PTH-related peptide (PTHrP) improves the bone marrow micro-environment to activate the bone-remodelling, but the coordinated regulation of PTHrP and transforming growth factor-β (TGFβ) signalling in TMJ-OA remains incompletely understood. We used disordered occlusion to establish model animals that recapitulate the ordinary clinical aetiology of TMJ-OA. Immunohistochemical and histological analyses revealed condylar fibrocartilage degeneration in model animals following disordered occlusion. TMJ-OA model animals administered intermittent PTHrP (iPTH) exhibited significantly decreased condylar cartilage degeneration. Micro-CT, histomorphometry, and Western Blot analyses disclosed that iPTH promoted subchondral bone formation in the TMJ-OA model animals. In addition, iPTH increased the number of osterix (OSX)-positive cells and osteocalcin (OCN)-positive cells in the subchondral bone marrow cavity. However, the number of osteoclasts was also increased by iPTH, indicating that subchondral bone volume increase was mainly due to the iPTH-mediated increase in the bone-formation ability of condylar subchondral bone. In vitro, PTHrP treatment increased condylar subchondral bone marrow-derived mesenchymal stem cell (SMSC) osteoblastic differentiation potential and upregulated the gene and protein expression of key regulators of osteogenesis. Furthermore, we found that PTHrP-PTH1R signalling inhibits TGFβ signalling during osteoblastic differentiation. Collectively, these data suggested that iPTH improves OA lesions by enhancing osteoblastic differentiation in subchondral bone and suppressing aberrant active TGFβ signalling. These findings indicated that PTHrP, which targets the TGFβ signalling pathway, may be an effective biological reagent to prevent and treat TMJ-OA in the clinic.
Animals ; Osteoclasts ; Osteogenesis ; Parathyroid Hormone-Related Protein/pharmacology* ; Temporomandibular Joint ; Transforming Growth Factor beta/pharmacology*

OBJECTIVETo observe the effects of different human parathyroid hormone 1-34 (hPTH1-34) administration on SaoS-2 cells, and explore the mechanism of bone formation improvement.

METHODSEach cycle covered 48 h. SaoS-2 cells were continuously or intermittently stimulated by 50 ng/ml hPTH1-34 for 1, 3, 6, 12, and 24 h in each cycle. Total RNA was extracted by Trizol kit. Alkaline phosphatase (ALP), osteocalcin or bone Gla-containing protein (BGP) and cyclic adenosine monophosphate (cAMP) levels were measured by chemical method, radioimmunoassay and competitive protein binding method, respectively. c-fos gene expression was semi-quantified by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSALP level was time-dependently increased in 1, 3 and 6 h stimulation, especially in 3 and 6 h (compared with control, P < 0.01; P < 0.05 or P < 0.01 compared with continuous stimulation). The cAMP level was time-dependently increased in 3 and 6 h incubation (P < 0.05 compared with control and continuous stimulation). Intermittent hPTH1-34 stimulation had more effects on cAMP level than continous action (P < 0.001). hPTH1-34 intermittent stimulation of 1, 3, and 6 h enhanced c-fos gene expression time-dependently.

CONCLUSIONSIntermittent hPTH1-34 stimulation has a stronger effect on osteoblast than continuous action, especially in 3, 6 h in each cycle intermittent stimulation. The synchronous responses of c-fos, ALP and cAMP to hPTH1-34 suggest that hPTH1-34 affect Saos-2 cells through cAMP dependent protein kinase A (PKA) pathway and c-fos gene paly an important role.

Alkaline Phosphatase ; analysis ; Cells, Cultured ; Humans ; Osteoblasts ; cytology ; Osteocalcin ; analysis ; Osteogenesis ; drug effects ; Osteosarcoma ; genetics ; pathology ; Parathyroid Hormone ; pharmacology ; Parathyroid Hormone-Related Protein ; pharmacology ; Peptide Fragments ; pharmacology ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

BACKGROUND: The objective of this study was to evaluate the role of proteases on the degradation of parathyroid hormone (PTH) in blood samples. METHODS: Protease inhibitors with specificity against serine proteases (aprotinin), cysteine proteases (E-64), serine and cysteine proteases (leupeptin), metalloproteases (EDTA), or a protease inhibitor cocktail with a broad spectrum of inhibitory activity were added to blood samples. After storage at room temperature (0-48 hr), PTH levels were measured. RESULTS: PTH levels in samples with the protease inhibitor cocktail did not change significantly after 48 hr of storage at room temperature, but the average PTH levels decreased by 40.7% and 20.1%, in samples stored at room temperature and stored at 4degrees C without protease inhibitors, respectively. PTH levels in samples with leupeptin were stable for up to 24 hr. After 48 hr, the mean PTH levels decreased by 17.1%, 16.0%, 26.2%, and 32.1%, with 500 KIU/mL aprotinin, 100 micro mol/L leupeptin, 10 micro mol/L E-64, and 10 micro mol/L EDTA, respectively, in the samples stored at room temperature. CONCLUSIONS: The decrease in PTH levels in blood samples seemed to be due to the degradation of PTH by proteases. Various proteases, including especially serine proteases, would act together to degrade PTH in blood specimen. The PTH degradation may be inhibited in blood specimen with protease inhibitor cocktail.
Aprotinin/pharmacology ; Blood Specimen Collection ; Edetic Acid/pharmacology ; Female ; Humans ; Leucine/analogs & derivatives/pharmacology ; Leupeptins/pharmacology ; Male ; Parathyroid Hormone/*blood/metabolism ; Protease Inhibitors/*pharmacology ; Time Factors

OBJECTIVETo assess the effect of intermittent subcutaneous injections of signal-selective parathyroid hormone (PTH) peptide analog on fracture healing in orchiectomized mouse models.

METHODSThirty-six 7-week-old C57/BL male mice were orchiectomized and injected with hPTH(1-34), the signal-selective PTH peptide analog [Gly(1), Arg(19)]hPTH (1-34), or an identical volume of vehicle 1 week after induction of femoral fracture. At 14 and 28 days after the operation, the mice were sacrificed for measurement of bone mineral density (BMD) and bone mineral content (BMC) of the callus using by dual energy X-ray absorptiometry. The bone healing was evaluated by radiography, biomechanical testing, micro-computed tomography (Micro-CT) and histological examination.

RESULTSAt 14 days after the operation, BMD in PTH peptide analog group was significantly increased (P<0.05). The mouse models treated with the PTH peptide analog showed significantly lower ultimate bending force and bending rigidity than those with hPTH(1-34) treatment. X-ray and Micro-CT scanning showed that callus transformation and remodeling was better in PTH peptide analog group than in the vehicle control group but poorer than in hPTH(1-34) group.

CONCLUSIONThe signaling-selective PTH peptide analog G1, R19 (1-28) can accelerate fracture healing in orchiectomized mouse models, in which process cAMP/PKA pathway plays an important role.

Animals ; Bone Density ; Fracture Healing ; drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Orchiectomy ; Parathyroid Hormone ; analogs & derivatives ; pharmacology ; Signal Transduction

BACKGROUNDRecombinant human parathyroid hormone (1-34) (rhPTH (1-34)) given by injection is a new seventh class drug of biological products, which is prepared by adopting gene recombination technique. rhPTH (1-34) is mainly used to treat osteoporosis, especially for postmenopausal women. This study compared the clinical efficacy and safety of rhPTH (1-34) with elcatonin for treating postmenopausal women with osteoporosis in 11 urban areas of China.

METHODSTwo hundred and five women with osteoporosis were enrolled in a 6-month, multicenter, randomized, controlled study. They were randomized to receive either rhPTH (1-34) 20 microg (200 U) daily or elcatonin 20 U weekly. Lumbar spine (L1-4) and femoral neck bone mineral density (BMD), as well as biochemical markers of bone turnover were measured. Adverse events were recorded.

RESULTSrhPTH (1-34) increased lumbar BMD significantly more than did elcatonin at 3 months and 6 months (2.38% vs 0.59%, P < 0.05; 5.51% vs 1.55%, P < 0.01), but there were no significant increases of BMD in these two groups at femoral neck. There were larger mean increases in bone markers in the rhPTH (1-34) group than in the elcatonin group at 3 months and 6 months (serum bone-specific alkaline phosphatase (BSAP) 36.79% vs 0.31%; 92.42% vs -0.17%; urinary N-telopeptide/creatinine (NTX/Cr) 48.91% vs -5.32%; 68.82% vs -10.86%). Both treatments were well tolerated and there were no significant differences detected between the two groups in the proportion of any adverse events and any serious adverse events (67.0% vs 59.0%; 0 vs 0).

CONCLUSIONSrhPTH (1-34) has more positive effects on bone formation, as shown by the larger increments of lumbar BMD and bone formation markers, than elcatonin, with only mild adverse events and no significant change in the liver, kidney or hematological indices.

Aged ; Calcitonin ; analogs & derivatives ; pharmacology ; therapeutic use ; Female ; Humans ; Middle Aged ; Osteogenesis ; drug effects ; Osteoporosis, Postmenopausal ; drug therapy ; Parathyroid Hormone ; pharmacology ; therapeutic use ; Recombinant Proteins ; pharmacology ; therapeutic use

OBJECTIVETo investigate the effect of the non-PLC-dependent protein kinase C (PKC) pathway of parathyroid hormone (PTH) on the apoptosis and proliferation of osteoblast MC-3T3E1 cells.

METHODSMC-3T3E1 cells were seeded in 96-well plates at the density of 1.5×10(4) cells/mL and incubated for 3 day. The cells were then exposed to 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-28), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34), 100 nmol/L of [Gly(1), Arg(19)]hPTH(1-34)+1 µmol/L Go6983, 1 µmol/L Go6983, or deionized water (control) for 1, 24 or 48 h. After the treatments, cell counting kit-8 (CCK-8) and Caspase-Glo® 3/7 Assay (Caspase-3) were used to examine the proliferation and apoptosis of MC3T3-E1 cells.

RESULTSCCK-8 results showed that hPTH(1-34) increased the number of MC3T3-E1 cells compared with hPTH(1-34)+Go6983 at 1 h and 24 h, but this difference was not statistically different. At 48 h, treatment with hPTH(1-34), as compared with hPTH(1-28), significantly increased the number of MC3T3-E1 cells (P<0.05), and this effect was blocked by the PKC inhibitor Go6983 (P<0.05). hPTH(1-34) did not result in significant inhibition of MC3T3-E1 cell apoptosis at 1 h and 24 h as compared with hPTH(1-34)+Go6983, but significantly inhibited the cell apoptosis as compared with hPTH(1-28) (P<0.05); this inhibitory effect was blocked by Go6983 (P<0.05).

CONCLUSIONs A relatively long time (for 48 h) of exposure to PTH can inhibit apoptosis and promote the proliferation of MC3T3-E1cells through a non-PLC-dependent PKC pathway.

3T3 Cells ; Animals ; Apoptosis ; Cell Proliferation ; Indoles ; pharmacology ; Maleimides ; pharmacology ; Mice ; Osteoblasts ; Parathyroid Hormone ; pharmacology ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Signal Transduction

Drynariae Rhizoma has great significance in the clinical practice of osteoporosis treatment. Based on the perspective of integrative pharmacology, the study explored the mechanism of action of Drynariae Rhizoma in the treatment of osteoporosis. Six active components in Drynariae Rhizoma were obtained, mainly including glycosides and sterols. Taking the median of 2 times of "node connectivity" as the card value, the core node of the Chinese medicine target disease gene interaction network was selected. Based on this, three topological structural eigenvalues, such as "node connectivity" "node tightness" and "node connectivity" were calculated, thereby screening out four core targets of Drynariae Rhizoma treatment for osteoporosis, including thyroid parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), calcitonin receptor gene (CALCR), and SPTBN1 gene (SPTBN1). Based on the gene ontology database GO and KEGG pathway database, the molecular function, intracellular localization, and biological reactions and pathways of proteins encoded by drug target genes were determined. Combined with enrichment calculation, it is predicted that osteoporosis may play a role in biosynthetic processes, such as circulatory system, nervous system, energy metabolism, prolactin signal pathway, GnRH signaling pathway, neurotrophic factor signaling pathway and other pathway. The conclusion of this study is certain with the existing research results, and the new target and new pathway could also be used as a theoretical basis for the further verification of osteoporosis.
Drugs, Chinese Herbal ; pharmacology ; Humans ; Osteoporosis ; drug therapy ; Polypodiaceae ; chemistry ; Receptor, Parathyroid Hormone, Type 1 ; metabolism ; Receptor, Parathyroid Hormone, Type 2 ; metabolism ; Receptors, Calcitonin ; metabolism ; Rhizome ; chemistry ; Spectrin ; metabolism

OBJECTIVETo explore the effect of recombinant human parathyroid hormone 1-34 [rhPTH(1-34)] on bone regeneration rabbit mandible during distraction osteogenesis (DO).

METHODS40 Japanese white rabbit (weight 2.0-2.5 kg) were randomly divided into control group and groups. The experimental groups were divided inito 12.5, 25 and 50 µg/kg group according to the dosage of rhPTH (1-34) in each group. Each group involved 10 rabbits, and unilateral DO models were established at the right mandible of the rabbits. From the first day of distraction to the day of execution, the rabbits in the experimental groups were injected subcutaneously rhPTH (1-34) of the corresponding dose respectively, and the rabbits in the control group were injected subcutaneously 2% heat inactivated rabbit serum 1 ml respectively.. Five rabbits in each group were executed respectively at 1 week and 3 weeks after completion of distraction, and the specimens of DO were harvested. The gross observation, X-ray examination, and histological study were performed.

RESULTSGross appearance: At the first week of consolidation, the dense and opaque white tissue was seen in the distraction gap of the 50 µg/kg group, and the white translucent tissue was seen in the distraction gaps of the rest groups. At the third week of consolidation, the greyish white tissue was seen in the distraction gap of the control group, while the cartilage-like tissue was seen in the buccal side of the distraction gap of the 12.5 µg/kg group, the color of new-formed tissues was close to that of normal bone tissue in the lingual side. The buccal tissue at the edge of the distraction gap of the 25 µg/kg group fitted together with the primary bone tissue in its two sides. It was difficult to distinguish the boundaries between the distraction gap and the bone tissues in its two sides in the 50 µg/kg group. X-ray findings: At the first week of consolidation, a sparse opaque image was seen in the distraction gap of the 50 µg/kg group, and a low-density image was seen in the distraction gap of the rest groups. At the third week of consolidation, a sparse bone image was seen in the control group, and the edge of the bone was not continuous. With the increase of the dose in the experimental groups, the image of the distraction gap became more and more opaque, and the image of the distraction gap in the 50 µg/kg group was close to that of the normal bone tissue. HISTOLOGICAL FINDINGS: At the first week of consolidation, few osteoblasts were present at the edge of the distraction gap of the control group. A large number of bone cells and bone trabecular were present in the distraction gap of the 12.5 µg/kg group, the network of the bone trabecula was present in the 25 µg/kg group, and a few new bones were found in the 50 µg/kg group. At the third week of consolidation, the network of the trabecular bone was present in the distraction gap of the control group, while the network of the bone trabecula was present in the 12.5 µg/kg group, a lot of bone-like tissues in the 25 µg/kg group, and near-mature bone in the 50 µg/kg group.

CONCLUSIONSrhPTH(1-34) can promote the formation of new bone in the distracted gap during mandibular DO in rabbits.

Animals ; Bone Density ; Bone Regeneration ; drug effects ; physiology ; Humans ; Mandible ; drug effects ; surgery ; Osteogenesis, Distraction ; methods ; Parathyroid Hormone ; pharmacology ; Rabbits ; Random Allocation ; Recombinant Proteins ; pharmacology

OBJECTIVETo probe into the mechanisms of thread implantation at Zusanli(ST 36) and Chinese herbs in treatment of chronic renal failure(CRF).

METHODSCRF rat model was made by Platt subtotal nephrectomy. They were divided into 5 groups, sham operation group, model group, Chinese herbs group, thread implantation group and thread implantation plus Chinese herbs group. After treatment of 8 weeks, serum parathyroid hormone (PHT), transforming growth factor beta1 (TGF-beta1) expression in residual renal cells, malondialdehyde(MDA) content in the residual renal tissue, serum urea nitrogen(BUN) and creatinine(Scr), protein in urine and pathological changes were investigated.

RESULTSThe above indexes after treatment by thread implantation at acupoint, Chinese herbs, and acupoint thread implantation plus Chinese herbs showed begin reversion, especially, the most obviously improvement in the acupoint thread implantation plus Chinese herbs treatment group.

CONCLUSIONThe mechanism of acupoint thread implantation and Chinese herbs in improvement of CRF is related with decrease of PTH, inhibition of TGF-beta1 expression, decrease of MDA content and resisting lesion of renal fibrosis.

Acupuncture Points ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Kidney Failure, Chronic ; blood ; therapy ; Male ; Parathyroid Hormone ; blood ; Rats ; Rats, Wistar ; Transforming Growth Factor beta ; blood ; Transforming Growth Factor beta1

OBJECTIVETo construct the expression vector of siRNA targeting parathyroid hormone 1 receptor (PTH1R) gene and evaluate its effect on the cell cycle of INS-1 cells.

METHODSThe sequences of PTH1R gene was retrieved from Genbank, and 4 pairs of oligonucleotides were synthesized and inserted into pSUPERretro RNAi, which was identified by RT-PCR and sequence analysis. The vectors were then transfected into INS-1 cells, in which the expression of PTH1R was observed by Western blotting to evaluate the transfection efficiency. The cell cycle of INS-1 cells in high glucose medium was detected by flow cytometry.

RESULTSRT-PCR and sequence analysis confirmed the correct construction of the siRNA recombinant expression vector targeting PTH1R gene. The vectors were successfully transfected into INS-1 cells, and the most effective vector was selected by Western blotting. Transfection with the siRNA for PTH1R gene silencing resulted in the inhibition of INS-1 form entering the S phase.

CONCLUSIONThe successful construction of the recombinant PTH1R-siRNA vectors establishes a basis for further study of protective role of the PTH1R gene in INS-1 cells in high glucose medium.

Cell Cycle ; drug effects ; Genetic Vectors ; genetics ; Glucose ; pharmacology ; Humans ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, Parathyroid Hormone, Type 1 ; genetics ; metabolism