Human osteoarthritic chondrocytes up-regulate the expression of osteoprotegerin in osteoblasts via the Indian hedgehog signaling pathway / 中国组织工程研究

BACKGROUND:Upregulation of hedgehog protein signaling can increase the expression of osteoarthritis markers,Runx2,a disintegrin and metalloproteinase with thrombospondin motifs,collagen type X alpha 1,and matrix metalloproteinase 13,while inhibition of hedgehog proteins attenuates the severity of osteoarthritis.It is speculated that osteoarthritic chondrocytes can influence bone formation by affecting osteoblasts through the Indian hedgehog protein(IHH)signaling pathway. OBJECTIVE:To investigate the effect of human osteoarthritic chondrocytes on subchondral osteoblasts. METHODS:Tibial plateau specimens from patients with osteoarthritis were collected.Chondrocytes were extracted using enzymatic digestion,and osteoblasts were extracted using enzymatic pre-digestion + bone block method.Chondrocytes were identified by toluidine blue staining and immunofluorescence and osteoblasts were identified by alkaline phosphatase staining and immunofluorescence.Chondrocytes were cultured in sodium alginate beads to maintain chondrocyte phenotype and co-cultured with osteoblasts.The co-culture system was added with IHH signaling pathway inhibitor(cyclopamine,10 nmol/L)and activator(purmorphamine,10 nmol/L)separately.After 48 hours of co-culture,osteoblasts from each group were collected,mRNA expressions of Gli1,osteoprotegerin,Runx2,parathyroid hormone-related peptide,alkaline phosphatase,receptor activator of nuclear factor-kB ligand(RANKL)and osteocalcin were detected by qRT-PCR,and protein expressions of GLi1,oseoprotegerin and RANKL in osteoblasts were detected by western blot. RESULTS AND CONCLUSION:The mRNA expression levels of GLi1,osteoprotegerin and RUNX2 in osteoblasts were significantly increased,while the mRNA expression levels of parathyroid hormone-related peptide were decreased(P＜0.05)when co-cultured with human osteoarthritic chondrocytes.The mRNA and protein levels of Gli1 were significantly decreased after the addition of IHH signaling pathway inhibitor(cyclopamine)(P＜0.05),and the mRNA and protein levels of Gli1 were significantly increased after the addition of IHH signaling pathway activator(purmorphamine)(P＜0.05).Osteoprotegerin showed the same trend as Gli1 in the experiment.The osteoprotegerin/RANKL ratio followed the same trend as osteoprotegerin.To conclude,human osteoarthritic chondrocytes can promote the expression of Gli1,osteoprotegerin,Runx2 and other proteins in osteoblasts.The upregulation of osteoprotegerin is related to the IHH signaling pathway.Osteoarthritic chondrocytes can up-regulate the expression of osteoprotegerin in osteoblasts through the IHH signaling pathway and thus up-regulate the osteoprotegerin/RANKL ratio,which will contribute to bone formation in subchondral bone.

Effects of Lipoic Acid on Bone Metabolism in Osteoporosis Rat and Its Mechanism / 中国医科大学学报

Objective To investigate the effects of oxidative stress and lipoic acid(antioxidant)on bone metabolism and explore the underlying mechanism. Methods A total of 24 Wistar rats aged 8 weeks were randomly divided into three groups. Osteoporosis rats model was established by bilateral ovaries deleted. Rat in lipoic acid group was injected with lipoic acid(60 mg/kg)for 8 weeks. The bone mineral density(BMD),steo?calcin,ALP,Ca,P,MDA,SOD and GSH?Px were detected. The levels of OPG and RANKL in serum were measured by Western blotting. OPG and RANKL mRNA were detected by real?time PCR. Results The level of BMD level in blood,SOD,GSH?Px,OPG mRNA and protein level in femur of osteoporosis group were significantly lower than the control group(P<0.05). On the other hand,steocalcin,ALP,MDA,RANKL mRNA and protein level were significantly higher than the control group(P<0.05). The level of BMD level in blood,SOD,GSH?Px,OPG mRNA and protein level of lipoic acid group were significantly higher than the osteoporosis group(P<0.05). The steocalcin,ALP,MDA,RANKL mRNA and protein level were significantly lower than the osteoporosis group(P<0.05). Conclusion Oxidative stress may increase osteoporosis through the upregulation of OPG/RANKL pathway in rats ,and antioxidant lipoic acid can alleviate the progress of osteoporosis.

The Relationship between Wnt Antagonist Genes Polymorphisms and Changes in Production of Osteoprotegerin and Soluble Receptor Activator of NF-kB by Whole Blood Cells after Hormone Therapy / 대한골다공증학회지

OBJECTIVES: To investigate the relationship between single nucleotide polymorphism (SNP)s in Wnt antagonist genes, and production of osteoprotegerin (OPG) and soluble receptor activator of NF-kappaB ligand (sRANKL) by whole blood cells after hormone therapy (HT) in postmenopausal Korean women. MATERIALS AND METHODS: The Dkk1 c.318A>G, Dkk2 c.437G>A, Dkk3 c.1003A>G polymorphisms and sFRP3 c.970C>G, sFRP4 c.958C>A, and c.1019G>A polymorphisms, and sFRP5 c.20G>C polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), direct sequencing, and Taqman assay in 75 postmenopausal Korean women receiving estrogen-progestogen therapy. The production of OPG and sRANKL by lipopolysaccharide-stimulated whole blood cells (WBC) before and after HT of 6 months were also measured. RESULTS: Changes in the production of OPG and sRANKL by lipopolysaccharide-stimulated WBC, and in ratios of sRANKL(x1,000)/OPG after HT of 6 months were not different according to SNPs in Wnt signal pathway genes except Dkk1 c.318A>G SNP. The AA genotype of Dkk1 c.318A>G SNP showed significantly higher changes (pA, and c.1019G>A polymorphisms after HT. CONCLUSIONS: Dkk1 c.318A>G SNP are related with changes in ratios of sRANKL(x1,000)/OPG in terms of the production of OPG and sRANKL by lipopolysaccharide-stimulated whole blood cells after HT.

RANKL mRNA expression induced by Porphyromonas endodontalis in rat osteoblasts / 实用口腔医学杂志

Objective:To study the effects of receptor activator of nuclear factor-kB ligand-RANKL in osteoblasts (OBs) activiated by Porphyromonas endodontalis(Pe),and to investigate the bone resorptive pathogenesis induced by Pe.Methods:Primary rat calvarial osteoblasts were cultured, then were infected with Pe ATCC 35406 at the density of 107 and 109 CFU/ml respectively for 24 h.To determine the kinetic effect of Pe on OBs,107 CFU/ml of Pe was used to stimulate the cells for 0,6,12,18 and 24 h respectively, RT-PCR were used to examine the RANKL mRNA expression.Results:The grey level ratio of RANKL mRNA expression to ?-actin in untreated OBs was 0.32,that in the cells infected by 107 and 109 CFU/ml of Pe for 24 h was increased to 1.73 and 2.24 respectively.Infected by 107 CFU/ml of Pe for 6,12 and 18 h,the ratio was 0.93,2.01 and 1.97 respectively.Conclusion:Pe may stimulate RANKL expresson in osteoblasts.

Effects of icariin on the expressions of osteoprotegerin and RANKL in rat osteoblasts / 中华内分泌代谢杂志

Objective To investigate the effects of icariin on the expressions of osteoprotegerin(OPG)and receptor activator of nuclear factor-kB ligand(RANKL)mRNA in rat osteoblasts cultured in vitro.Methods Calvarial osteoblasts were obtained from newborn SD rats aged＜24 h by trypsin-collagenase digestion method. After treatment with different concentrations of icariin(10~(-10)-10~(-4) mol/L)for 48 h,total RNA was isolated from osteoblasts.Expressions of OPG and RANKL mRNA were detected by semiquantitative RT-PCR.Results Icariin remarkably increased OPG mRNA levels and decreased RANKL mRNA levels in calvarial osteoblasts in a dose- dependent fasion with a maximum effect at a concentration of 10~(-7)mol/L(P＜0.05).Osteoblasts pre-treated with icariin prevented the inhibitory effect of dexamethasone on OPG mRNA(0.570?0.093 vs 1.083?0.081)and the stimulative effect on RANKL mRNA(1.079?0.050 vs 0.718?0.082)(both P＜0.05).Conclusion Icariin upregulates OPG expression and downregulates RANKL expression in osteoblasts,which may explain the beneficial role of icariin in preventing and treating osteoporosis.