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BACKGROUND: Carnosic acid, a bioactive compound found in rosemary, has been shown to reduce inflammation and reactive oxygen species (ROS). However, its mechanism of action in osteoclast differentiation remains unclear. OBJECTIVE: To investigate the effects of carnosic acid on osteoclast activation, ROS production, and mitochondrial function. METHODS: Primary bone marrow-derived macrophages from mice were extracted and cultured in vitro. Different concentrations of carnosic acid (0, 10, 15, 20, 25 and 30 μmol/L) were tested for their effects on bone marrow-derived macrophage proliferation and toxicity using the cell counting kit-8 cell viability assay to determine a safe concentration. Bone marrow-derived macrophages were cultured in graded concentrations and induced by receptor activator of nuclear factor-κB ligand for osteoclast differentiation for 5-7 days. The effects of carnosic acid on osteoclast differentiation and function were then observed through tartrate-resistant acid phosphatase staining, F-actin staining, H2DCFDA probe and mitochondrial ROS, and Mito-Tracker fluorescence detection. Western blot and RT-PCR assays were subsequently conducted to examine the effects of carnosic acid on the upstream and downstream proteins of the receptor activator of nuclear factor-κB ligand-induced MAPK signaling pathway. RESULTS AND CONCLUSION: Tartrate-resistant acid phosphatase staining and F-actin staining showed that carnosic acid dose-dependently inhibited in vitro osteoclast differentiation and actin ring formation in the cell cytoskeleton, with the highest inhibitory effect observed in the high concentration group (30 μmol/L). Carnosic acid exhibited the most significant inhibitory effect during the early stages (days 1-3) of osteoclast differentiation compared to other intervention periods. Fluorescence imaging using the H2DCFDA probe, mitochondrial ROS, and Mito-Tracker demonstrated that carnosic acid inhibited cellular and mitochondrial ROS production while reducing mitochondrial membrane potential, thereby influencing mitochondrial function. The results of western blot and RT-PCR revealed that carnosic acid could suppress the expression of NFATc1, CTSK, MMP9, and C-fos proteins associated with osteoclast differentiation, and downregulate the expression of NFATc1, Atp6vod2, ACP5, CTSK, and C-fos genes related to osteoclast differentiation. Furthermore, carnosic acid enhanced the expression of antioxidant enzyme proteins and reduced the generation of ROS during the process of osteoclast differentiation. Overall, carnosic acid exerts its inhibitory effects on osteoclast differentiation by inhibiting the phosphorylation modification of the P38/ERK/JNK protein and activating the MAPK signaling pathway in bone marrow-derived macrophages. [ABSTRACT FROM AUTHOR]

BACKGROUND: Liuwei Dihuang Wan takes “three tonifying and three reducing effects” as its compatibility feature to nourish yin and tonify the kidneys, while Zuogui Wan takes “seeking yin in yang” as its compatibility feature to nourish yin and tonify the kidneys by promoting yang. Both of them belong to the same method of nourishing yin and tonifying the kidneys, and have better curative effects at the symptomatic and cellular molecular levels. OBJECTIVE: To observe the effects of Liuwei Dihuang Wan and Zuogui Wan in bone metabolism, and to explore their mechanism of action in the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) osteoblastic pathway. METHODS: Thirty-two Sprague-Dawley rats were randomized into model, Liuwei Dihuang Wan, Zuogui Wan, and sham operation group, with eight rats in each group. Osteoporosis models were prepared using removal of both ovaries in the first three groups. Starting at 30 days postoperatively, rats in the Liuwei Dihuang Wan group were gavaged with Liuwei Dihuang Wan 1.125 g/kg/d; rats in the Zuoqui Wan group were gavaged with Zuogui Wan 2.25 g/kg/d; and rats in the sham operation group and the model group were gavaged with saline 10 mL/kg/d. After 12 weeks of gavage, the rat tibia was taken to measure bone mineral density. The serum levels of estrogen, bone alkaline phosphatase, and cAMP/cGMP were measured using ELISA, and the expression of OPG/RANKL in the femur was detected using western blot. RESULTS AND CONCLUSION: Compared with the sham operation group, the model group showed a decrease in bone mineral density and levels of estrogen and bone alkaline phosphatase (P < 0.05) and an increase in cAMP/cGMP level (P < 0.05). Compared with the model group, the Liuwei Dihuang Wan group and the Zuogui Wan group significantly increased bone mineral density (P < 0.05) and bone alkaline phosphatase levels (P < 0.05); the Zuogui Wan group significantly decreased cAMP/cGMP levels (P < 0.05) and upregulated OPG expression (P < 0.05); the Liuwei Dihuang Wan group upregulated OPG expression and downregulated RANKL expression (P < 0.05); and both groups were unable to significantly increase estrogen levels (P > 0.05). To conclude, Zuogui Wan, which seeks yin from yang, can effectively increase the expression of OPG but cannot downregulate the expression of RANKL. However, Liuwei Dihuang Wan, which has three tonifying and three reducing effects, can bidirectionally regulate the expression of OPG and RANKL. This result suggests that Liuwei Dihuang Wan can significantly inhibit osteoclastic function compared with Zuogui Wan, and further research is needed to verify this conclusion. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cistanoside A has the effects of anti-inflammation, antioxidation, antioxidation, reducing renal damage and anti-osteoporosis, but its effect on osteoclast differentiation, function and its underlying molecular mechanisms remain unclear. OBJECTIVE: To investigate the effect of Cistanoside A on osteoclast differentiation and bone resorption induced by receptor activator of nuclear factor kappa-B ligand (RANKL) in vitro and its mechanism. METHODS: Bone marrow macrophages were obtained from the femur and tibia of 4-6-week-old C57BL/6 mice. The cytotoxic effect of Cistanoside A (5, 10, 20, 40, 80, and 160 μmol/L) on bone marrow macrophage viability was examined using the cell counting kit-8 assay kit. Tartrate-resistant acid phosphatase staining was performed to observe the effect of different concentrations of Cistanoside A on osteoblast differentiation and its effective intervention concentration was determined. There was positive control group, Cistanoside A low, medium, and high dose groups (40, 80, and 160 μmol/L). After cell attachment, 50 ng/mL RANKL was added to induce osteoblast differentiation, and the corresponding dose of Cistanoside A was added to the Cistanoside A low, medium, and high dose groups, respectively. F-actin ring and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride staining were performed to detect the effects of Cistanoside A on the formation of osteoclasts. Toluidine blue staining of bone abrasion slices was used to observe the effects of Cistanoside A on bone resorption function of osteoclasts. The expression of upstream and downstream proteins of the JNK/MAPK pathway was detected by Western blot. The expression of genes related to osteoclast differentiation and bone resorption function such as tartrate-resistant acid phosphatase, DC-STAMP, Nfatc-1, Ctsk and c-Fos was detected by RTqPCR. RESULTS AND CONCLUSION: Tartrate-resistant acid phosphatase staining, F-actin ring staining and resorption pit assay showed that Cistanoside A significantly inhibited RANKL-induced osteoclast differentiation and bone resorption in a dose-dependent manner compared with the positive control group. The results of RT-qPCR showed that compared with the positive control group, both high and low dose groups of Cistanoside A could significantly downregulate the mRNA expression of tartrate-resistant acid phosphatase, DC-STAMP, Nfatc-1, Ctsk and c-Fos in a dosedependent manner. The results of western blot assay showed that the high dose group of Cistanoside A significantly inhibited the expression of p-JNK protein at 10, 20, 30 and 60 minutes of intervention; compared with the positive control group, Cistanoside A significantly inhibited the expression of Nfatc1 and c-Fos proteins in a dose-dependent manner. To conclude, Cistanoside A could inhibit the formation and bone resorption of osteoclasts by reducing the level of p-JNK protein, inhibiting the activation of MAPK pathway and the expression of key genes in osteoclasts. [ABSTRACT FROM AUTHOR]

Objective To investigate the molecular mechanisms by which silencing of the Periostin gene affects the expression of Runx2, receptor activator of nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) in murine osteoblasts and its impact on bone resorption activity. Methods Murine osteoblast cell line MC3T3-El was divided into an experimental group and a control group. The experimental group was transfected with LVpFU - GW 016PSC66473-1 virus, while the control group received pFU-GW-016PSC53349-1 virus to ensure equivalent cellular conditions. The efficiency of gene silencing was assessed using MDC method, and the protein expression of Runx2, RANKL, and OPG in both groups was evaluated by Western blotting. Results Following Periostin silencing, the experimental group showed significantly enhanced fluorescence expression compared to the control group (P<0.01). Protein expression of Runx2 and RANKL was significantly decreased (P<0.05), whereas OPG expression was markedly in- creased (P<0.05) in the experimental group compared to the control group. Conclusion Silencing of Periostin alters RANKL and OPG expression in murine osteoblasts, potentially influencing the Runx2 gene via the nuclear factor kappa B (NF-kB) signaling pathway. This enhancement of osteoblast function may contribute to the inhibition of bone resorption. [ABSTRACT FROM AUTHOR]

Objective To study the effect of different masticatory pressures on alveolar bone remodeling in rats with orthodontic moving teeth. Methods Forty-five male SD rats aged 8 weeks were randomly divided into 3 groups: the baseline group (n=5), the soft diet group (n=20) and the hard diet group (n=20). Rats in the baseline group were sacrificed at the beginning of the experiment. The experimental rat models of right upper first molar movement were established in the soft diet group and the hard diet group. Left side of the maxillary was used as the control group. Each group was fed with corresponding diet. Five rats were sacrificed in each group on the day 3, day 5, day 7 and day 14 after orthodontic treatment respectively. Micro CT was used to measure the mesial movement distance of molar, bone volume/tissue volume (BV/TV), trabecular separation (Tb. Sp) and trabecular thickness (Tb. Th) of the alveolar bone on the pressure side. The number of osteoclasts in alveolar bone was counted after TRAP staining. In situ hybridization was used to detect the expression of RANKL and OPG. Results The amount of tooth movement was smaller on day 14 in the soft diet group than that of the hard diet group (P＜0.05). There were no significant differences in BV/TV, Tb.SP and Tb.Th between the soft diet group and the hard diet group. TRAP staining and in situ hybridization showed that the number of osteoclasts and the RANKL/OPG ratio on day 5 and day 7 were lower in the soft diet group than those of the hard diet group (P＜0.05). Conclusion The osteoclast activity in the alveolar bone on the pressure side of the orthodontic tooth is decreased and the tooth movement is inhibited under lower masticatory pressure. [ABSTRACT FROM AUTHOR]

To investigate the regulatory effect of adiponectin on RANKL/OPG in peripheral circulation and its therapeutic effect on diabetic osteoporosis. Methods High fat diet combined with streptozomycin was used to construct diabetic osteoporosis model mice, which were treated with adiponectin and alendronate sodium as positive control. They were divided into control group, model group, treatment group and positive control group. After the intervention, the weight of the mice was measured, the venous blood of the mice was collected, and the fasting glucose level, glucose tolerance test, and serum RANKL/ OPG were detected. The primary bone marrow mesenchymal stem cells were extracted from the femur of mice in each group, induced osteogenic differentiation and stained with alizarin red to detect the expression levels of Opg and Rankl genes. The lumbar 3 vertebrae of mice were obtained, and the changes of bone microstructure in each group were detected. Results After adiponectin treatment, compared with the model group, the body weight, fasting blood glucose level and glucose tolerance test were significantly decreased, serum OPG concentration increased, serum RANKL concentration decreased, and serum RANKL/OPG decreased in the treatment group. Compared with the control group, the bone formation ability of bone marrow mesenchymal stem cells in the model group was significantly weakened, and RANKL/OPG was significantly up-regulated. Compared with the model group, the osteoblastic differentiation ability of bone marrow mesenchymal stem cells in the treatment group was significantly enhanced, and RANKL/OPG was significantly down-regulated. After adiponectin treatment, the number of osteoblasts per unit length, the percentage of trabecular area and the thickness of trabecular were significantly increased. Conclusion Adiponectin can reduce the body weight of diabetic mice, reduce fasting blood glucose, partially restore the body's blood glucose regulation ability, up-regulate Opg gene expression and inhibit Rankl gene expression, promote bone marrow mesenchymal stem cell osteogenic differentiation, and improve bone microstructure, which is a potential choice for the treatment of diabetic osteoporosis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Overactive osteoclasts disrupt bone homeostasis and play a bad role in the pathological mechanisms of related skeletal diseases, such as osteoporosis, fragility fractures, and osteoarthritis. Studies have confirmed that ellagic acid and ellagtannin have the potential to inhibit osteoclast differentiation. As their natural metabolites, urolithin A has antioxidant, anti-inflammatory, anti-proliferative and anti-cancer effects, but its effect on osteoclast differentiation and its underlying molecular mechanisms remain unclear. OBJECTIVE: To explore the effect of urolithin A on osteoclast differentiation induced by receptor activator for nuclear factor-κB ligand and its mechanism. METHODS: Mouse mononuclear macrophage leukemia cells (RAW264.7) that grew stably were cultured in vitro. Toxicity of urolithin A (0, 0.1, 0.5, 1.5, 2.5 μmol/L) to RAW264.7 cells were detected by cytotoxic MTS assay to screen out the safe concentration. Different concentrations of urolithin A were used again to intervene with receptor activator for nuclear factor-κB ligand-induced differentiation of RAW264.7 cells in vitro. Then, tartrate-resistant acid phosphatase staining and F-actin ring and nucleus staining were performed to observe its effect on the formation and function of osteoclasts. Finally, the expressions of urolithin A on upstream and downstream genes and proteins in the MAPK signaling pathway were observed by western blot and RT-qPCR assays. RESULTS AND CONCLUSION: Urolithin A inhibited osteoclast differentiation and F-actin ring formation in a concentration-dependent manner and 2.5 μmol/L had the strongest inhibitory effect. Urolithin A inhibited the mRNA expression of Nfatc1, Ctsk, Mmp9 and Atp6v0d2 and the protein synthesis of Nfatc1 and Ctsk, related to osteoclast formation and bone resorption. Urolithin A inhibited the activity of osteoclasts by downregulating the phosphorylation of p38 protein to inhibit the mitogen-activated protein kinase signaling pathway. [ABSTRACT FROM AUTHOR]

Objective To investigate the mechanisms of 1, 25(OH)2D3 on joint inflammation and bone destruction in a mouse model of collagen-induced arthritis (CIA). Methods Thirty DBA/1J mice were equally allocated into control, CIA and intervention (VD) groups. The CIA model was established in the mice in the CIA group and VD group, meanwhile the VD group was treated with 1, 25(OH)2D3. The thickness of the hindfoot paws was measured with vernier caliper. Micro-CT scanning was used to measure bone mineral density (BMD) of the hindfoot paw, ELISA was employed to detect the serum levels of 1, 25(OH)2D3 and proinflammatory cytokines (IL-1β, IL-6, TNF-α), and flow cytometry was performed to calculate the ratio of Th17 to Treg cells in the spleen. The pathological changes of the knee joint were observed with HE staining. Western blotting was applied to detect the expression levels of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), NF-κB, and vitamin D receptor (VDR) in mouse ankle joints. Results 1, 25(OH)2D3 did not reduce the incidence of CIA in the of mice in the VD group, but significantly reduced joint inflammation. Obviously decreased thickness of the hindfoot paw, arthritis score, and knee histological synovitis score (HSS) were observed in the VD group than the CIA group (all P < 0.05). The CIA group presented more serious bone destruction and lower BMD and bone volume fraction (BV/TV) when compared with the VD group (both P < 0.05). The serum levels of IL-1β, IL-6 and TNF-α and ratio of Th17/Treg cells in the VD group were statistically lower than those in the CIA group (all P < 0.05). Compared with the CIA group, the VD group had notably higher protein levels of VDR and OPG, lower levels of NF-κB and RANKL, and reduced RANKL/OPG ratio in the ankle joint (all P < 0.05). Conclusion 1, 25(OH)2D3 may alleviate arthritic inflammation and suppress bone destruction in the mouse CIA model via regulating the Th17/Treg ratio and the RANKL/OPG axis.

BACKGROUND: Naringin, a monomer compound of Traditional Chinese medicine, has anti-inflammatory and osteogenic effects, but its effect on the healing rate of tooth extraction wounds is not clear. OBJECTIVE: To investigate the effect of naringin on the healing rate of tooth extraction wounds in rat models of osteoporosis. METHODS: Forty-eight female Sprague-Dawley rats were randomly divided into sham-operation group (n=14) and ovariectomized group (n=34). In the ovariectomized group, bilateral ovaries were removed to establish osteoporosis models. After 6 weeks, four rats in each group were randomly selected for MicroCT detection to confirm the modeling results. After successful modeling, the remaining rats were subject to tooth extraction and randomly divided into tooth extraction group (the remaining rats in the sham-operation group), control group, naringin group, and estradiol group, and the second molar of left maxillary was extracted. Naringin group was given naringin 300 mg/kg/d by gavage, estradiol group was given 17-β estradiol 20 μg/kg/d by subcutaneous injection, and tooth extraction group and control group were treated with the same volume of normal saline. After 4 weeks of intervention, the rat maxilla was separated for hematoxylin-eosin staining, Masson staining, and MicroCT detection. The expressions of osteoprotegerin and receptor activator of nuclear factor-κB ligand (RANKL) at mRNA and protein levels in the bone tissue around the tooth extraction wounds were also detected. RESULTS AND CONCLUSION: After 6 weeks of castration, the bone volume fraction of the ovariectomized group decreased, indicating that the modeling of osteoporosis was successful. Four weeks after tooth extraction, compared with the control group, the naringin group and estradiol group had smaller trabecular separation and less loss of alveolar bone height, indicating that naringin could promote bone healing of tooth extraction wounds. Compared with the control group, the expression level of osteoprotegerin and RANKL in bone tissues around tooth extraction was increased and decreased respectively in the naringin and estradiol groups, indicating that naringin could up-regulate osteoprotegerin expression and down-regulate RANKL expression. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of osteocyte-like cell MLO-Y4-derived exosomes on osteoclast differentiation. Methods Osteocyte-like cell MLO-Y4-derived exosomes (MLO-Y4-Exo) were obtained by ultrahigh speed centrifugation. Their characteristics were identified using Western blotting and transmission electron microscopy. MLO-Y4-Exo were co-cultured with bone marrow macrophages. TRAP staining was conducted to detect the osteoclast differentiation. Mice were subjected to subcutaneous supra-calvarial injection of MLO-Y4-Exo for 5 consecutive days to detect the osteoclast differentiation in vivo. The mechanism of MLO-Y4-Exo promoting osteoclast differentiation was detected with small interfering RNA. Results The size and morphology of MLO-Y4-Exo met the exosome characteristics and highly expressed CD63 and Alix exosome proteins. MLO-Y4-Exo promoted osteoclast differentiation in vitro and in vivo (P<0.05). RANKL small interfering RNA assay confirmed that MLO-Y4-Exo promoted osteoclast differentiation by transmitting RANKL protein to osteoclast precursors (P<0.05). Conclusion MLO-Y4-derived exosomes promote osteoclast formation by transferring RANKL to osteoclast precursors. [ABSTRACT FROM AUTHOR]

Objective To explore the biological role of receptor activator of nuclear factor kappa-B ligand (RANKL) in the activation of hepatic stellate cells (HSCs) and the process of hepatic fibrosis. Methods Twenty-six male C57BL/6 mice (6~8 weeks old) were randomly assigned to control group and liver fibrosis group (n=13).The mice in the latter group were injected intraperitoneally with 5 μL/g 10% carbon tetracholoride (CCL4, twice/week) to induce liver fibrosis, while those of the former group were given same volume of sterile oil solution.In 6 weeks later, the expression of RANKL, α-SMA and Collagen-Ⅰ at mRNA and protein levels in liver tissue, primary hepatocytes (HCs) and HSCs was detected by quantitative real-time PCR (RT-qPCR), immunohistochemical assay and Western blotting, respectively.After primary HSCs were treated with different concentrations of exogenous recombinant RANKL, the mRNA and protein levels of above molecules were measured with RT-qPCR and immunofluorescence assay. Results The mRNA and protein levels of RANKL were significantly increased in the liver tissue from the model group than the control group (P < 0.05), and the protein was in a diffuse distribution.In the control mice, the mRNA and protein levels of RANKL was obviously higher in HCs than HSCs (P < 0.001), while CCL4 treatment induced liver fibrosis resulted in the expression level of RANKL was further increased in HCs, while decreased in HSCs.After different concentrations of recombinant RANKL were added to stimulate the activation of primary HSCs, the expression of RANKL, α-SMA and Collagen-Ⅰat mRNA and protein levels in HSCs activated by recombinant RANKL was decreased in a dose-dependent manner (P < 0.05, P < 0.01 or P < 0.001). Conclusion In the process of liver fibrosis in mice, HCs express a large amount of RANKL, and RANKL can inhibit the activation of HSCs.

Objective　To analyze the correlation between serum receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG), fibroblast growth factor 23 (FGF23) with renal function and calcium and phosphorus metabolism in patients with chronic kidney disease (CKD). Methods　A total of 62 patients who met the diagnostic criteria of CKD in our hospital from January 2021 to February 2022 were enrolled as the research group, and 26 healthy persons were included in the control group. Renal function and calcium and phosphorus metabolism were measured, and serum levels of RANKL, OPG and FGF23 were measured by enzyme-linked immunosorbent assay (Elisa). Results　The serum levels of RANKL, OPG and FGF23 in the research group were significantly increased, and the serum levels of RANKL, OPG and FGF23 in the CKD5 stage group were higher than the CKD2-3 stage group and the CKD4 stage group. Correlation analysis showed that the serum levels of RANKL, OPG and FGF23 were negatively correlated with eGFR, serum RANKL levels were positively correlated with PTH, serum FGF23 levels were positively correlated with PTH and P levels, and negatively correlated with Ca. Conclusions　The levels of serum RANKL, OPG and FGF23 are increasing with the progression of CKD disease. Serum RANKL and FGF23 are sensitive indicators for early detection of calcium and phosphorus metabolism disorders in patients with CKD. [ABSTRACT FROM AUTHOR]

BACKGROUND: Orthodontic treatment can change the levels of various cytokines in periodontal tissue, which promotes bone resorption on the tooth pressure side and bone formation on the tooth tension side. Changes in high glucose level and other inflammatory factor levels in diabetic patients induce an imbalance in bone metabolism where absorbed bone tissue is more than that formed. OBJECTIVE: To observe the effects of interleukin-10 and mechanical stimulation on the expressions of osteoprotegerin and receptor activator of nuclear factor kappa B ligand in human periodontal ligament cells under high glucose environment, and to explore the effect of interleukin-10 on bone remodeling of alveolar bone in diabetic patients under orthodontic force. METHODS: Human periodontal ligament cells were cultured by tissue explant method. According to the mechanical stimulation the cells received, the cells were divided into stress group (A, B, C, D subgroups) and tension group (E, F, G, H subgroups). Both stress and tension groups included four subgroups (lowglucose DMEM group, low-glucose DMEM group+interleukin-10 group, high-glucose DMEM group, high-glucose DMEM+interleukin-10 group). Cell supernatant was collected at 0, 12, 24, 48, and 72 hours after culture. The expressions of osteoprotegerin and receptor activator of nuclear factor kappa B ligand in the supernatant were detected by ELISA. RESULTS AND CONCLUSION: Pressure could decrease the expression of osteoprotegerin and increase the expression of receptor activator of nuclear factor kappa B ligand in human periodontal ligament cells, and decreased their ratio. High glucose could promote this process, while the effect of interleukin-10 was opposite to that of high glucose. Tension could increase the expression of osteoprotegerin, decrease the expression of receptor activator of nuclear factor kappa B ligand in human periodontal ligament cells, and increase their ratio. High glucose could inhibit this process, and interleukin-10 had an effect that was opposite to high glucose. To conclude, high glucose environment can reduce the expression of osteoprotegerin, increase the expression of receptor activator of nuclear factor kappa B ligand in human periodontal ligament cells, and decrease their ratio under mechanical stimulation. Whereas, interleukin-10 can antagonize the effect of high glucose. [ABSTRACT FROM AUTHOR]

To analyze the correlation between gingival crevicular fluid C-reactive protein (CRP), interleukin-17 (IL-17), receptor activator of NF-KB ligand (RANKL) and periodontal clinical indicators in patients with peri-implantitis (PI) and to explore the evaluation value of combined detection for prognosis after treatment. 80 patients with PI who were admitted to our hospital from January 2018 to January 2022 were selected as the inflammation group. Another 80 healthy implant patients in the same period were selected as the healthy group. The levels of gingival crevicular fluid CRP, IL-17 and RANKL and periodontal clinical indicators were measured and compared between the two groups. Pearson correlation was used to analyze the correlation between gingival crevicular fluid CRP, IL-17, RANKL and periodontal clinical indicators. In addition, all patients with PI were divided into good prognosis group with 27 cases and poor prognosis group with 53 cases according to the difference between acute physiology and chronic health status score II (APACHE II). The levels of gingival crevicular fluid CRP, IL-17 and RANKL were compared between the two groups. Through the receiver operating characteristic (ROC) curve, the efficacy of the above three indicators combined to predict the poor prognosis of patients with PI after treatment was analyzed. The levels of gingival crevicular fluid CRP, IL-17 and RANKL in inflammatory group were higher than those in healthy group (all P＜0.05). The scores of modified gingival index (mGI), modified gingival sulcus bleeding index (mSBI) and probing depth (PD) in the inflammatory group were higher than those in the healthy group (all P＜0.05). Pearson correlation analysis showed that the levels of gingival crevicular fluid CRP, IL-17 and RANKL were positively correlated with mGI, mSBI scores and PD (all P＜0.05). The levels of gingival crevicular fluid CRP, IL-17 and RANKL in the poor prognosis group were higher than those in the good prognosis group (all P＜0.05). The ROC curve analysis showed that the area under the curve (0.95CI) of gingival crevicular fluid CRP, IL-17 and RANKL combined to predict the poor prognosis of patients with PI after treatment was 0.851 (0.752 ~ 0.944), which was better than the above three indicators alone. The gingival crevicular fluid CRP, IL-17 and RANKL are closely related to mGI, mSBI scores and PD in patients with PI. The detection of these three indicators is helpful to evaluate the patient's condition and prognosis after treatment. [ABSTRACT FROM AUTHOR]

Objective: To study the effects of Qianghuo Dihuang decoction on helper T cells/regulatory T cells (Th17/Treg) imbalance, serum matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-9 (MMP-13) and vascular endothelial growth factor (VEGF), receptor activator of nuclear factor-资B ligand (RANKL) mRNA expression in synovium tissue of adjuvant rheumatoid arthritis (RA) rats. Methods: 40 SD rats were taken for study, they were randomly divided into normal control group, model group, Qianghuo Dihuang decoction group and methotrexate group by random number table, with 10 rats in each group. Except for normal control group, adjuvant RA animal model was established in other groups. The Qianghuo Dihuang decoction group was given gavage intervention of Qianghuo Dihuang decoction, methotrexate group was given gavage intervention of methotrexate, normal control group and model group were given gavage intervention of normal saline. Each group was intervened for 28 days, the body weight, arthritis index, joint swelling degree, peripheral blood Th17/Treg related indicators, serum MMP-3 and MMP-13 levels, VEGF and RANKL mRNA expressions in synovial tissues were compared among all groups. Results: Body weight in model group was lower than that in normal control group, arthritis index and joint swelling degree in Qianghuo Dihuang decoction group and methotrexate group were lower than those in model group, but body weight was higher than that in model group (all P＜0.05). The peripheral blood CD4+IL-17+T and Th17/Treg in model group, Qianghuo Dihuang decoction group and methotrexate group were higher than those in normal control group, while Foxp3+CD4+CD25+Treg were lower than that in normal control group. The peripheral blood CD4+IL-17+T and Th17/Treg in Qianghuo Dihuang decoction group and methotrexate group were lower than those in model group, while Foxp3+CD4+CD25+Treg were higher than that in model group (all P<0.05). The serum MMP-3 and MMP-13 levels in model group, Qianghuo Dihuang decoction group and methotrexate group were all higher than those in normal control group, while the serum MMP-3 and MMP-13 levels in Qianghuo Dihuang decoction group and methotrexate group were lower than those in model group (all P<0.05). The VEGF and RANKL mRNA expression levels in synovium in model group, Qianghuo Dihuang decoction group and methotrexate group were higher than those in normal control group, while the VEGF and RANKL mRNA expression levels in synovium in Qianghuo Dihuang decoction group and methotrexate group were lower than those in model group (all P<0.05). Conclusion: Qianghudihuang Decoction can improve the clinical symptoms and Th17/Treg imbalance of adjuvant RA rats, and effectively improve the levels of MMP-3 and MMP-13 in serum, as well as the expressions of VEGF and RANKL mRNA in synovial tissues [ABSTRACT FROM AUTHOR]

BACKGROUND: The anti-osteoporotic mechanism of salvianolic acid B is different from traditional anti-bone resorption drugs. It has a multi-target pharmacological effect, which has not yet been fully elucidated. OBJECTIVE: To study the effect of salvianolic acid B on bone density of femur and lumbar vertebra in rats with osteoporosis, and to explore its anti-osteoporotic mechanism. METHODS: Totally 36 SPF SD rats aged 6 months were randomly divided into three groups: sham operation group, osteoporosis group, and salvianolic acid B group. Bilateral ovaries were removed and the osteoporosis model was replicated in the osteoporosis group and salvianolic acid B group. In the sham operation group, intact ovaries were kept and part of adipose tissue was removed. At 4 weeks after surgery, 50% salvianolic acid B 25 mg/kg was given by gavage for 6 days per week for 12 consecutive weeks in the salvianolic acid B group. In the other two groups, the same amount of normal saline was given for the same time. At 16 weeks postoperatively, all the rats were sacrificed after anesthesia. The bone density of the right posterior femur and lumbar vertebra was scanned by dual-energy X-ray absorptiometry. The levels of osteoprotegerin and receptor activator of nuclear factor-kB ligand in serum were detected by enzyme-linked immunosorbent assay. The protocols were approved by the Animal Management Committee of Bengbu Medical College (approval No. [2021]255). RESULTS AND CONCLUSION: (1) The bone mineral density of femur in the osteoporosis group was lower than that in the sham group (P < 0.05). The femoral bone mineral density of salvianolic acid B group was significantly higher than that of osteoporosis group (P > 0.05). The bone mineral density of lumbar vertebrae in osteoporosis group was not significantly different from that in sham operation group (P > 0.05). The lumbar vertebrae bone density of salvianic acid B group was higher than that of osteoporosis group, but the difference was not significant (P > 0.05). (2) Osteoprotegerin level of osteoporosis group was lower than that of sham operation group (P < 0.05). Osteoprotegerin level of salvianolic acid B group was higher than that of osteoporosis group, but the difference was not significant (P > 0.05). The level of receptor activator of nuclear factor-kB ligand of osteoporosis group was significantly higher than that of sham operation group (P < 0.05). The level of receptor activator of nuclear factor-kB ligand of salvianolic acid B group was lower than that of osteoporosis group, but the difference was not significant (P > 0.05). (3) These findings suggest that in the early stage of postmenopausal osteoporosis, bone mineral density of femur decreased significantly, but there was no significant change in bone mineral density of the lumbar spine. At present, it is not believed that salvianolic acid B alone can effectively treat osteoporosis in the short term. [ABSTRACT FROM AUTHOR]

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.

Objective To Explore significance of the signaling pathways related to nuclear factor receptor activator icI3 ligand (RANKL), phosphorylated extracellular signal-regulated kinase-1/2(p-ERK1/2) and phosphorylated mammalian target of rapamycin ( p-mTOR ) signaling in extramammary Paget disease (EMPD). Methods The expressions of RANKL,p-ERK1/2 and p-mTOR in 80 patients with EMPD ( from 2008 to 2017, the patient went to the Dermatology Department of the First Affiliated Hospital of Fujian Medical University and Fujian Provincial Dermatology and Venereology Hospital) were detected by immunohistochemical staining. Results RANKL was localized in Paget cytoplasm,66 cases were positive and 14 cases were negative. The positive rate was 82.5%. p-ERK1/2 was located in Paget cytoplasm,69 cases were positive and 11 cases were negative. The positive rate was 86. 25%. p-mTOR was located in Paget cytoplasm, positive in 71 cases and negative in 9 cases. The positive rate was 88. 75%. There was a positive correlation among them (correlation coefficients were 0. 907,0. 931 and 0. 998, P <0. 05). Conclusion The RANK-RANKL signaling pathway, RAS-RAF-MEK-ERK signaling pathway and PI3K-Akt-mTOR signaling pathway are activated in EMPD, which may promote the growth and proliferation of tumor cells and increase the invasiveness of the disease. [ABSTRACT FROM AUTHOR]

To investigate the effect of minocycline combined with tea polyphenols on periodontal index, inflammatory factors, gingival crevicular fluid nuclear factor kappa β receptor activator ligand (RANKL), Hedgehog factor (Shh) levels in patients with early peri implant soft tissue inflammation. A total of 106 patients with early peri-implant soft tissue inflammation admitted to our hospital were included in this study, they were divided into control group (n=53, treated with minocycline) and study group (n=53, treated with tea polyphenol on the basis of minocycline) by envelope lottery. The periodontal index, curative effect, inflammatory factors, RANKL and Shh levels in gingival crevicular fluid of the two groups before treatment and after 4 weeks of treatment were compared, adverse reactions of the two groups during treatment were recorded. The total clinical effective rate of the study group at 4 weeks after treatment was higher than that of the control group (P<0.05). The modified plaque index (mPLI), modified gingival sulcus bleeding index (mSBI), periimplant pocket depth (PPD), serum interleukin-1β(IL-1β), monocyte chemoattractant protein-1 (MCP-1), RANKL and Shh levels in gingival sulcus fluid decreased in the two groups at 4 weeks after treatment, and study group was lower than the control group (P<0.05). There was no significant difference between incidence of adverse reactions of the two groups (P>0.05). Minocycline combined with tea polyphenols in the treatment of patients with early periimplant soft tissue inflammation are significant efficacy, which can effectively improve the periodontal situation of patients, reduce the serum IL-1β, MCP-1levels and RANKL, Shh levels in gingival crevicular fluid, which is safe and reliable. [ABSTRACT FROM AUTHOR]

Osteoporosis is a chronic systemic bone disease characterized by loss of bone mass and degeneration of bone microstructure, which eventually leads to increased fragility of the bone and an increased risk of fracture. At present, the aging population in China is gradually increasing and we have entered an aging society. The incidence of osteoporosis and brittle fractures caused by osteoporosis are increasing year by year, which greatly affect the safety and quality of life of patients and have become an urgent problem to be solved. Before the emergence of the concept of bone immunology, bone was believed to be vital to the immune system because of the formation of immune cells in bone marrow. However, the role of the immune system in bone regulation is not well understood. In 2000,Arron proposed the concept of bone immunology in the Nature and pointed out the complex interaction between the bone and immune system. Since then, bone immunology has gradually become the research hot spot of metabolic osteoarthropathy. Bone immunology plays an important regulatory role in the process of bone reconstruction. Its main research content is to elucidate the close and complex mutual regulatory mechanism between the immune system and the bone. During the occurrence and development of osteoporosis, OPG/RANK/RANKL signaling system, immune cells, and cytokines produced by them play a decisive role. Bone immunology has laid the theoretical foundation for the research and development of novel antiosteoporosis drugs. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect and potential molecular mechanisms of isorhamnetin (ISO) extracted from Ginkgo biloba on the differentiation of osteoclasts. Methods Osteoclast precursor RAW264.7 cells were induced with RANKL to differentiate into mature osteoclasts. Different concentrations of ISO were added to RAW264.7 cells to determine its effect on osteoclast differentiation. CCK8 was used to evaluate the effect of ISO on cytotoxicity. The im⁃ pact of ISO on the osteoclast differentiation process was investigated by analyzing tartrate resistance and bone resorption lacuna. Real⁃time PCR was performed to analyze the levels of differentiation marker genes, including tartrate resistant acid phosphatase (Trap), cathepsin K (Ctsk), and matrix metalloproteinase 9 (MMP⁃9); differentiation⁃related transcription factors, including the proto⁃oncogene protein c⁃Fos, nuclear factor of activated T⁃cells cytoplasmic 1(NFATc1); and the levels of downstream NF⁃κB p65 signaling pathway phosphorylation. Using the above⁃described method, we verified that ISO exerted an inhibitory effect on osteoclast differentiation and explored related molecular mechanisms. Results Dif⁃ ferent concentrations of ISO (1⁃10 μM) had no cytotoxic effects on RAW264.7 cells, inhibited TRAP activity and decreased the number of bone resorption lacuna during osteoclast differentiation. When applied at a concentration of 10 μM, its inhibitory effect was significant. In addition, ISO significantly reduced the expression levels of Trap, Ctsk, MMP⁃9, c⁃Fos, NFATc1 and NF⁃ κB p65 mRNA. Conclusion ISO extracted from Ginkgo biloba extract exerted an inhibitory effect on osteoclast differentiation, and the mechanism underlying its activity may involve the inhibition of the classical NF⁃κB pathway.

BACKGROUND: Although zoledronic acid can effectively prevent bone loss in postmenopausal women, its effect and mechanism on the mandible are not clarified. OBJECTIVE: To study the morphological and pathological changes of mandibular tissue in ovariectomized rats treated with low-dose zoledronic acid and to investigate the regulatory effect and mechanism of RANKL/RANK/OPG signaling system in the inhibition of bone resorption by zoledronic acid. METHODS: Thirty-six adult female Sprague-Dawley rats were randomized into control, model and treatment groups. Animal models of osteoporosis were made by bilateral ovariectomy in the latter two groups, while the same amount of lipid tissues surrounding the ovary was removed in the control group. Three weeks after ovariectomy, the rats in the treatment group were given subcutaneous injection of 20 μg/kg zoledronic acid. The corresponding doses of saline were injected subcutaneously in the control and model groups. One week after treatment, the left mandibular molars were extracted from all the rats, and the rats’ jaws were separated and removed at 4 weeks after tooth extraction for detection. The residual alveolar bone of the extracted socket was observed by X-ray. The pathological changes of the mandibular cortex and cancellous bone were detected by hematoxylin-eosin staining. The number of apoptotic osteoblasts was detected by TUNEL apoptosis test. The expressions of receptor activator of nuclear factor-κ B ligand (RANKL), osteoprotegerin and nuclear transcription factor-κB (NF-κB) in the mandibular alveolar bone were detected by immunohistochemical technique. Western blot was finally used to detect the expression of RANKL and NF-κB at protein levels. RESULTS AND CONCLUSION: (1) At 4 week after tooth extract, subcutaneous injection of 20 μg/kg zoledronic acid effectively inhibited alveolar bone resorption and promoted new bone formation at the extraction socket. (2) Hematoxylin-eosin staining results showed that: in the model group, the cortical bone was thinned, and the trabecular bone was thinned and even ruptured. There were a large number of bone resorption lacunae but few osteoblasts in the model group. In the treatment group, the cortical bone was thickened and the trabecular bone had normal structure, with only a small amount of bone resorption lacunae and increased number of osteoblasts. (3) The number of apoptotic osteoblasts was significantly lower in the treatment group than the model group (P < 0.001). (4) Immunohistochemical staining results showed significantly decreased RANKL and NF-κB protein expressions (P < 0.001, P < 0.002) and significantly increased osteoprotegerin level (P < 0.001) in the treatment group than the model group. (5) Western blot results revealed that the protein expressions of RANKL and NF-κB in the model group were significantly higher than those in the control group, and treatment with zoledronic acid significantly reduced these protein levels (both P < 0.001). To conclude, zoledronic acid could inhibit the differentiation of osteoclasts by down-regulating the NF-κB signal pathway and meanwhile regulate the apoptosis of osteoblasts. [ABSTRACT FROM AUTHOR]

Objective To explore the molecular mechanism of 99Tc-MDP alleviating osteoporosis through miR-338-3p to regulate RANKL to affect the activity of osteoclast (OC). Methods After the cells were treated by 99Tc-MDP，the expression of miR-338- 3p and OC cells activity marker CD51 and MMP-9 were detected by qRT-PCR. CCK-8 assay，dual luciferase reporter gene assay and Western blotting were used to detect the proliferation，livability of OC and the targeted relationship of miR-338-3p and RANKL. Results 99 Tc-MDP inhibited the expression of OC cells activity marker CD51 and MMP-9 and suppressed the livability of OC cells.99Tc-MDP could up-regulate miR-338-3p and down-regulate RANKL. The result of dual luciferase reporter gene assay and Western blotting suggested that miR-338-3p could target RANKL and down-regulate it. Furthermore，99 Tc-MDP inhibited the proliferation and activity of OC cells through inhibiting RANKL by up-regulating miR-338-3p. Conclusion 99Tc-MDP alleviates osteoporosis by inhibiting the activity of osteoclast through miR-338-3p down-regulating RANKL. [ABSTRACT FROM AUTHOR]

Objective To investigate the application of osteo assay surface plate instead of bone grinding plate to identify the bone erosion ability of osteoclasts. Methods The osteoclast differentiation model was established by RANKL-induced osteoclast precursor cell RAW264. 7 cells. The degree of osteoclast differentiation was detected by TRAP staining. The osteo assay surface plate was used instead of the bone grinding plate to test the bone erosion ability of osteoclasts， and the bone erosion ability was reflected as the erosion area. Results Different concentrations of RANKL factor could effectively induce the differentiation of osteoclast precursor cells RAW264. 7 into mature multinucleated osteoclasts in a concentration-dependent manner. Mature osteoclasts formed irregular erosion rings of different areas on the surface of osteo assay surface plate， and the trend was consistent with the degree of differentiation of osteoclasts. Conclusion The osteo assay surface plate can effectively reflect the formation of osteoclasts and its bone erosion ability， consistent with the TRAP staining result ， and has the advantages of simple operation， intuitive result and convenient statistical analysis. [ABSTRACT FROM AUTHOR]

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). (2) The level of RANKL in serum and the expression of RANKL in bone tissue were measured, which were higher in the model group than the control group, higher in the OPG silencing group than the model group, lower in the psoralea corylifolia extract group than the model group, and lower in the psoralen corylifolia extract+OPG silencing group than the OPG silencing group, but higher than the psoralea corylifolia extract group (all P < 0.05). (3) There was no significant difference in each index between model group and no-load group (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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Denosumab is a monoclonal antibody agent for treating osteoporosis. It targets the RANKL/RANK signaling pathway, which is the core pathway of bone metabolism. Denosumab has been approved for listing in the US in 2010, and has been applied to the first-line treatment for postmenopausal osteoporosis currently. Denosumab-related osteonecrosis of the jaw is one of the serious adverse effects of denosumab, which severely affects patients’ quality of life. OBJECTIVE: To review the possible mechanisms and treatment of denosumab-related osteonecrosis of the jaw. METHODS: The first author retrieved the literature related to denosumab-related osteonecrosis of the jaw in the PubMed and WanFang databases for recent decade. The keywords were “denosumab-related osteonecrosis of the jaw, denosumab, osteonecrosis of the jaw, osteoclast, RANKL/RANK, bisphosphonate-related osteonecrosis of the jaw” in English and Chinese, respectively. Fifty-four eligible articles were finally reviewed. RESULTS AND CONCLUSION: As a new drug for osteoporosis, denosumab has a good clinical application prospect. The study of denosumab-related osteonecrosis of the jaw is still in the exploratory stage. The underlying mechanism of denosumab-related osteonecrosis of the jaw may be related to abnormal bone metabolism and immune dysfunction. Prevention is the main treatment for osteonecrosis of the jaw. Although conservative treatment is still the mainstream of treatment, surgery has greater chance to cure the disease. During treatment, discontinuing the drug and reducing the stimulus has positive effect. In the course of clinical drug use, we should strictly control the risk-benefit relationship, and communicate with patients about the advantages and disadvantages of treatment, so as to make the application of denosumab more reasonable, safe and effective. [ABSTRACT FROM AUTHOR]

Osteoclasts are multinucleated cells that differentiate from bone marrow mononuclear cells. Mature osteoclasts are located on the inner surface of the trabecular bone and cortical bone. Steady state of bone tissue is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. However, under pathological conditions, various factors including tumor necrosis factor superfamily ligands and inflammatory proteins, promote the formation of osteoclasts, which could cause imbalance of bone metabolism, leading to the excessive bone resorption and bone remodeling. Osteoclastic hyperactivity is commonly seen in osteoporosis, autoimmune arthritis, and other bone metabolic diseases. Dysfunction of osteoclasts also causes diseases such as osteopetrosis. Therefore, osteoclasts are important targets for the prevention and treatment of bone metabolism diseases. With the deepening of research, new researches on osteoclasts have been made in recent years. This article summarizes the latest research progress in osteoclasts. [ABSTRACT FROM AUTHOR]

BACKGROUND: Myeloma patients have less movement because of bone pain and osteolytic destruction, which result in bone loss and disease aggravation. OBJECTIVE: To explore the effect of myeloma cells (U266 cells) on osteocytes (Y4 cells), osteoblasts and osteoclasts as well as RANKL secreted by osteocytes under fluid shear stress simulating human mechanical movement by a fluid flow devise. METHODS: A flow and no-flow model was established. U266 cell culture fluid was used in the experimental group and Y4 standard culture fluid was used in the flow and control groups. Myeloma cell line U266 and mouse osteocytes cell line Y4 were cultured and passaged in vitro. Y4 cells were allotted and cultured for 48 hours, and the morphology of osteocytes and osteoclasts was observed and counted under microscope. The level of RANKL was quantitatively determined by ELISA and western blot assay. The RANKL, sample and standard culture system was established, RAW264.7 cells were cultured in vitro, and induced by RANKL. Number and morphology of RAW264.7 cells differentiating into osteoclasts were observed by TRAP staining. RESULTS AND CONCLUSION: Compared with the control group, Y4 cells in U266 culture supernatant exhibited morphological changes and their number was significantly decreased. The number of TRAP-positive osteoclasts was increased and the expression of RANKL protein in Y4 cells was significantly up-regulated (P < 0.05). Comparison between the flow model and the no-flow model showed that the number of Y4 cells was significantly increased, the expression of RANKL protein decreased and the number of TRAP-positive osteoclasts was significantly reduced (P < 0.05). Our study indicates that myeloma cells can inhibit the growth of normal osteocyte and might promote the expression of RANKL protein and the growth of osteoclasts. Compared with static state, fluid shear stress may promote the proliferation of osteocytes and might inhibit the expression of RANKL protein and the growth of osteoclasts. So, we hypothesize that mild exercise may prevent the progression of myeloma bone disease. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of interleukin-6 ( IL-6), a prototypical cytokine featuring pleiotropic and redundant activity, on the osteoclastic differentiation of RANKL-stimulated RAW264． 7 macrophages. Methods RAW264． 7 cells were pre-treated with 50ng / mL RANKL for 1 day and followed by IL-6 treatment. Then they were incubated and divided into 4 groups: control group (50ng / mL RANKL + PBS), low (50ng / mL RANKL + 50ng / mL IL-6), medium (50ng / mL RANKL + 100ng / mL IL-6), and high concentration (50 ng / mL RANKL+ 150ng / mL IL-6) group for 9 consecutive days. These cells were harvested and stained with hematoxylin-eosin (HE) and tartrate-resistant acid phosphatase (TRAP). The resorption pits of bone slices were observed using scanning electron microscope (SEM). Results HE stains showed that there were increased mature osteoclast cells in the control compared to 100 and 150ng / mL of IL-6 treatment, with a difference being statistically significant (P< 0． 05). However, the difference did not reach statistical significance between the control and 50ng / mL IL-6 treatment (P>0． 05). Similarly, compared to the control, TRAP-positive multinucleated osteoclasts (more than 3 nucleus) significantly decreased under the constant stimuli of 100 and 150ng / mL of IL-6 concentration (P <0． 05). Under SEM observation, multiple bone resorption formation was clearly visualized in control group. In contrast, few formations of bone resorption and mature osteoclasts were found when the addition of IL-6 concentration was more than 50ng / mL ( P < 0． 05). Conclusion IL-6 at 100 or 150ng / mL concentrations can powerfully suppress the differentiation of mature osteoclasts, thereby resulting in a remarkably reduction of osteolysis. [ABSTRACT FROM AUTHOR]

Objective: To explore the effect of insulin on the serum and bone OPG (osteoprotegerin) and RANKL (OPG receptor activator nuclear factor-kB) expressions in the type 2 diabetic rats with osteoporosis. Methods: A rat model of type 2 diabetes with osteoporosis was established by high-fat diet and 4%fructose water for 4 weeks, and then the rats were treated with streptozocin. The expression of serum and bone OPG and RANKL between control and model group were analyzed with ELISA, bone density was detected by bone mineral density analyzer and the level of blood glucose were analyzed by blood glucose meter. Results: The expression of OPG, blood glucose, glycosylated hemoglobin ,bone density of serum and bone in model group were significantly lower than those control group(P<0.05), while the expression of RANKL was significantly higher than that of the control group (P<0.05), and insulin can increase the expression of OPG in serum and bone of model group, and decrease the expression of RANKL in serum and bone in model group (P<0.05). Conclusions: Insulin can decrease the expression of RANKL and increase the expression of OPG in serum and bone in type 2diabetic rats with osteoporosis. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of chronic intermittent hypoxia (CIH) on bone metabolism and the mRNA expression of OPG and RANKL in bone tissue using Wistar rat CIH model． Methods 20 healthy Wistar rats were randomly divided into normal control group and CIH group，with 10 rats in each group． The control group was placed in the air circulation cabin，and the CIH group was placed in the intermittent hypoxic capsule for 8 hours per day for 8 weeks． Bone mineral density (BMD) of whole body，femur and lumbar spine were determined using DXA． Serum levels of rat bone specific alkaline phosphatase (BALP) ，osteocalcin (BGP) ，type I collagen N terminal peptide (PINP) ，type I collagen carboxy-terminal peptide， beta degradation products (beta-CTX) ，tartrate resistant acid phosphatase (TRAP) of rats were measured by ELISA． Real-time PCR was applied to determinate the mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) in the femur of rats． Results Compared with the control group，the CIH group's BMD of whole body and femur decreased ［(0. 141 ± 0. 028) vs (0. 122 ± 0. 027) and (0. 143 ± 0. 026) vs (0. 125 ± 0. 034) ］，serum BGP decreased ［(26. 42 ± 3. 71) vs (22. 05 ± 2. 16) ］，serum beta-CTX and TRAP increased ［(132. 24 ± 26. 25) vs (173. 82 ± 22. 16) and (20. 12 ± 2. 43) vs (23. 58 ± 2. 36) ］，mRNA expression of OPG in the femoral tissue decreased ［(1. 031 ± 0. 125) vs (0. 924 ± 0. 141) ］，while mRNA expression of RANKL increased ［(0. 856 ± 0. 068) vs (1. 113 ± 0. 134) ］． These differences were all statistically significant (P < 0. 05 ) ． Conclusion Chronic intermittent hypoxia might reduce OPG mRNA，increase RNAKL mRNA of bone tissue，affect bone metabolism，reduce bone mineral density and increase the risk of osteoporosis． [ABSTRACT FROM AUTHOR]

Chronic kidney disease-mineral and bone disorder (CKD-MBD)-rnduced vascular calcification has been proved to be an independent risk factor of cardiovascular events in patients with chronic kidney disease (CKD). Impaired calcium and phosphate homeostasis, increased oxidative stress, loss of calcification inhibitors and increased expression of RANKL are related to the vascular calcification in CKD. Additionally, impaired bone may be implicated in the pathogenesis of vascular calcification through perturbing serum calcium phosphate and parathyroid hormone, thus contributing to increased vascular calcification in CKD. Therapeutic approaches for CKD. such as phosphate binders and bisphosphonates. have been shown to ameliorate bone loss as well as vascular calcification. This article reviews recent progress of the pathogenesis of vascular calcification in CKD-MBD. [ABSTRACT FROM AUTHOR]

Objective To observe the effects of electroacupuncture on bone mineral density and bone mineral content in ovariectomy rats and the effects of serum from acupunctured rats on the expression of OPG and RANKL in osteoblasts cultured in vitro. Method Forty-eight female two-month old SD rats from the grade of SPF were selected and divided randomly into four groups, in which three groups undergone the surgery of bilateral ovariectomy and then named the groups of electroacupuncture, the group of western medicine and the group of model. The other group were blank group which did not receive any procedure. Rats in the western medicine group were administrated estradiol valerate by means of intragastrical administration for three courses. Rats in the electroacupuncture group were treated by acupuncture and received the same volume distilled water for three months. Rats in the model group were administrated with distilled water for three months. Rats in the blank group were not treated. After three months when the rats were raised in the laboratory of SPF by the common way, they were assessed and the result showed that the models were established. Then the all groups of rats were anesthetized by the chloral hydrate and hemospasia from heart was performed.Blood samples of each group undergone filter mesh and water bath, and the serum was added to osteoblasts cultured in vitro. After three days osteoblasts were fixed using paraformaldehyde. The mRNA of OPG and RANKL were detected by means of immunofluorescence cytochemistry. Results Compared with the blank group, the group of model had significantly lower bonemineral density and bone mineral content. After the treatment of western medicine or electroacupuncture, they were increased, and the differences were significant(P<0.01) compared with the group of model. After the osteoblasts were cultured in the serum, the blank group was the highest, the model group was the lowest and lower than the western group and the electroacupuncture group on the mRNA of OPG(P<0.01). The model group was the highest on the mRNA of RANKL, but after the treatment, that of the western group and the electroacupuncture group decreased(P<0.01). Conclusion The treatment of electroacunpuncture could mostly raise bone mineral density and bone mineral content in rats. After the treatment of electroacupuncture, the serum could increase bone formation and decreased bone resorption. [ABSTRACT FROM AUTHOR]

Objective By studying the changes of bone mineral density (BMD) and serum OPG and RANKL in obstructive sleep apnea hypopnea syndrome (OSAHS) patients, to explore the relationship between OSAHS and osteoporosis preliminarily. Methods According to their apnea hypopnea index (AHI), 88 patients with OSAHS were collected and divided into 3 groups: mild (5≤AHI<15times/h, n=30), moderate (15≤AHI<30times/h, n=30), and severe (AHI≥30times/h, n=28). Thirty healthy persons were selected as control. The general information of the subjects was recorded. Bone metabolic markers and BMD of L1-4 and the femoral neck (Neck) were measured. The expressions of serum OPG and RANKL were determined using ELISA kits. The difference among the groups was compared and the correlation with AHI was analyzed. P<0.05 was considered statistically significant. Results BMD of L1-4 and Neck of patients with moderate and severe OSAHS was significantly lower than that in normal control group and the differene between severe and mild OSAHS is also significant statistically. The serum OPG of patients in each OSAHS group was lower than that in normal control group, and the difference was statististically significant between severe OSAHS patients and mild OSAHS patients, and normal control group, respectively. The serum RANKL of patients in each OSAHS group was higher than that in normal control group, and the differences were statististically significant between moderate OSAHS patients, severe OSAHS patients, and normal control group, and between severe OSAHS patients with normal control group, respectively. The result of Pearson correlation analysis showed that BMD and OPG were negatively correlated with AHI, but RANKL was positively correlated with AHI. Conclusion The risk of suffering from osteoporosis is higher in OSAHS patients than in healthy people, and it is closely related to the degree of hypoxia. The imbalance of OPG and RANKL levels may be one of the main causes of the occurrence and development of OSAHS with osteoporosis. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of shikonin on osteoclastogenesis in vitro and amelioration of bone loss in ovariectomized-induced osteoporosis in mouse model. Methods: The optimal concentration of shikonin treating were evaluated in vitro depending on its effect on the viability of C57BL/6J mouse bone-marrow-derived macrophages by CCK-8 method. To establish the osteoclastogenesis cell model, macrophages were cultured with RANKL and M-CSF treatment, and TRAP staining was used to observe the generation of osteoclasts after treating with different concentration of shikonin solution. Expressions of osteoclast marker genes, including TRAP, c-Fos and NFATcl were detected with real-time PCR. Fifthteen mice were randomly allocated into sham operation group, ovariectomized model group and shikonin treatment group. After the modeling, mice in treatment group were received the intraperitoneal injection of shikonin, while the other two groups treated with normal saline. After thirty days treatments, all animals' tibias were dissected for micro-CT analysis. Results: ①The macrophages viability was significantly inhibited when the concentration of shikonin was higher than 250 nmol/(P＜0.01). ②The osteoclastogenesis was significantly suppressed by different dose of shikonin (P＜0.01). ③ The expression of the osteoclastic marker genes (TRAP, c-Fos and NFATc1) were suppressed by addition of shikonin comparing to control group (P＜0.01). ④ Shikonin effectively prevented ovariectomy-induced bone loss (P＜0.05). Conclusion: Shikonin suppresses osteoclastogenesis in vitro and ameliorates ovariectomized-induced osteoporosis in mouse model. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect of different dosage of icariin on the RANKL and Wnt3a expressions in the pressure side of periodontal tissue during orthodontic tooth movement (OTM). Methods: Twenty four male SD rats were randomly divided into four groups according to the different dosage of icariin as follows: physiological saline group (control group); 1 mg/kg icariin group; 3 mg/kg icariin group: 5 mg/kg icariin group (experience group). 50 g force was used to move the left maxillary first molar. Immunohistochemical staining technology was used to detect the expression of RANKL and Wnt3a in the pressure side of periodontal tissue. Results: Rats in the physiological saline group showed crude surfaces of the root of tooth and alveolar bone at the pressure area of the maxillary first molar and narrowed space of the periodontium, and bone resorption pits and osteoclasts could be observed. Rats in various dosage of icariin group displayed that the space of the periodontium tended to recover normally, and significantly reduced bone resorption pits could be noted. RANKL and Wnt3a expressed on both the physiological saline and icariin groups in the pressure zone of periodontal tissue. Compared with the saline group, the expression of RANKL was significantly decreased in the pressure side of periodontal tissue in the different dosage of icariin groups, and the expression of Wnt3a were significantly increased. Furthermore, the expression of RANKL was decreased gradually with the increase of dosage (P<0.05). And the expression of Wnt3a was increased gradually with the increase of dosage (P<0.05). Conclusions: Different dosage of icariin could reduce the expressions of RANKL and increase the expression of Wnt3a in the periodontal tissue remodeling during OTM. and its effect has certain correlation with its dosage. [ABSTRACT FROM AUTHOR]

Objective: To select ssDNA aptamers with high affinity and specificity for RANKL which could efficiently inhibit RANKL-induced osteoclastogenesis in vitro. Metiiods: Firstly, a 93nt single stranded DNA (ssDNA) random library was subjected to 14 rounds of selection against RANKL expressed and purified from the prokaryotic expression system by SELEX method. And then the structure of ssDNA aptamers was analyzed by RNAfolding server software and the affinities of aptamers to RANKL were determined by ELISA. Lastly, the inhibition effects of ssDNA aptamers on RANK-induce osteoclastogenesis were verified in vitro. Results:① Mouse RANKL was successfully expressed and purified by the prokaryotic expression system .② Twelve ssDNA aptamers with high affinity and specificity for RANKL were selected out.③ the numbers of the TRAP+ multinucleated osteoclasts were decreased in different groups of ssDNA aptamers comparing with control group(P<0.05). Conclusions: The ssDNA aptamers of RANKL have been successively selected by the SELEX method for the first time and the selected ssDNA aptamers can effectively inhibit RANKL-induced osteoclastogenesis in vitro. [ABSTRACT FROM AUTHOR]

BACKGROUND: Previous studies have proved platelet-rich fibrin (PRF) with osteoinduction ability, and the centrifugal speed and time to prepare rabbit advanced PRF (A-PRF) with the most similar structure to that of human PRF have been determined. OBJECTIVE: To observe the histological changes during A-PRF-induced osteogenesis. METHODS: Thirty Japanese white rabbits were randomly divided into A-PRF and blank control groups (n=15 per group). The full-thickness defect models were established on the rabbit parietal bone, followed by implanted with A-PRF or nothing, respectively. The model rabbits were killed immediately, at 2, 4, 8 and 12 weeks after modeling, to grossly observe the bone formation, and the histological changes in the defect region were observed through hematoxylin-eosin staining, Masson staining and immunohistochemistry. RESULTS AND CONCLUSION: Unhealed defects were observed in the blank control group. Gross and histological observations showed that the speed, amount and maturity of bone formation in the A-PRF group were significantly better than those in the blank control group immediately, 4, 8 and 12 weeks after modeling (P < 0.05). Our findings suggest that the rabbit skull bone defect is successfully established. A-PRF can induce osteogenesis, and more mature newly born bones appear with time. Additionally, osteoclasts can act with osteoblasts synergically under the A-PRF induction to promote the bone formation. [ABSTRACT FROM AUTHOR]

Objective To observe the effect of electroacupuncture on bone mineral density (BMD) and bone mineral content (BMC) in ovariectomized rats，and on the expression of OPG and RANKL in cultured osteoblasts in vitro. Methods Forty 2-month-old female SPF-grade SD rats were selected and were randomly divided to blank group，electroacupuncture group，Western medicine group，and model group，with 10 rats in each group. Rats were bilaterally ovariectomized except in the blank group. The model was established in 3 months according to the BMD results. Rats in the western group were then administrated with estradiol valerate by means of intragastrical administration for 3 course. Rats in the electroacupuncture group were treated with acupuncture and intragastrical administration of same volume of distilled water for 3 month. Rats in the model group were administrated with distilled water for 3 month. Rats in the blank group were fed normally. After 3 months in the SPF laboratory，BMD and BMC were detected using dual energy X-ray absorptiometry. The rats of all the groups were anesthetized with chloral hydrate and blood were collected from the heart. The blood was mesh filtered and dealt with 56 C water bath. Then it was added in the cultured osteoblasts. The osteoblasts were fixed with paraformaldehyde after 3 days. The protein expression of OPG and RANKL in the osteoblasts were detected using immunofluorescence cytochemistry. Results Comparing to those in the blank group，BMD and BMC in the model groups decreased significantly (P < 0.05). After the medicine or acupuncture treatment，BMD and BMC increased significantly，comparing to those in the model group (P < 0. 01). The protein expression of OPG was low in the osteoblasts cultured with the model group serum. It increased after the medicine or acupuncture treatment (P < 0.01) . The expression of RANKL was the highest in the model group. After the treatment，it decreased in the western group and the electroacupuncture group (P < 0. 01) . Conclusion The treatment of electroacupuncture increases BMD and BMC in rats. After the treatment with electroacupuncture，the rat serum has good capacity of increasing bone formation and decreasing bone resorption. [ABSTRACT FROM AUTHOR]

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Objective To observe the change situation of serum OPG/RANKL of patients with type 2 diabetes in Yunnan district，and study the corresponding relationship between the patient's serum concentration and bone density. Method 250 cases of patients with type 2 diabetes were selected， divided into two groups according to the detected bone density data: the control group and bone mass reduction group. The control group was composed of T2DM patients with normal bone mass. The BMD data of lumbar 1-lumbar 4 （L1～L4） rear-front part， lumbar 1-4 （L1L-4）， proximal femur near-end， all parts of the body， left and right forearm were detected in both groups by using dual-energy X-ray absorption measures. The value of plasma OPG/RANKL without eating was measured by enzyme-linked immunoassay method. Result（1） OPG/RANKL value in control group of diabetes bone mass was significantly higher than that in bone mass reduction group， and RANKL concentration was remarkably lower than that in bone mass reduction group. （2） According to the correlation analysis of double variant in low bone mass group， it was known that the OPG was in direct proportion to the BMD data of L1～L4 and L1L-4. Besides，OPG/RANKL was also in direct proportion to the BMD data of L1～L4， L1L-4， left and right forearm.RANKL was in inverse proportion to the BMD data of L1-L4， L1L-4， left and right forearm. Conclusion OPG/RANKL systemplayed a crucial role in the osteoporosis occurrence of patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

Objective To build an animal model to investigate the effect of Muscles′ Imbalance on the bilateral condylar subchondral bone．Methods 32 male rats were randomly divided into excise the coracoid and botulinum neurotoxin A masseter injection group and control group. Each group of 16 SD rats were performed the corresponding operation on the right side. The rats were killed at the end of the 2th， 4th， 8th and 12th weeks， acquire TMJ condylar and observe its Histological changes. The expression of OPG and RANKL in condylar subchondral bones was observed by immunohistochemical staining．Results Immunohistochemical showed: After 2 weeks， the expression of OPG in the experimental groups were higher thanthat ofblank control group （P < 0.05）， and had no statistical difference with blank control group after 4， 8， 12 weeks （P > 0.05） . The expression of RANKL were higher than that of blank control group after 2 and 4 weeks（P < 0.05）， and had no statistical difference with blank control group after 8， 12 weeks （pP > 0.05） ;The ratio of OPG/RANKL in experimental groups were lower than those ofthe blank control group after 2，4 weeks （P < 0.05）， and had no statistical difference with the blank control group after 8，12 weeks（P > 0.05） .Conclusions By OPG/RANKL signaling pathways， excise the coracoid and botulinum neurotoxin A masseter injection involved in pathologic reconstruction of the bilateral TMJ condylar subchondral bone. [ABSTRACT FROM AUTHOR]

The RAW264. 7 cell line was obtained from a tumor of BALB /c mouse induced by Abelson murine leukemia virus. RAW264. 7 cell was broadly applied in medical sciences: it is the mostly adopted cell line in many leukemia-related research. It is also commonly used in immunity and microbiology research. In addition，it is the murine source pre-osteoclast which could be stimulated with cytokines and differentiate into mature osteoclasts in vitro，thus becoming an in vitro model applied in many skeletal diseases. However，its cellular morphology and differentiation stage are variable and hard to handle and therefore brings some difficulties to practical work. As to the methods of induction of RAW264. 7 cells to mature osteoclasts，researchers`opinions are also different. In this article，we explored the culture condition with combination of literature study，figured out the methods of cryopreservation，recovery and subculture of RAW264. 7 cells，and stimulated it with the receptor activator for nuclear factor-κB ligand (RANKL) . The aims of this article are to summarize experience of culturing RAW264. 7 cell and its differentiation into mature osteoclast，and to explore the related skills，thus providing reference to researchers. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of GLP-1 analogue liraglutide on the serum levels of osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B ligand (RANKL), and bone mineral density in diabetic rats. Methods Healthy male SD rats were randomly divided into normal control group (NC), diabetes mellitus (DM) group, low dose liraglutide group (LR-L), and high dose liraglutide group (LR-H). Type 2 diabetic rat model was induced with high fat and sugar diet combined with low-dose streptozotocin (STZ). Rats in LR-L group received 400 ug/kg/d of liraglutide with subcutaneous injection. Rats in LR-H group received 800 ug/kg/d of liraglutide subcutaneously. After 4 weeks, rats were sacrificed. The concentrations of blood glucose, serum calcium, phosphorus, alkaline phosphatase, total cholesterol, and triglyceride were measured. OPG and RANK were detected using ELISA method. Bone mineral density was detected with DEXA. Results Comparing with those in NC group, 1) The body weight reduced in DM, LR-L, and LR-H group. The body weight in LR-L and LR-H group was lighter than that in the DM group. It was not different between LR-L group and LR-H group. 2) Blood glucose increased in DM, LR-L, and LR-H group. Blood glucose in LR-L and LR-H group was lower than that in DM group. It was not different between LR-L and LR-H group. 3) The serum concentrations of OPG and RANKL increased significantly in DM, LR-H, and LR-L group, and the difference was statistically significant (P<0.05). The serum concentration of OPG in LR-L group is higher than that in DM group, and the difference was statistically significant (P<0.05). The serum concentration of OPG in LR-H group was higher than that in DM group, but the difference was not statistically significant. The serum concentration of RANK in LR-H and LR-L group was significantly lower than that in DM group. 4) The serum OPG/RANKL ratio in DM group was lower than that in NC group. It was higher in LR-L and LR-H group than that in DM group, and the difference was statistically significant (P<0.05). 5) The whole body BMD was lower in DM, LR-I, and LR-H group, and the difference was statistically significant (P<0.05). There was no significant difference among the 3 groups. Conclusion Liraglutide plays a role in reducing the body weight in type 2 diabetic rats, reducing the serum RANKL, and elevating serum OPG. However, bone mineral density does not increase after liraglutide treatment. [ABSTRACT FROM AUTHOR]

Objective To study the effect of Xuling invigorating bone prescription on bone mineral density (BMD) in rat osteoporosis model. Methods SD female rats were randomly divided into Caltrate D group, Xuling invigorating bone prescription group, osteoporosis model group, and sham-operated control group. Drug administration lasted for 12 weeks. BMD of the left tibia was detected. Serum bone alkaline phosphatase (BALP) level was detected using ELISA method. Protein expression levels of OPG and RANKL were measured using Western blot method. Results (1) BMD was significantly higher in Xuling invigorating bone prescription group than that in other three groups,and the difference between the two groups was statistically significant (compared with Caltrate D group, P < 0. 05, compared with the sham-operated control, P < 0. 01, compared with the model group, P <0. 000). (2) BALP was higher in Xuling invigorating bone prescription group than that in osteoporosis model group, and the difference was statistically significant between the two groups (P < 0. 01). However, there was no significant difference compare with the sham-operated group and Caltrate D group (P > 0. 05). (3) OPG protein expression level was higher in Xuling invigorating bone prescription group than that in osteoporosis model group, with statistical significance (P < 0. 05). RANKL protein expression level was lower than in osteoporosis model group,with statistical significance (P < 0. 01). RANKL protein expression level in osteoporosis model group was higher than that in sham-operated control group (P < 0. 05). Conclusion Xuling invigorating bone prescription significantly improves BMD in osteoporotic rats. The mechanism is related to up-regulation of OPG and down-regulation of RANKL. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of regulating the expression of osteoprotegerin (OPG) and receptoractivator of nuclear factor-KB ligand (RANKL) on early fracture by local application of OPG-Fc to rats with femoral fracture in the process of early fracture, and so as to explore its expression regulation mode and related mechanism. Methods 48 female SD rats were randomly divided into the experimental group and the control group respectively, and 24 pieces per group. And then open fracture model of rats was established, the animals were sacrificed at 4 time points (7, 14, 21 and 28 day) after operation, after callus specimens were sectioned, the information of fracture healing was observed by HE staining and the change of Osteoclast number was detected by immunohistochemical staining. Results HE staining indicated that the process of fracture healing in the pure fracture group was classical, but the callus formation and reforming in the fracture group of local injections of OPG were ahead of schedule, and fracture healing was accelerated. Immunohistochemical staining showed that the osteocyte count values of the pure fracture group were higher than that of the OPG application fracture group at all time points of 7, 14, 21 and 28 day, the difference was statistically significant (P < 0.05). Conclusion when regulating RANKL expression in the early stage of fracture, with the decreasing of RANKL/OPG, the number of local osteoclast declines, callus formation increases,and fracture healing is accelerated. [ABSTRACT FROM AUTHOR]

Objective To assess the RANKL、OPG, inflammatory cytokines and bone metabolism among postmenopausal women with RA,and to investigate their relationships. Methods A total of 45 RA patients and 25 healthy controls are involved, both are postmenopausal women. Bone mineral density (BMD) were measured by dual energy X-ray absorptiometry. The level of RANKL, OPG and IL-6 were detected by ELISA. The clinical and lab data were also recorded in details. T-test were used for data analysis, and Pearson's test were adsopted for correlation analysis. Results ① Compared with controls, the level of BMD and OPG /RANKL in RA patients decreased significantly, while the level of RANKL and IL-6 increaesd obviously (P < 0.05). OPG had no statistical difference between two groups.② RA patients determined as OP had higher anti-CCP (P < 0.05) than the Non-OP patients. The level of RANKL, OPG, DAS28 score and others had no statistical difference between two groups( P > 0.05). ③The level of RANKL was positively correlated with DAS28 score, PINP, β-CTX and IL-6 (r = 0.357, 0.381, 0.370, 0.330,P < 0.05). The level of OPG was positively correlated with PINP, β-CTX and IL-6 (r = 0.561,0.511, 0.328,P < 0.05). Conclusion RANKL levels of Postmenopausal women with RA is closely related to the inflammatory cytokines IL-6.However, it just reflects recent bone metabolism,does not reflect the changes of bone mineral density for a long time as a dynamic index. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the effects of Danshensu (DSS) on the RANKL-induced osteoclastogenesis and the mechanism in vitro. Methods: Bone marrow cells were obtained by flushing the femurs and tibiae of mice, the non-adherent cells were collected to differentiate in the presence or absence of Danshensu and RANKL. Count the number of the TRAP+ multinucleated osteoclasts, western bolt analysis and quantitative real-time PCR analysis. Results: ①The number of osteoclast decreased comparing differernt dose danshensu group with control group. ② The phosphorylation of Akt upregulated by RANKL-induced degraded by danshensu with different dose comparing to control group, while no significant difference were found in phosphorylation of ERK, p38 MAPK and JNK.③We found the expression of mRNA of the osteoclastic marker genes (TRAP, CTSK) were attenuated by addition of danshensu comparing to control group, as well as c-fos, but not seen in NFATc1. Conclusions: Danshensu suppress RANKL-induced osteoclastogenesis by downgrading phosphorylation of Akt. [ABSTRACT FROM AUTHOR]