OBJECTIVE: To determine whether resveratrol (Res) can correct osteoporosis induced in a rat model of male hypogonadism. METHODS: Thirty-two rats were randomly divided into 4 groups, 8 in each group; 1) a control sham group: underwent a similar surgical procedure for induction of orchiectomy (ORCD) without ligation of any arteries or veins or removal of the testis and epididymis; 2) a control + Res-treated group (Con+Res): underwent sham surgery similar to the control, but was then treated with Res, as described below; 3) an ORCD-induced group: bilateral ORCD surgery as described above, and 4) a ORCD+Res-treated group: bilateral ORCD surgery followed by Res treatment. Res treatment began 4 weeks after ORCD and continued for 12 weeks. After 12 weeks, bone mineral density (BMD) and bone mineral content (BMC) were measured in the tibia and femur of each rat's right hind leg. Blood levels of bone turnover indicators such as deoxypyridinoline (Dpd), N-telopeptide of type I collagen (NTX I), alkaline phosphatase (ALP), and osteocalcin (OC), as well as receptor activator of nuclear factor kappa B (RANK) and osteoprotegerin (OPG) were assessed. RESULTS: ORCD significantly decreased BMD (P<0.01) and significantly increased bone resorption, manifested by increased RANK. In addition, it inhibited serum levels of OPG and OC. Res treatment after ORCD effectively increased serum levels of bone formation markers such as OPG and OC, compared with testisectomized rats (P<0.05). CONCLUSION Res could ameliorate bone loss induced by male hypogonadism, possible via restoration of the normal balance between RANK and OPG.
Rats ; Male ; Animals ; Bone Density ; Resveratrol/pharmacology* ; Osteoporosis ; Osteoprotegerin/pharmacology* ; Bone Remodeling ; Hypogonadism ; RANK Ligand/pharmacology*

OBJECTIVES: This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients. METHODS: HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm²) and C2 and D2 (energy density: 5.625 J/cm²). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay. RESULTS: 1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05). CONCLUSIONS 1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm². Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.
Humans ; Osteoprotegerin ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/pharmacology* ; RANK Ligand/pharmacology* ; Periodontal Ligament/metabolism* ; Lasers ; Glucose/pharmacology*

OBJECTIVETo investigate the effects of 17beta-estradiol on the expression of alkaline phosphatase (ALP) and osteoprotegerin in human periodontal ligament cells.

METHODSHuman periodontal ligament cells (hPDLC) were obtained from periodontal tissue explants of teeth extracted for orthodontic treatment ALP activity was determined by PNPP, and OPG protein and corresponding mRNA levels were quantitatively detected by ELISA and RT-PCR RESULTS: ALP activity was significantly increased at 14 days and 21 days (P <0.05). 17beta-E2 of physiological concentration promoted secretion of OPG protein and expression of OPG mRNA (P <0.05). 17beta-E2 with high-dose showed no effect on OPG protein secretion and decrease OPG mRNA expression.

CONCLUSIONS17beta-E2 may have a positive impact on periodontium through promoting expression of ALP and OPG in hPDLC.

Alkaline Phosphatase ; metabolism ; Cells, Cultured ; Estradiol ; pharmacology ; Humans ; Osteoprotegerin ; metabolism ; Periodontal Ligament ; cytology ; drug effects ; metabolism

OBJECTIVETo observe serum osteoprotegerin (OPG) level in children with nephrotic syndrome (NS) and changes in serum OPG level after glucocorticoid therapy, with the aim of studying the role of OPG in the bone metabolism of children with NS.

METHODSForty-four children with idiopathic NS were randomly selected as the study group, including 24 newly diagnosed, untreated patients and 20 who had relapsed during the process of glucocorticoid reduction (cumulative dose of glucocorticoid 28327±5879 mg/m2). Twenty-three age- and sex-matched healthy children served as the control group. Serum osteoprotegerin (OPG) level was measured using ELISA. Serum N-terminal midfragment of osteocalcin (N-MID osteocalcin) was determined using electrochemical luminescence immunoassays (ECLIA).

RESULTSSerum levels of OPG (211±55 ng/L) and N-MID osteocalcin (46±14 ng/mL) in the untreated NS group were reduced compared with 470±57 ng/L (OPG) and 73±9 ng/ml (N-MID osteocalcin) in the control group (P<0.05). Serum levels of OPG (176±42 ng/L) and N-MID osteocalcin (29±10 ng/mL) in the NS relapsed group were lower than in the untreated NS and control groups (P<0.05).

CONCLUSIONSBone metabolism disorders are found in children with NS. High-doses of glucocorticoid therapy can aggravate these disorders. Serum OPG levels in children with NS may be affected by both the renal disease itself and steroid therapy, suggesting that OPG is expected to become a new biochemical indicator for predicting changes to the bone metabolism of children with NS.

Child ; Glucocorticoids ; pharmacology ; Humans ; Nephrotic Syndrome ; blood ; drug therapy ; Osteocalcin ; blood ; Osteoprotegerin ; blood

OBJECTIVES: The expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) was investigated by cell culture under continuous static pressure. METHODS: HPDLCs were primarily cultured by tissue explant method and divided into three groups: group A (13-18 years old), group B (19-29 years old), and group C (30-44 years old). CCK-8 was used to detect the proliferation of HPDLCs. The senescence of HPDLCs was detected by senescence-associated β-galactosidase staining. Cells in the three groups were respectively given 0, 1.5, 3, 6, 12, 24, 48, and 72 h of continuous static pressure in vitro. The expression of OPG and RANKL in the supernatant was detected by enzyme-linked immunosorbent assay. RESULTS: After continuous static pressure in vitro for stimulation, the expression of OPG and RANKL changed. The expression of OPG increased with time and age (P<0.01). The expression of RANKL increased with time and decreased with age (P<0.01). The ratio of OPG/RANKL initially decreased, increased with time, and then continued to rise with age (P<0.01). CONCLUSIONS Aging could increase the expression of OPG and the ratio of OPG/RANKL and decrease the expression of RANKL in HPDLCs under continuous static pressure in vitro.
Humans ; Adolescent ; Young Adult ; Adult ; Osteoprotegerin ; RANK Ligand/pharmacology* ; Periodontal Ligament/metabolism* ; Cells, Cultured ; Aging

OBJECTIVETo investigate the effect of alendronate on the expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) in mouse osteoblasts.

METHODSMouse calvarial osteoblasts cultured in vitro were identified by alkaline phosphatase (ALP) staining and immunofluorescence assay of OPG and RANKL expressions. The second passage of the osteoblasts were treated with different concentrations of alendronate (10(-4) to 10(-7) mol/L) for 48 h, and the changes in OPG and RANKL mRNA and protein expressions were examined using real-time PCR and Western blotting, respectively.

RESULTSThe isolated osteoblasts were positive for ALP and expressed OPG and RANKL. Real-time PCR and Western blotting showed that at the concentration of 1×10(-4) mol/L, alendronate caused an obvious down-regulation of OPG and RANKL expressions in the cells, whereas at lower concentrations, alendronate increased the expressions of both genes with the highest expressions occurring after treatment with 1×10(-5) mol/L.

CONCLUSIONHigh concentrations of alendronate (>1×10(-4) mol/L) decrease the expressions of OPG and RANKL, whereas low concentrations (1×10(-5) to 1×10(-7) mol/L) increase their expressions in mouse osteoblasts cultured in vitro.

Alendronate ; pharmacology ; Animals ; Cells, Cultured ; Mice ; Mice, Inbred BALB C ; Osteoblasts ; drug effects ; metabolism ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism

OBJECTIVETo study the effect of combination rhOPG-Fc and alendronate on mature osteoclasts.

METHODSRecombinant human osteoprotegerin secretory expression in P. pastoris was performed. Osteoblasts were got from new born mouse skeletal bone and proved by ALP staining and incubated together with osteoclasts precursor cell line Raw 264.7 in 96 well plate. After 9 d, 10 micromol/L ALN, 10(-5) g/L rhOPG-Fc, 10 micromol/L ALN + 10(-5) g/L rhOPG-Fc, 5 micromol/L ALN + 5 x 10(-6) g/L rhOPG-Fc were added to these coculture systems. Osteoblasts cultured without the drugs mentioned above served as controls. TRAP stain positive cells counting and cortical bone pit formation counting were preformed in the following the 3rd and 7th d.

RESULTSSDS-PAGE and Western blot showed that molecular weight of the expressed protein was about 55 KD, and it could reach specifically with anti-IgG antibody. Many multi-nuclear TRAP stain positive cells were found in the coculture control group after 9 d incubation, and proved to be mature osteoclasts by TRAP stain. In the 3rd and 7th d after the addition of rhOPG-Fc, ALN or both, TRAP stain positive cells counting and cortical bone pit formation counting decreased significantly in the rhOPG-Fc, ALN or both groups than in the control group, and the combine group (10(-5) g/L rhOPG-Fc + 10 micromol/L ALN) decreased most significantly when compared with rhopG-FC or ALN single.

CONCLUSIONSrhOPG-Fc can decrease the number of osteoclasts and inhibit their function. The combination of both rhOPG-Fc and ALN shows the significant inhibition effect on mature osteoclasts.

Alendronate ; pharmacology ; Animals ; Drug Synergism ; Humans ; In Vitro Techniques ; Mice ; Mice, Inbred Strains ; Osteoclasts ; cytology ; drug effects ; Osteoprotegerin ; biosynthesis ; pharmacology ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; pharmacology

OBJECTIVETo investigate the effect of zoledronic acid on proliferation and osteogenic differentiation of osteoblasts.

METHODSOsteoblasts were obtained from newly born rabbit jaw bones and cultured by the method of bone-tissue cultivation. Primary cultivated osteoblast was identified by alkaline phosphatase (ALP) and the mineralization nodes. Zoledronic acid at various concentrations was added to six groups of media with serial subcultivated cells (the final concentration: 0, 10(-5), 10(-6), 10(-7), 10(-8) and 10(-9) mol/L). At different time, ALP, osteoprotegerin and osteocalcin were observed and calculated.

RESULTSThe concentration of 10(-6), 10(-7), 10(-8) and 10(-9) mol/L zoledronic acid significantly increased ALP activity [(5.91 ± 0.35), (7.62 ± 0.33), (10.00 ± 0.38), (8.91 ± 0.29) U/L]. Protein expression of osteoprotegerin and osteocalcin was enhanced. The differences among the groups were significant (P < 0.01). Peak level was attained at a concentration of 10(-8) mol/L.

CONCLUSIONSZoledronic acid promotes osteoblast proliferation and maturation and modulates osteoprotegerin production.

Alkaline Phosphatase ; Animals ; Bone and Bones ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Diphosphonates ; pharmacology ; Imidazoles ; pharmacology ; Osteoblasts ; drug effects ; Osteocalcin ; drug effects ; metabolism ; Osteogenesis ; Osteoprotegerin ; drug effects ; metabolism ; Rabbits

OBJECTIVETo explore the feasibility of in vitro differentiation of human umbilical cord blood cells (HUCBC) into neural cells induced by receptor activator of NF-KappaB ligand (RANKL) and brain-derived neurotrophic factor (BDNF).

METHODSNormal fresh HUCBC were cultured as the following: (1) Control group cultured by differentiation medium only; (2) BDNF group, cultured by differentiation medium + BDNF; (3) RANKL group, cultured by differentiation medium + human soluble RANKL (sRANKL); (4) BDNF + RANKL group, cultured by differentiation medium + BDNF and sRANKL. Cultured cells were observed with invert microscope. After ten-days culture, the expression of glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN) of the cultured cells were detected by immunocytochemical staining.

RESULTSAfter 10 day's culture, the NeuN positive cells were (97.0 +/- 13.5), (85.0 +/- 5.6), (167.0 +/- 19.7) in RANKL, BDNF and BDNF + RANKL groups, respectively, with 1.7, 1.5, 3.0 fold in crease than that of control (55.7 +/- 8.5), the GFAP positive cells were (114.7 +/- 18.0), (233.3 +/- 21.7), (289.0 +/- 24.7), respectively, with 1.4, 2.9, 3.6 fold increase compared with the control group. The differentiation ratio of neurons in RANKL group was similar to that of the BDNF group, but the differentiation ratio of glial cells was lower than that in the BDNF group. In the RANKL + BDNF group, the differentiation of HUCBC into neurons and glial cells were enhanced obviously, the differentiated neural cells were typical with longer axons and dendrites.

CONCLUSIONRANKL and BDNF could induce HUCBC into neurons and glial cells, and they have synergistic effect on the induced differentiation. It is hopeful that HUCBC might be an source of stem cells for the treatment of central nervous system injury.

Brain-Derived Neurotrophic Factor ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Fetal Blood ; cytology ; Glycoproteins ; pharmacology ; Humans ; Neurons ; cytology ; Osteoprotegerin ; Receptors, Cytoplasmic and Nuclear ; Receptors, Tumor Necrosis Factor

OBJECTIVETo study the effects of Zhengqing Fengtongning Tablet (ZFT) and methotrexate (MTX) on the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB ligand (RANKL), and interleukin 17 (IL-17) in collagen-induced arthritis (CIA) rats, thus addressing their bone protection.

METHODSThe CIA rat model was established by intradermally injecting type II collagen emulsion from the rats' back and tail. Totally 28 successfully modeled rats [with the arthritis index (AI) more than 2] were randomly divided into the model group, the Chinese medicine (CM) treatment group, the MTX group, and the ZFT + MTX treatment group, 7 rats in each group. Another 7 rats were recruited as the normal control group. Rats were administered from the 7th day of modeling. Rats in the MTX group were treated with MTX at 3.8 mg/kg once a week. Those in the CM group were treated with ZFT at the daily dose of 130 mg/kg, once a day. Those in the ZFT + MTX treatment group were treated with both MTX (at 3.8 mg/kg once a week) and ZFT (at the daily dose of 130 mg/kg, once a day). Those in the model group and the normal control group were administered with normal saline of the equal volume by gastrogavage. All the intervention lasted for 26 days. The destruction of joints in the four limbs were observed using X-ray. The AI was recorded. The expression levels of serum OPG, RANKL, and IL-17 were detected at the end of the experiment.

RESULTSDuring the whole process, all rats except those in the model group were in a good condition. On the 21st day of modeling the AI of all rats reached the peak, but it decreased after treatment. Compared with the model group, the AI decreased in the CM treatment group, the MTX group, and the ZFT + MTX treatment group with statistical difference (P < 0.05). Compared with the model group, the OPG increased and RANKL decreased in the MTX group; the OPG and OPG/RANKL increased in the CM treatment group; the OPG, RANKL, and OPG/RANKL increased, and IL-17 decreased in the ZFT + MTX treatment group, all showing statistical difference (P < 0.05). Compared with the MTX and the ZFT + MTX treatment group, OPG/RANKL increased and IL-17 decreased in the ZFT + MTX treatment group (both P < 0.05).

CONCLUSIONZFT + MTX could synergistically elevate peripheral OPG/RANKL and down-regulate IL-17 in CIA model rats.

Animals ; Arthritis, Experimental ; blood ; chemically induced ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Interleukin-17 ; blood ; Methotrexate ; pharmacology ; therapeutic use ; Osteoprotegerin ; blood ; RANK Ligand ; blood ; Rats