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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.
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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.
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BACKGROUND: Long-term use of dexamethasone as a glucocorticoid will destroy the dynamic balance of osteogenesis and osteoclastation, reduce bone mineral density, damage bone biomechanics. A regulation of receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) protein pathway may be an approach to improve bone mineral density and bone biomechanics in dexamethasone-induced osteoporosis rats. OBJECTIVE: To investigate the effects of psoralen corylifolia extract on bone mineral density and bone biomechanics in dexamethasone-induced osteoporosis rats based on RANKL/OPG pathway. METHODS: Osteoporosis models were established in Wistar rats, SPF grade, using intramuscular injection of dexamethasone. Lentiviral vectors containing OPG gene interference fragments at concentration of 1×107 TU/mL were selected for subsequent experiments. The model rats were randomly divided into model group, no-load group (lentivirus empty vector), OPG silencing group (lentivirus vector containing OPG gene interference fragment), psoralen corylifolia extract group, psoralen corylifolia extract+OPG silencing group, with 12 rats in each group. Another 12 rats were selected as the control group. After drug treatment, bone mineral density of the left femur in rats was measured by bone densitometer; the elastic modulus, maximum load and yield load of the right femur of rats were measured by mechanical testing machine; the mineral salt content in the femur of rats was determined; the levels of RANKL and OPG in the serum of rats were detected by ELISA kit; and the expression levels of RANKL and OPG in bone tissues of rats were detected by western blot. The study protocol was approved by the Animal Ethics Committee of Qinghai University Medical School with an approval No. 2017081501. RESULTS AND CONCLUSION: (1) Bone mineral density, modulus of elasticity, maximum load, yield load, bone mineral salt content, serum OPG level and OPG protein expression in bone tissues were measured, which were lower in the model group than the control group, lower in the OPG silencing group than the model group, higher in the psoralea corylifolia extract group than the model group, and higher in the psoralen corylifolia extract+OPG silencing group than the OPG silencing group, but lower than the psoralea corylifolia extract group (all P 0.05). (4) All these findings indicate that psoralea corylifolia extract can increase bone mineral density and improve bone biomechanics in dexamethasone-induced osteoporosis rats, possibly by up-regulating the expression of OPG and down-regulating the expression of RANKL.
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Abstract The aim of this study was to evaluate markers of bone loss and immune response present in evolution of periodontal disease. One hundred and two Wistar rats were divided into three animals groups: PD0, without ligation and PD15 days and PD60 days, submitted to ligation placement with a sterile 3-0 silk cord in the cervical region of the upper first molar on both sides. Samples were obtained from the gingival tissue for histomorphometric analysis, immunohistochemical analysis of RANK, RANKL, OPG, characterization of the inflammatory infiltrate, quantification of nitric oxide, MCP-1, RANTES, IP10 chemokines, and expression of the TGF-b1, VEG, and bFGF. The number of inflammatory cells in gingival tissue was higher in PD60 samples. The collagen content and the area occupied by birefringent collagen fibers were lower for PD60. Differential leukocyte counting showed that there was a significantly higher polymorphonuclear influx in group PD15, while PD60 showed a greater number of lymphocytes. PD60 showed higher RANTES, IP-10, MCP-1 gene transcripts, as well as a higher nitric oxide concentration. Clinical evaluation revealed that the PD60 group presented an increase in furcal area. In conclusion, in this animal model the increase of RANK/RANKL and HGF markers is related to a specific immune response, and probably contributed to the evolution of periodontal disease. Investigating the effect of these biomarkers can help in targeted therapy for bone resorption, since blocking these can inhibit bone loss.
Resumo Este estudo avaliou marcadores de perda óssea e da resposta imune presentes na evolução da doença periodontal. Cento e dois ratos Wistar foram divididos em três grupos de animais: PD0, sem ligadura e PD15 dias e PD60 dias, submetidos a colocação de ligadura com um fio de seda estéril 3-0 na região cervical do primeiro molar superior em ambos os lados. Foram obtidas amostras de tecido gengival para análise histomorfométrica, análises imunohistoquímicas de RANK, RANKL, OPG, caracterização do infiltrado inflamatório, quantificação de óxido nítrico, expressão de quimiocinas MCP-1, RANTES, IP10 e do TGF-b1, VEGF e bFGF . O número de células inflamatórias no tecido gengival foi maior nas amostras PD60. O teor de colágeno na área ocupada pelas fibras de colágeno birrefringentes foram menores para PD60. A contagem diferencial de leucócitos mostrou que houve um influxo polimorfonuclear significativamente maior no grupo PD15, enquanto que PD60 mostrou número maior de linfócitos. PD60 apresentou transcritos de genes RANTES, IP-10, MCP-1 mais elevados, bem como uma maior concentração de óxido nítrico. A avaliação clínica revelou que o grupo PD60 apresentou aumento da área óssea exposta na região da furca. Em conclusão, neste modelo animal o aumento dos marcadores RANK/RANKL e HGF está relacionado a uma resposta imunológica específica e provavelmente contribuiu para a evolução da doença periodontal. Investigar o efeito destes biomarcadores pode ajudar na terapia dirigida para a reabsorção óssea, uma vez que bloquear estes pode inibir a perda óssea.
Subject(s)
Animals , Male , Rats , Periodontal Diseases/immunology , RANK Ligand/metabolism , Receptor Activator of Nuclear Factor-kappa B/metabolism , Osteoprotegerin/metabolism , Periodontal Diseases/metabolism , Immunohistochemistry , Blotting, Western , Rats, Wistar , Chemokines/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Inflammation/metabolismABSTRACT
Objective To explore the correlation between HIF-1α expression and RANKL/OPG pathway activation of fibroblast-like synoviocytes(FLSs)induced by Ti particles in aseptic loosening interfacial membrane. Method FLSs were extracted from the synovial tissue collected in surgeries and then co-cultured with Ti particles. QRT-PCR and Western blotting were conducted to measure the mRNA and protein expression of RANKL ,OPG and HIF-1αin FLSs at different concentration and time. Results Genes and protein expression levels of RANKL/OPG and HIF-1α were up-regulated with the increase of concentration of Ti particles. Expression of HIF-1α gene and protein increased time-dependently;the mRNA and protein expression of RANKL/OPG increased firstly and then declined alongside the increase of HIF-1αexpression. Conclusions Ti particles induce the up-regulation of HIF-1α expression and activate RANKL/OPG pathway. Up-regulation of HIF-1α may suppress the activation of RANKL/OPG pathway in FLSs of the interfacial membrane induced by Ti particles.
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ABSTRACT Objective The aim of this study was to investigate the in vitro and in vivo biological responses to nanostructured carbonated hydroxyapatite/calcium alginate (CHA) microspheres used for alveolar bone repair, compared to sintered hydroxyapatite (HA). Material and Methods The maxillary central incisors of 45 Wistar rats were extracted, and the dental sockets were filled with HA, CHA, and blood clot (control group) (n=5/period/group). After 7, 21 and 42 days, the samples of bone with the biomaterials were obtained for histological and histomorphometric analysis, and the plasma levels of RANKL and OPG were determined via immunoassay. Statistical analysis was performed by Two-Way ANOVA with post-hoc Tukey test at 95% level of significance. Results The CHA and HA microspheres were cytocompatible with both human and murine cells on an in vitro assay. Histological analysis showed the time-dependent increase of newly formed bone in control group characterized by an intense osteoblast activity. In HA and CHA groups, the presence of a slight granulation reaction around the spheres was observed after seven days, which was reduced by the 42nd day. A considerable amount of newly formed bone was observed surrounding the CHA spheres and the biomaterials particles at 42-day time point compared with HA. Histomorphometric analysis showed a significant increase of newly formed bone in CHA group compared with HA after 21 and 42 days from surgery, moreover, CHA showed almost 2-fold greater biosorption than HA at 42 days (two-way ANOVA, p<0.05) indicating greater biosorption. An increase in the RANKL/OPG ratio was observed in the CHA group on the 7th day. Conclusion CHA spheres were osteoconductive and presented earlier biosorption, inducing early increases in the levels of proteins involved in resorption.
Subject(s)
Humans , Animals , Male , Alginates/pharmacology , Biocompatible Materials/pharmacology , Bone Regeneration/drug effects , Durapatite/pharmacology , Nanostructures/therapeutic use , Cell Count , Glucuronic Acid/pharmacology , Hexuronic Acids/pharmacology , Materials Testing , Osteoblasts/drug effects , Osteoprotegerin/blood , Rats, Wistar , Receptor Activator of Nuclear Factor-kappa B/blood , Reproducibility of Results , Time Factors , Tooth Socket/drug effects , X-Ray DiffractionABSTRACT
Objective: To explore the effect of sika pilose antler type I collagen (SPC-I) on osteoclast and its molecular mechanism. Methods: The osteoclasts and osteoblasts were cultured by the induction method of whole bone marrow cells. The control (with full medium), osteoclasts (with HG-DMEM inducing medium), and SPC-I (2.5, 5, and 10 g/L) groups were set up. Except the control group, others were given the HG-DMEM inducing medium with each 40 ng/mL of both RANKL and macrophage colony-stimulating factor (M-CSF), then conditioned cultured for 7 d, every other 3 d to replace medium for the complement of the drug concentration. By HE and tartrate-resistant acid phosphatase (TRAP) stainings, the cell morphology was observed under inverted microscope. The TRAP activity was detected using spectrophotometer, the gene expression of TRAP, receptor activator of NF-κB (RANK), receptor activator of NF-κB lig and (RANKL), and osteoprotegerin (OPG) was measured by RT-PCR, and the RANK protein expression was detected by Western blotting. Results: Compared with the osteoclast group, SPC-I (5 and 10 g/L) groups could make TRAP positive cells and TRAP activity decreased, TRAP, RANK, and RANKL expression in gene level reduced, and RANK expression in protein level down-regulated also (P<0.01); Compared with the control group, SPC-I (2.5 and 10 g/L) could make the OPG expression in gene level increased and the RANKL/OPG ratio declined (P<0.01). The effect of 5 g/L SPC-I was the most significant (P<0.01). The effect of 2.5g/L SPC-I was not significant. Conclusion: SPC-I has the inhibitory effect on the osteoclast formation and differentiation; The effect of implementation is through RANKL/OPG signal transduction pathway to regulate the expression of TRAP and RANK genes.