas proteínas responsáveis pela perda óssea. Os dados de docking molecular entre o resveratrol e o ligante do receptor ativador do fator nuclear kappa- [Receptor Activator of Nuclear Factor kappa-B Ligant (RANKL)] provaram que o resveratrol se liga fortemente aos receptores, mostraram as afinidades de ligação mais altas de 6,9, 7,6, 7,1, 6,9, - 6,7 e -7,1 kcal / mol. De acordo com dados in-vitro, o resveratrol reduziu os osteoclastos após o tratamento de macrófagos derivados da medula óssea [Bone Marrow-derived Macrophage (BMM)] com fator estimulador de colônias de macrófagos [Macrophage Colony-Stimulating Factor (MCSF)] 20ng / ml e RANKL 50ng / ml, com diferentes concentrações de resveratrol (2,5, 10 g / ml). Durante sete dias, as células foram tratadas com MCSF (20 ng / ml) e RANKL (40 ng / ml) juntamente com éter trimetílico concentrado e resveratrol (2,5, 10 g / ml) em 12 horas, processo que não afeta a sobrevivência celular. Após a fixação de células de osteoclastos com fixador de formaldeído em lamela de vidro seguido de incubação com 0,1% Triton X-100 em PBS por 5 min, foi realizado posteriormente o procedimento para corar com rodamina faloidina a actina e Hoechst os núcleos. A microscopia de fluorescência foi realizada para ver a distribuição dos filamentos de actina [F.actina]. Finalmente, o resveratrol reduziu a formação do anel de actina. O resveratrol é o melhor composto bioativo para o preparo de medicamentos contra a perda óssea.

Traditional Chinese Medicine in Prevention and Treatment of Osteonecrosis of Femoral Head Based on OPG/RANK/RANKL Signaling Pathway： A Review / 中国实验方剂学杂志

Osteonecrosis of the femoral head （ONFH） is a painful and debilitating disease caused by impaired blood supply to the femoral head and cellular and tissue degeneration， leading to gradual destruction of the bone structure and progressive collapse of the femoral head. The main pathological mechanism of ONFH is the disruption of the balance between bone absorption and the reconstruction of new bone， resulting from microcirculation damage and decreased cellular tissue ability. This imbalance leads to biomechanical changes and accelerates the pathological progression of ONFH. In the early stages， clinical manifestations may not be obvious， mainly presenting as pain or discomfort in the hip or groin area， which can be relieved after rest. In the later stage of the disease， pain intensifies， and limb shortening， lower limb weakness， difficulty walking， or limping may occur. Currently， western medicine commonly uses osteogenic agents， anticoagulants， and artificial joint replacement for treatment， but there are also many issues such as prosthesis loosening and infection. Research has shown that traditional Chinese medicine （TCM） treatment of ONFH takes a holistic approach and employs multi-functional， multi-target， and multi-system Chinese medicine therapies， ensuring the safety and effectiveness of the treatment. The osteoprotegerin （OPG）/receptor activator of nuclear factor-κB （RANK）/RANK ligand （RANKL） signaling pathway plays a crucial role in maintaining the dynamic balance of bone remodeling. TCM treatments utilize this pathway to promote apoptosis of osteoclasts， reduce bone resorption， and accelerate bone formation， thereby playing an important role in the prevention and treatment of ONFH. This paper reviewed the role of OPG/RANK/RANKL signaling pathway and related cytokine expression in ONFH by reviewing relevant literature in China and abroad and research status of Chinese medicinal monomers， Chinese medicinal formulations， and combinations with physical therapy in increasing osteoblast secretion， promoting OPG expression， enhancing cytokine expression levels， and inhibiting osteoclast activity for the prevention and treatment of ONFH. This paper is expected to provide new ideas and directions for TCM in the prevention and treatment of ONFH.

Effect of Jingui Shenqiwan on Diabetic Osteoporosis in Mice via AGEs/RANKL/NF-κB Pathway Based on Theory of "Kidneys Governing Bones" / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Jingui Shenqiwan on diabetic osteoporosis （DOP） in mice by regulating the advanced glycation end products （AGEs）/receptor activator of nuclear factor-κB ligand （RANKL）/nuclear factor-κB （NF-κB） signaling pathway based on the theory of "kidneys governing bones". MethodForty 6-week-old male and female skeletal-muscle-specific， dominant negative insulin-like growth factor-1 receptor （MKR） mice were selected and fed on a high-fat diet for eight weeks to establish the DOP model. The model mice were randomly divided into a model group， low- and high-dose Jingui Shenqiwan group （1.3， 2.6 g·kg-1）， and an alendronate sodium group （0.01 g·kg-1）， with 10 mice in each group. Additionally， 10 FVB/N mice of the same age were assigned to the normal group. The corresponding drugs were administered orally to each group once a day for four weeks. After the administration period， fasting blood glucose （FBG） measurement and oral glucose tolerance test （OGTT） were conducted. Kidney function and kidney index were measured. Renal tissue pathological changes were observed through hematoxylin-eosin （HE） and Masson staining. Immunohistochemistry was performed to assess the protein expression levels of AGEs， phosphorylated NF-κB （p-NF-κB）， and RANKL in renal tissues. Western blot analysis was conducted to measure the expression of proteins related to the AGEs/RANKL/NF-κB signaling pathway， osteoprotegerin （OPG）， and Runt-related transcription factor 2 （RUNX2） proteins in femoral bone tissues. ResultCompared with the normal group， mice in the model group exhibited significantly increased FBG （P<0.01）， trabecular bone degeneration， abnormal bone morphological parameters， significantly increased area under the curve （AUC） of OGTT （P<0.01）， enlarged kidney volume， significantly increased kidney function indicators and kidney index （P<0.01）， disrupted renal glomeruli and renal tubule structures， significantly increased expression of AGEs， RANKL， and p-NF-κB/NF-κB in renal tissues （P<0.05）， and significantly decreased expression of OPG and RUNX2 in femoral bone tissues （P<0.01）. Compared with the model group， mice in the Jingui Shenqiwan groups showed a significant decrease in OGTT AUC （P<0.01）. Histopathological analysis revealed alleviated structural lesions in renal glomeruli and renal tubules. Furthermore， the expression of AGEs， RANKL， and p-NF-κB/NF-κB in renal tissues was significantly reduced （P<0.05， P<0.01）， and the expression of RUNX2 and OPG in femoral bone tissues was significantly increased （P<0.05， P<0.01）. ConclusionJingui Shenqiwan can improve kidney function and downregulate the AGEs/RANKL/NF-κB signaling pathway to inhibit inflammatory reactions， thereby alleviating the symptoms of DOP in mice， demonstrating a therapeutic effect on DOP from the perspective of the kidney.

Traditional Chinese Medicine Intervenes in Signaling Pathways Associated with CKD-MBD： A Review / 中国实验方剂学杂志

Traditional Chinese medicine （TCM） has certain advantages in the treatment of chronic kidney disease-mineral and bone disorder （CKD-MBD）. In recent years， there have been many studies on the treatment of CKD-MBD by Chinese medicinal compounds and monomers. As revealed by literature retrieval， the research on the mechanism of Chinese medicine in intervening in signaling pathways related to CKD-MBD was mainly based on self-made Chinese medicinal compounds， and the action pathways involved fibroblast growth factor 23/Klotho （FGF23/Klotho） signaling pathway， Wnt/β-catenin signaling pathway， receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin （RANK/RANKL/OPG） system， and other signaling pathways. TCM can improve calcium and phosphorus metabolism and bone metabolism disorder， and regulate inflammatory reaction， oxidative stress， apoptosis， and autophagy by regulating this series of signaling pathways for the treatment of CKD-MBD. This paper introduced the research results of these signaling pathways and the mechanism of TCM in the treatment of CKD-MBD in order to provide ideas and references for the related research of Chinese medicine in the treatment of CKD-MBD.

Effects of bone marrow mesenchymal stem cell transplantation on bone metabolism and RANKL/OPG/TRAF6 signaling pathway in osteoporotic rats / 中华内分泌外科杂志

Objective:To investigate the effect of bone marrow derived mesenchymal stem cells (BMSC) transplantation on bone metabolism and its mechanism in ovariectomized osteoporosis rats.Methods:Forty clean SD female rats aged 7 weeks were divided into 4 groups according to the random number table method: sham operation group, model group, the transplantation group, positive control group, in addition to control the rest of the group were performed bilateral oophorectomy build osteoporosis rats model, after 2 months of model establishment, rats in transplantation group were injected with 80 μl/kg PBS solution containing bone marrow mesenchymal stem cells through tail vein, rats in sham operation group and model group were injected with the same amount of PBS solution through tail vein, and rats in positive control group were given Xianlinggubao (0.5 g/100 g) by gavage every day. Serum and femur were collected 14 days after treatment. Hematoxylin and eosin staining (HE) was used to observe the histopathological changes of femur. Micro-CT was used to measure bone mineral density and bone parameters. The expression levels of osteocalcin, osteoprotegerin, alkaline phosphatase and insulin-like growth factor 1 were detected by enzyme-linked immunosorbent assay (ELISA) kit. The serum levels of calcium, phosphorus and magnesium were measured by spectrophotometer. The protein expressions of RANKL, OPG, TRAF6 and NF-KB1 in femur of each group were detected by Western blot.Results:Compared with the sham operation group, the bone mineral density (BMD) of the model group was decreased by (0.28±0.01) g/cm 3, bone volume fraction (BMD) was decreased by (0.28±0.01) g/cm 3. BV/TV) decreased by (19.73±2.02) %, trabecular thickness (Tb.Th) decreased by (0.082±0.008) mm, trabecular number (Tb.N) decreased by (1.60±0.17) mm -1 and trabecular separation/spacing (Tb.Sp) increased (0.273±0.024) mm, osteoprotegerin (489.49±55.29) ng/L, alkaline phosphatase (229.13±15.05) U/L, insulin-like growth factor-1 (236.64±14.32) μg/L, and osteocalcin were decreased (1.866±0.109) μg/L, calcium (11.98±1.09) mg/dl, phosphorus (6.85±0.68) mg/dl, and magnesium decreased (0.62±0.04) mg/dl) , the relative expression level of RANKL increased (1.05±0.09) , the relative expression level of OPG decreased (0.58±0.08) , the relative expression level of RANKL increased (0.74±0.10) , and the relative expression level of NF-kB1 increased (1.01±0.11) ( P<0.05) ; bone mineral density, bone mineral density, bone mineral density BMD (0.38±0.04 g/cm 3, BV/TV (26.73±2.74) %, Tb.Th (0.094±0.006) mm, Tb.N (2.67±0.09) mm-1 and Tb.Sp were decreased (0.241±0.026) mm) , osteoprotegerin (720.09±67.41) ng/L, alkaline phosphatase (269.48±14.15) U/L, insulin-like growth factor 1 (IGF-1) decreased (335.95±24.13) μg/L, and osteocalcin increased (1.392±0.153) μg/L, calcium (7.12±0.53) mg/dl, phosphorus (4.54±0.32) mg/dl, magnesium (0.87±0.08) mg/dl. RANKL relative expression level increased (0.59±0.05) , OPG relative expression level decreased (0.97±0.10) , RANKL relative expression level increased (0.45±0.06) , NF-kB1 relative expression level increased (0.72±0.06) ( P<0.05) ;bone mineral density, bone mineral density, bone mineral density BMD (0.36±0.05) g/cm 3, BV/TV (28.72±3.20) %, Tb.Th (0.096±0.011) mm, Tb.N (2.85±0.24) mm -1 Tb.Sp was basically unchanged (0.241±0.027) mm, osteoprotegerin was decreased (716.78±36.90) ng/L, alkaline phosphatase was basically unchanged (270.65±18.59) U/L, and insulin-like growth factor 1 was decreased (336.94±17.50) μg/L, osteocalcin (1.377±0.101) μg/L, calcium (7.13±0.80) mg/dl, phosphorus (4.58±0.71) mg/dl, and magnesium (0.89±0.04) remained unchanged mg/dl, the relative expression level of RANKL increased (0.55±0.08) , the relative expression level of OPG decreased (0.98±0.13) , the relative expression level of RANKL was basically unchanged (0.40±0.05) , and the relative expression level of NF-kB1 increased (0.65±0.09) ( P<0.05) . Conclusion:Bone marrow mesenchymal stem cell transplantation can improve osteoporosis in ovariectomized rats by regulating bone metabolism and serum levels of calcium, phosphorus and magnesium, which may be related to RANKL/OPG/TRAF6 pathway.

Comparative study of the synovial levels of RANKL and OPG in rheumatoid arthritis, spondyloarthritis and osteoarthritis

Abstract Introduction In chronic arthropathies, there are several mechanisms of joint destruction. In recent years, studies have reported the implication of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in the process of activation and differentiation of osteoclasts, a key cell in the development of bone erosion. The RANKL/OPG ratio is increased in the serum of patients with malignant diseases and lytic bone disease, as well as rheumatoid arthritis (RA). The objective of this study was to measure and compare the concentrations of OPG and RANKL in the synovial fluid (SF) of patients with rheumatoid arthritis, spondyloarthritis (SpA) and osteoarthritis (OA). Methods This was an observational and cross-sectional study with 83 patients, 33 with RA, 32 with SpA and 18 with OA, followed up regularly in the outpatient clinics of the Rheumatology Department of the Clinics Hospital of the Ribeirão Preto Medical School-USP. All patients were assessed for indications for arthrocentesis by the attending physicians at the time of SF collection and were evaluated for demographic variables and medication use. Disease activity was assessed in individuals with RA and SpA. The quantification of SF OPG and RANKL levels was performed by ELISA, and the correlations of the results with clinical, laboratory and radiological parameters were assessed. Results We found no statistically significant difference in the RANKL and OPG levels among the groups. Patients with RA showed a positive correlation between the SF cell count and RANKL level (r = 0.59; p < 0.05) and the RANKL/ OPG ratio (r = 0.55; p < 0.05). Patients with OA showed a strong correlation between C-reactive protein (CRP) and the RANKL/OPG ratio (r = 0.82; p < 0.05). There was no correlation between the OPG and RANKL levels and markers of inflammatory activity or the disease activity index in patients with RA or SpA. Conclusion Within this patient cohort, the RANKL/OPG ratio was correlated with the SF cell count in patients with RA and with serum CRP in patients with OA, which may suggest a relationship with active inflammation and more destructive joint disease.

Receptor Activator of NF-kB Ligand Induced Osteoclastogenesis in Cervical Spondylosis - Role of Balamula Ghritam (A Powerful Antioxidant Drug) as Uttarabhaktika Sneha

The discs which are located in between the vertebras to act as shock absorbers during jolts to prevent spine injuries get either dislocated, bulged out or get thinned on continuous pressure being exerted upon them either by travel or a posture that is unsuitable to their alignment. In cases of discs getting thinned out and the height of these discs get reduced due to various causes produce undue pressure over the cervical spine resulting in oxidative stress and production of free radicals in the body causing deterioration of bone tissue and bone mass leading to regulation of RANKL/OPG ratio levels causing degeneration of bone. It is possible to resurrect the dehydrated discs with rehydration using Balamula (Sida cordifolia root) with the help of its properties and actions that it can positively act upon the area by refilling the gaps of erosions and assure a recovery with the proper administration of it in the form of ghee which has been processed with it and its intake being after food. Material & Methods: Various Ayurvedic classical textbooks and published journal articles were reviewed and analysed. Results: Evidences from various studies show that the phytochemicals obtain from Balamula (Sida cordifolia root) and Go- Ghritam (cow’s ghee) acts as antioxidant, reduces the activity of osteoclast and bone resorption by inhibiting RANKL receptor pathway. It also has anti-inflammatory, anti-arthritic, anti-analgesic effect that found to be effective in reducing the symptoms of cervical spondylosis.

Duchenne muscular dystrophy: RANK/RANKL/OPG (receptor activator of nuclear factor-kB/RANK ligand/osteoprotegerin) system and glucocorticoids / Distrofia muscular de Duchenne: el sistema RANK/RANKL/OPG (receptor activador del factor nuclear-kB/ligando de RANK/osteoprotegerina) y glucocorticoides

Abstract Duchenne muscular dystrophy (DMD) is an X-linked inherited disorder. Patients present with decreased bone mineral density (BMD) due to glucocorticoid therapy and progressive muscle weakness. Bone remodeling allows bone volume and structure to be maintained and controlled by local and systemic factors. These include the receptor activator of the nuclear factor-kB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, a determining pathway in the balance between bone formation and resorption. Disruptions in this complex, caused by factors such as glucocorticoids, can affect bone metabolism. The extensive action of the RANK/RANKL/OPG pathway suggests an influence on dystrophic muscle pathophysiology. This review aimed to highlight some aspects of the RANK/RANKL/OPG system, the effect of glucocorticoids on this pathway, and the pathophysiology of the patient with DMD.

Resumen La distrofia muscular de Duchenne (DMD) es un trastorno hereditario ligado al cromosoma X. Los pacientes presentan una disminución de la densidad mineral ósea (DMO) debido a los efectos adversos del tratamiento con glucocorticoides y a la debilidad muscular progresiva. El remodelado óseo permite mantener el volumen y la estructura ósea, proceso controlado por factores locales y sistémicos. Entre ellos destaca el sistema del receptor activador del factor nuclear-kB (RANK), su ligando natural RANKL (RANKL) y la osteoprotegerina (OPG), una vía determinante en el equilibrio entre la resorción y formación ósea. Las alteraciones en este complejo, originadas por factores como los glucocorticoides, pueden afectar el metabolismo óseo. La amplia acción de RANKL y OPG ha sugerido una influencia en la fisiopatología de la DMD. El objetivo de esta revisión fue destacar algunos aspectos del sistema RANK/RANKL/OPG, el efecto de los glucocorticoides en esta vía y la fisiopatología del paciente con DMD.

Experimental study on the regulation of subchondral bone plate osteoprotegerin/receptor activator of nuclear factor kappa-B ligand system by berberine to retard the development of osteoarthritis in rabbits / 中国骨伤

OBJECTIVE@#To investigate the effect of intra-articular berberine injection on the structural remodeling of subchondral bone plate and osteoprotegerin/receptor activator of nuclear factor kappa-B ligand(OPG/RANKL) system expression in rabbits with osteoarthritis(OA).@*METHODS@#Forty 12-month-old male rabbits with an average of(2.73±0.18) kg of body weight, underwent left anterior cruciate ligament transection(ACLT), and were divided into berberine group and placebo groups after operation, 20 rabbits in each group. The berberine group received intra-articular injection of 100 μmol/L berberine 0.3 ml every week for 6 weeks. In placebo group, the same dose of 0.9% sodium chloride injection was injected into the left knee joint cavity every week for 6 weeks. Another 20 12-month-old male rabbits, weighing (2.68±0.18) kg, underwent sham operation on the left knee joint without intra-articular injection intervention (sham operation group). On the last day of the sixth week after operation, three groups of animals were sacrificed to obtain knee joint specimens. The femoral medial condyle samples were obtained for histological evaluation of cartilage and subchondral bone, Mankin scoring system was used to evaluate articular cartilage structure. Image-Pro Plus(IPP) software was used to evaluate subchondral bone plate bone volume(BV), bone volume/total volume(BV/TV), trabecular circumference(TC), mean trabecular thickness (Tb.Th). Real-time quantitative reverse transcription polymerization Enzyme chain reaction(reverse transcription-polymerase chain reaction, RT-PCR) was used to detect the mRNA expression levels of OPG and RANKL in subchondral bone tissue at 6 weeks after operation.@*RESULTS@#The cartilage structure evaluation showed that the surface of cartilage tissue in the sham operation group was smooth and flat, and the safranin coloration was full in the full thickness of the cartilage;the cartilage tissue in the berberine group showed uneven surface layer, and the staining of safranin O was mildly decreased;the surface layer fibrosis was seen in placebo group, Safranin O faded significantly. The Mankin score in the berberine group was lower than that in placebo group(P<0.01), but higher than that in sham operation group(P<0.01). The structural evaluation of subchondral bone plate showed that the trabecular bone in sham-operated group was densely arranged;after berberine intervention, the trabeculae were closely arranged;the subchondral bone trabeculae in placebo group were relatively sparse, and the distance between trabeculae was wider. Subchondral bone plate IPP software evaluation showed that BV, BV/TV, TC, Tb.Th in berberine group were higher than those in placebo group(P<0.01), BV, BV/TV, TC, Tb.Th in berberine group were higher than those in placebo group(P<0.01), while lower than the sham operation group (P<0.01). PCR test results showed that the expression of OPG mRNA in the berberine group was significantly higher than that in placebo group(P<0.01), and OPG mRNA in the berberine group was lower than that in sham operation group (P<0.01). There was no significant difference in mRNA expression of RANKL among three groups(P>0.05);the ratio of OPG/RANKL in berberine group was higher than that in placebo group(P<0.01), but lower than that in sham operation group(P<0.01).@*CONCLUSION@#Intra-articular injection of berberine can effectively inhibit the resorption of subchondral bone in the early stage of OA and delay the development of the disease. The specific mechanism may be that berberine maintains the balance of OPG/RANKL system by up-regulating the expression of OPG gene in subchondral bone.

Caracterização de polimorfismos nos genes OPG, RANK e RANKL em uma população brasileira / Characterization of OPG, RANK and RANKL gene polymorphisms in a Brazilian population

Introdução: o sistema RANKL (receptor-ativador do fator nuclear-ligante κB)/RANK (receptor ativador do NF-kB)/OPG (osteoprotegrina) Introdução: o sistema OPG (osteoprotegrina)/RANK (receptor ativador do NF-kB)/RANKL (receptor-ativador do fator nuclear-ligante κB) regula os processos fisiológicos e patológicos da remodelação óssea. Polimorfismos genéticos nos genes OPG, RANK e RANKL têm sido associados a doenças, em diferentes populações. Objetivo: Descrever a frequência e o potencial regulatório dos polimorfismos do sistema OPG, RANK e RANKL em uma população brasileira; avaliar o seu potencial como marcadores genéticos informativos de ancestralidade; comparar com patologias associadas em outras populações. Metodologia: neste estudo, 506 indivíduos adultos, participantes de uma coorte acometidos de asma e periodontite, tiveram o DNA genômico extraído e genotipado, utilizando-se a plataforma Illumina. As plataformas NCBI, RegulomeDB, Haploview 4.2 e rSNPBase foram consultadas e utilizadas para análises. Resultados e Discussão: os polimorfismos mais frequentes na população estudada foram o rs3102724 no gene OPG, com frequência de menor alelo (MAF) de 46%; o rs4941129 em RANK, MAF 50%; e o rs9525641 em RANKL, MAF 46%. Os rs3134063 (1f) em OPG, rs17069898 (1f) em RANK e rs2200287 (1d) em RANKL apresentaram maior impacto funcional. Em OPG e RANK, nove polimorfismos se caracterizaram como marcadores genéticos informativos de ancestralidade, com predomínio nas populações YRI (africanos) e CEU (europeus). Os nove polimorfismos, com função intrônica, apresentaram MAF entre 2 a 46% na população-alvo e foram associados a patologias do metabolismo ósseo em outras populações. Conclusão: polimorfismos dos genes estudados se mostraram frequentes na população estudada e tiveram seus alelos mais frequentes associados a doenças em populações ancestrais. Sugere-se que sejam realizados mais estudos.

Introduction: The OPG (osteoprotegerin)/ RANK (NF-kB activating receptor)/ RANKL (nuclear-binding factor κB receptor-activating system regulates the physiological and pathological processes of bone remodeling. Genetic polymorphisms (SNPs) in OPG, RANK and RANKL genes have been associated with diseases in different populations. Objective: Describe the regulatory frequency and potential of SNPs in OPG, RANK and RANKL in a Brazilian population; assess their potential as informative genetic markers of ancestry; compare with pathologies associated with these polymorphisms in other populations. Methods: in this study, 506 adult individuals, participating in a cohort involving asthma and periodontitis, had genomic DNA extracted and genotyped using the Illumina platform. The NCBI, RegulomeDB, Haploview 4.2 and rSNPBase platforms were consulted and used for analysis. Results and discussion: the most frequent polymorphisms in the studied population were the rs3102724 in the OPG gene, with the lowest allele frequency (MAF) of 46%; rs4941129 in RANK, MAF 50% and rs9525641 in RANKL, MAF 46%. The rs3134063 (1f) in OPG, rs17069898 (1f) in RANK and rs2200287 (1d) in RANKL, had greater functional impact. In OPG and RANK, 9 SNPs were characterized as informative genetic markers of ancestry, predominantly in YRI (African) and CEU (European) populations. These 9 SNPs, with intronic function, presented MAF between 2 and 46% in our population, and were associated with pathologies in bone metabolism in other populations. Conclusion: SNPs of the studied genes were found to be frequent in the studied population and had their most frequent alleles associated with diseases in ancestral populations. It is suggested that further studies be carried out

miR-187-5p alleviates osteoporosis through inhibiting activity of osteoclast via RANKL/NFκB / 中华内分泌外科杂志

Objective:To investigate the role of microRNA-187-5p (miR-187-5p) on osteoporosis (OP) as well as the related molecular mechanism.Methods:The level of miR-187-5p and tartrate-resistant acid phosphatase (TRAP, the osteoclast marker) was quantitatively detected in TRANKL-induced OP group and control (con) group by reverse transcription-quantitative PCR (RT-qPCR) ; Cell counting kit 8 (CCK8) was used to measure the viability of murine macrophage cell line (RAW264.7) ; Western blot was used to detect the expression of p65 and TNFα. Moreover, the dual luciferase reporter assays was applied to detect the interaction between miR-187-5p and p65.Results:The osteoclast proliferation determined by CCK8 and the mRNA level of TRAP detected by RT-qPCR demonstrated that the growth of osteoclast was inhibited in OP group compared with that in con group ( P=0.008; P=0.017) , which reversed by miR-187-5p mimic stimulation ( P=0.023; P=0.037) . The miR-187-5p was lowly expressed in OP group compared with that in con group ( P=0.031) ,which was increased by miR-187-5p mimic treatment compared with miR-187-5p NC treatment ( P=0.041) . Moreover, western blot indicated that the protein level of p65 and TNFα in OP group were up-regulated compared with that in con group ( P=0.034; P=0.024) , which restored by the miR-187-5p mimic co-incubation ( P=0.028; P=0.036) . Moreover, the RT-qPCR indicated that the mRNA level of p65 in OP group was also increased compared with that in con group ( P=0.039) , and the miR-187-5p mimic co-incubation decreased the mRNA level of p65, compared with miR-187-5p NC group ( P=0.025) . The dual luciferase reporter assays indicated that there was an interaction between miR-187-5p and p65. Conclusion:miR-187-5p alleviates OP through inhibiting the activity of osteoclast via RANKL/NFκB signaling

Effect of Kidney-tonifying Method on OPG/RANK/RANKL Signaling Pathway in Postmenopausal Osteoporosis Animal Model： A Meta-analysis / 中国实验方剂学杂志

Objective:As the problem of global aging intensifies，postmenopausal osteoporosis （PMOP） has become a global health problem among females. At present，the commonly used biological agents have been proved not suitable for long-term use due to multiple adverse reactions. Several Meta-analyses have confirmed the good safety and effectiveness of kidney-tonifying method against PMOP，but its therapeutic mechanism remains unclear. The purpose of this Meta-analysis was to evaluate the effect of kidney-tonifying method on osteoclastogenesis inhibitory factor（OPG）/receptor activator of nuclear transcription factor （NF）-<italic>κ</italic>B （RANK）/receptor activator of NF-<italic>κ</italic>B ligand （RANKL） signaling pathway in PMOP animal model，so as to provide an experimental basis for the treatment of PMOP with kidney-tonifying method. Method:The related articles were retrieved from PubMed，Ovid Medline，Embase，China National Knowledge Infrastructure （CNKI），Chongqing Weipu Database for Chinese Technical Periodicals （VIP），and Wanfang Data Knowledge Service Platform with the retrieval time set from their inception to January 2020. The quality of each included article was evaluated using the SYRCLE's risk of bias tool. Then RevMan 5.3 was utilized for Meta-analysis according to the Cochrane systematic review methodology. Result:Thirty-two studies involving 619 rats were included. The quality score of these studies ranged from 3 to 5 points. The results of the Meta-analysis indicated obvious advantages of kidney-tonifying method in increasing bone mineral density （BMD）［standardized mean difference （SMD）=2.01，95% confidence interval（CI）=1.50-2.52，<italic>P</italic><0.000 01］），serum OPG level （SMD=3.33，95% CI=2.59-4.07，<italic>P</italic><0.000 01），and OPG mRNA expression （SMD=11.81，95% CI=7.49-16.13，<italic>P</italic><0.000 01），promoting OPG protein production （SMD=4.95，95% CI=3.09-6.81，<italic>P</italic><0.000 01），reducing serum RANKL（SMD=-4.88，95% CI=-6.01--3.75，<italic>P</italic><0.000 01） and RANK levels （SMD=-7.30，95% CI=-9.53--5.07，<italic>P</italic><0.000 01），and down-regulating RANKL （SMD=-6.22，95%CI=-8.95--3.49，<italic>P</italic><0.000 01） and RANK mRNA （SMD=-3.18，95% CI=-6.19--0.18，<italic>P</italic><0.05） expression and RANKL protein expression in bone tissue （SMD=-3.99，95% CI=-5.47--2.50，<italic>P</italic><0.000 01）. Conclusion:The kidney-tonifying method has been proved to possess potential advantages in regulating the balance of OPG/RANK/RANKL signaling pathway in PMOP animal model. Nevertheless，more large-sample sized，properly designed，and high-quality animal experiments are still needed for further verification.

Therapeutic Effect of Guizhi Shaoyao Zhimutang on Osteoporosis in Rats Based on RANKL/OPG / 中国实验方剂学杂志

Objective:To investigate the therapeutic effect of Guizhi Shaoyao Zhimutang （GSZT） on the osteoporosis （OP） in SD female rats induced by ovariectomy （OVX） combined with glucocorticoid injection and its related mechanisms. Method:The rats were divided into a sham operation group and an experimental group for OVX. One week later， the experimental rats were divided into an OP model group， a calciferol （positive control， 0.1 mg·kg^-1） group， and low（0.8 g·kg^-1）， medium（1.6 g·kg^-1）， and high-dose （3.2 g·kg^-1） GSZT groups. Except for those in the sham operation group， the rats received an intramuscular injection of dexamethasone （1 mg·kg^-1） twice per week for six weeks. After the rats were treated correspondingly for eight weeks， the rats were sacrificed for thighbone sample collection. The computer microtomography （Micro CT） was used to analyze the parameters of bone mineral density （BMD）， bone volume （BV）， tissue volume （TV）， structure model index （SMI）， connectivity density （Conn.D）， trabecular number （Tb.N）， trabecular thickness （Tb.Th）， trabecular pattern factor （Tb.Pf）， and trabecular separation （Tb.Sp）. In addition， hematoxylin-eosin （HE） and Masson staining was used for the histopathological examination of the thighbone. Tartrate-resistant acid phosphatase （TRAP） staining was used to analyze osteoclasts in bone tissues. The enzyme-linked immunosorbent assay （ELISA） was used to determine matrix metalloproteinase-9 （MMP-9）， cathepsin K （CTSK）， and TRAP5b in bone tissues， and Western blot was carried out to determine receptor activator of NF-kappaB ligand （RANKL）， osteoprotegerin （OPG）， alkaline phosphatase （ALP） in bone tissues. Result:Compared with normal group， the number of bone trabeculae in cancellous part of OP model rats decreased significantly， the spacing widened， the thickness became thinner， and the continuity was poor. Compared with model group， GZST medium and high-dose groups could improve the changes of femoral bone trabecular cancellous bone. Masson staining showed that compared with the normal group， the growth plate thickness of the distal femur epiphysis was thinner and the area of new bone was smaller in the OP model group， while the reduction of new bone was inhibited in the high dose group of calcitol and GZST. TRAP staining results showed that the number of osteoclasts in OP group was significantly increased compared with the normal group， and the number of osteoclasts in medium-high dose GZST group was reduced. Micro CT results showed that compared with normal group， BMD， BV， Tb.N， Tb.Th and Conn.D of OP rats were decreased， SMI and Tb.Sp were significantly increased（<italic>P</italic><0.01）. Compared with model group， medium and high dose of GZST could improve the degree of osteoporosis in OP rats， BV， Tb.N were significantly increased， SMI， Tb.Sp were significantly decreased （<italic>P</italic><0.01）. ELISA results revealed that compared with the sham operation group， the OP model group showed decreased ALP content （<italic>P</italic><0.01） and increased MMP-9， TRAP5b， and CTSK （<italic>P</italic><0.01）. Compared with the OP model group， the high-dose GSZT group showed increased ALP content （<italic>P</italic><0.01） and decreased MMP-9， TRAP5b， and CTSK （<italic>P</italic><0.01）. Western blot results demonstrated that compared with the OP model group， the calciferol group and the high-dose GSZT group showed inhibited expression of the RANKL protein （<italic>P</italic><0.05）， and increased OPG expression （<italic>P</italic><0.05）. Conclusion:The findings suggest that GZST has a potential therapeutic effect on OP caused by OVX combined with glucocorticoids in rats， and the underlying mechanism is associated with the regulation of RANKL/OPG in bone tissues. This study is expected to lay a preliminary foundation for its clinical application and modern preparation development.

Norlichexanthone purified from plant endophyte prevents postmenopausal osteoporosis by targeting ER

Although different types of drugs are available for postmenopausal osteoporosis, the limitations of the current therapies including drug resistances and adverse effects require identification of novel anti-osteoporosis agents. Here, we defined that norlichexanthone (NOR), a natural product, is a ligand of estrogen receptor-alpha (ER

The effect of cigarette smoking and low-level laser irradiation in rank/rankl/opg expression

Abstract The objective of this study was to investigate the effects of low-level laser therapy (LLLT) and cigarette smoke on alveolar socket osteoclastogenesis signaling after tooth extraction, in rats. Sixty male Wistar rats were randomly assigned to four groups with 15 animals each: Control Group (with right maxillary molar extraction - ME), Experimental I (with ME and LLLT), Experimental II (with ME and cigarette smoke) and Experimental III group (with ME, LLLT and cigarette smoke). Euthanasia was performed at 3, 7 and 14 days postoperative. qRT-PCR was used to evaluate expression of Tnfrsf11a (RANK), Tnfsf11 (Rankl) and Tnfrsf11b (OPG). Data were submitted to statistical analysis using two-way ANOVA followed by Bonferroni test (α=0.05). There was an upregulation of RANK, RANKL and OPG genes over all the time of healing in Exp I group compared to control group. Exp II group showed a decreased expression of all genes over time, whereas Exp III genes expression were higher than Exp II values but lower than Control and Exp I values over time. The results of this study concluded that the LLLT had a positive effect, whereas cigarette smoke had a negative effect on RANK, RANKL and OPG gene expression in bone remodeling process.

Resumo O objetivo deste estudo foi investigar os efeitos da terapia a laser de baixo nível (LLLT) e a fumaça de cigarro na sinalização da osteoclastogênese do alvéolo após extração dentária, em ratos. Sessenta ratos Wistar machos foram distribuídos aleatoriamente em quatro grupos com 15 animais cada: Grupo de controle (com extração do molar superior direito (EM), Experimental I (EM e LLLT), Experimental II (EM e fumaça de cigarro) e Grupo Experimental III (EM, LLLT e fumaça de cigarro). A eutanásia foi realizada aos 3, 7 e 14 dias após a extração. O qRT-PCR foi utilizado para avaliar a expressão de Tnfrsf11a (RANK), Tnfsf11 (RANKL) e Tnfrsf11b (OPG). Os dados foram submetidos à análise estatística usando two-way ANOVA seguido do teste de Bonferroni (α=0,05). Houve um aumento na expressão dos genes RANK, RANKL e OPG ao longo do tempo de cicatrização no grupo Exp I em comparação com o grupo controle. O grupo Exp II mostrou uma expressão diminuída de todos os genes ao longo do tempo, enquanto a expressão dos genes do grupo Exp III foi superior aos valores observados em Exp II, mas inferiores aos valores do grupo Controle e Exp I ao longo do tempo. Os resultados deste estudo concluíram que o LLLT tem um efeito positivo, enquanto a fumaça de cigarro possui efeito prejudicial na expressão gênica de RANK, RANKL e OPG no processo de remodelação óssea.

Research progress on the relationship between osteocytes and periodontitis / 口腔疾病防治

@#Osteocytes, which develop from osteoblasts, are recognized as the main cells embedded in mature bone tissue. The traditional notion is that osteocytes exclusively play a structural role, however, with the development of related research in recent years, the role of osteocytes in bone metabolism has been explored. Periodontitis is a chronic inflammatory disease initiated by plaque biofilm, and is the main cause of adult tooth loss. Clinically, periodontitis primarily manifests as attachment loss, bleeding on probing and other symptoms. Alveolar bone resorption is the most characteristic pathological change. Current research demonstrated that osteocytes sense mechanical stress, participate in bone remodeling, regulate mineral balance, and participate in endocrine function. Thus, these cells play an important role in bone homeostasis and systemic metabolic balance. Osteocytes are actively involved in the development of periodontitis through the high expression of receptor activator of nuclear factor kappa B ligand (RANKL), secretion of sclerostin, and effect on apoptosis, senescence and autophagy. In the future, the detection of bone cell metabolism-related products will have certain application prospects for the clinical evaluation of periodontitis prevention and treatment. Therefore, this paper reviewed the role of osteocytes in bone homeostasis and the relationship between osteocytes and periodontitis, to provide new ideas for the prevention and treatment of periodontitis.