This paper aimed to explore the effects of Duzhong Decoction(DZD)-containing serum on the proliferation and osteoblast differentiation of MC3T3-E1 cells through L-type voltage-gated calcium channels(L-VGCCs). L-VGCCs inhibitors, nifedipine and verapamil, were used to block L-VGCCs in osteoblasts. MC3T3-E1 cells were divided into a control group, a low-dose DZD-containing serum(L-DZD) group, a medium-dose DZD-containing serum(M-DZD) group, a high-dose DZD-containing serum(H-DZD) group, a nifedipine group, a H-DZD + nifedipine group, verapamil group, and a H-DZD + verapamil group. The CCK-8 method was used for cell proliferation analysis, alkaline phosphatase(ALP) assay kits for intracellular ALP activity measurement, Western blot for protein expression level in cells, real-time fluorescence quantitative PCR technology for intracellular mRNA expression level determination, fluorescence spectrophotometer for free Ca~(2+) concentration determination in osteoblasts, and alizarin red staining(ARS) for mineralized nodule formation in osteoblasts. The experimental results show that compared to the control group, DZD groups can promote MC3T3-E1 cell proliferation, ALP activity, and mineralized nodule formation, increase intracellular Ca~(2+) concentrations, and upregulate the protein expression of bone morphogenetic protein 2(BMP2), collagen Ⅰ(COL1), α2 subunit protein of L-VGCCs(L-VGCCα2), and the mRNA expression of Runt-related transcription factor 2(RUNX2), and BMP2. After blocking L-VGCCs with nifedipine and verapamil, the intervention effects of DZD-containing serum were inhibited to varying degrees. Both nifedipine and verapamil could inhibit ALP activity, reduce mineralized nodule areas, and downregulate the expression of bone formation-related proteins. Moreover, the effects of DZD-containing serum on increasing MC3T3-E1 cell proliferation, osteoblast differentiation, and Ca~(2+) concentrations, upregulating the mRNA expression of osteoprotegerin(OPG) and protein expression of phosphorylated protein kinase B(p-Akt) and phosphorylated forkhead box protein O1(p-FOXO1), and upregulating phosphatase and tensin homolog(PTEN) expression were reversed by nifedipine. The results indicate that DZD-containing serum can increase the Ca~(2+) concentration in MC3T3-E1 cells to promote bone formation, which may be mediated by L-VGCCs and the PTEN/Akt/FoxO1 signaling pathway, providing a new perspective on the mechanism of DZD in treating osteoporosis.
Animals ; Osteoblasts/metabolism* ; Cell Proliferation/drug effects* ; Cell Differentiation/drug effects* ; Mice ; Drugs, Chinese Herbal/pharmacology* ; Calcium Channels, L-Type/genetics* ; Alkaline Phosphatase/genetics* ; Serum/chemistry* ; Cell Line ; Osteogenesis/drug effects* ; Bone Morphogenetic Protein 2/genetics*

OBJECTIVE: To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. METHODS: Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups ( n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. RESULTS: The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B ( P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A ( P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B ( P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A ( P<0.05). CONCLUSION Under the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Osteogenesis, Distraction/methods* ; Rats ; RNA, Long Noncoding/metabolism* ; Osteopontin/genetics* ; Osteogenesis ; Bone Regeneration ; RNA, Small Interfering/genetics* ; Osteocalcin/genetics* ; Alkaline Phosphatase/metabolism* ; Osteoblasts/cytology* ; Signal Transduction ; Femoral Fractures/surgery*

OBJECTIVE: To initially investigate the function of neuronal pentraxin 1 (NPTX1) gene on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). METHODS: hBMSCs were induced to undergo osteogenic differentiation, and then RNA was collected at different time points, namely 0, 3, 7, 10 and 14 d. The mRNA expression levels of key genes related with osteogenic differentiation, including runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and NPTX1, were detected on the basis of quantitative real-time polymerase chain reaction (qPCR) technology. In order to establish a stable NPTX1-knockdown hBMSCs cell line, NPTX1 shRNA lentivirus was constructed and used to infect hBMSCs. ALP staining, alizarin red (AR) staining, and qPCR were employed to assess the impact of NPTX1-knockdown on the osteogenic differentiation ability of hBMSCs. RESULTS: The results showed that during the osteogenic differentiation of hBMSCs in vitro, the mRNA expression levels of osteogenic genes RUNX2, ALP and OCN significantly increased compared with 0 d, while NPTX1 expression decreased markedly (P < 0.01) as the osteogenic induction period exten-ded. At 72 h post-infection with lentivirus, the result of qPCR indicated that the knockdown efficiency of NPTX1 was over 60%. After knocking down NPTX1 in hBMSCs, RNA was extracted from both the NPTX1-knockdown group (sh NPTX1 group) and the control group (shNC group) cultured in regular proliferation medium. The results of qPCR showed that the expression levels of osteogenic-related genes RUNX2 and osterix (OSX) were significantly higher in the sh NPTX1 group compared with the shNC group (P < 0.01). ALP staining revealed a significantly deeper coloration in the sh NPTX1 group than in the shNC group at the end of 7 d of osteogenic induction. AR staining demonstrated a marked increase in mineralized nodules in the sh NPTX1 group compared with the shNC group at the end of 14 d of osteogenic induction. CONCLUSION NPTX1 exerts a modulatory role in the osteogenic differentiation of hBMSCs, and its knockdown has been found to enhance the osteogenic differentiation of hBMSCs. This finding implies that NPTX1 could potentially serve as a therapeutic target for the treatment of osteogenic abnormalities, including osteoporosis.
Humans ; Mesenchymal Stem Cells/cytology* ; Osteogenesis/genetics* ; Cell Differentiation/genetics* ; Nerve Tissue Proteins/genetics* ; Cells, Cultured ; C-Reactive Protein/genetics* ; RNA, Small Interfering/genetics* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Bone Marrow Cells/cytology* ; Gene Knockdown Techniques ; Osteocalcin/metabolism* ; Alkaline Phosphatase/metabolism* ; RNA, Messenger/metabolism*

BACKGROUND: Congenital scoliosis (CS) is a complex spinal malformation of unknown etiology with abnormal bone metabolism. Fibroblast growth factor 23 (FGF23), secreted by osteoblasts and osteocytes, can inhibit bone formation and mineralization. This research aims to investigate the relationship between CS and FGF23. METHODS: We collected peripheral blood from two pairs of identical twins for methylation sequencing of the target region. FGF23 mRNA levels in the peripheral blood of CS patients and age-matched controls were measured. Receiver operator characteristic (ROC) curve analyses were conducted to evaluate the specificity and sensitivity of FGF23. The expression levels of FGF23 and its downstream factors fibroblast growth factor receptor 3 (FGFr3)/tissue non-specific alkaline phosphatase (TNAP)/osteopontin (OPN) in primary osteoblasts from CS patients (CS-Ob) and controls (CT-Ob) were detected. In addition, the osteogenic abilities of FGF23-knockdown or FGF23-overexpressing Ob were examined. RESULTS: DNA methylation of the FGF23 gene in CS patients was decreased compared to that of their identical twins, accompanied by increased mRNA levels. CS patients had increased peripheral blood FGF23 mRNA levels and decreased computed tomography (CT) values compared with controls. The FGF23 mRNA levels were negatively correlated with the CT value of the spine, and ROCs of FGF23 mRNA levels showed high sensitivity and specificity for CS. Additionally, significantly increased levels of FGF23, FGFr3, OPN, impaired osteogenic mineralization and lower TNAP levels were observed in CS-Ob. Moreover, FGF23 overexpression in CT-Ob increased FGFr3 and OPN levels and decreased TNAP levels, while FGF23 knockdown induced downregulation of FGFr3 and OPN but upregulation of TNAP in CS-Ob. Mineralization of CS-Ob was rescued after FGF23 knockdown. CONCLUSIONS Our results suggested increased peripheral blood FGF23 levels, decreased bone mineral density in CS patients, and a good predictive ability of CS by peripheral blood FGF23 levels. FGF23 may contribute to osteopenia in CS patients through FGFr3/TNAP / OPN pathway.
Humans ; Osteopontin/genetics* ; Alkaline Phosphatase/metabolism* ; Receptor, Fibroblast Growth Factor, Type 3/metabolism* ; Scoliosis/genetics* ; Osteoblasts/metabolism* ; Calcinosis ; RNA, Messenger/metabolism* ; Bone Diseases, Metabolic/metabolism* ; Fibroblast Growth Factors/genetics*

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.
Alkaline Phosphatase/metabolism* ; Animals ; Electromagnetic Fields ; Osteoblasts/metabolism* ; Osteogenesis/genetics* ; Rats ; TRPP Cation Channels/physiology*

OBJECTIVE: To explore effect of tobramycin (TOB) on healing of femoral fractures in rats. METHODS: Totally 32 male sprague-dawley (SD) rats were selected and randomly divided into sham group (group A), fracture group (group B), fracture with TOB group (group C) and fracture + TOB + IWR-1 group (group D), 8 rats in each group. Close femoral fracture model in rats were established in group B, C and D, group A was sham operation without otherwise process. Group D was intraperitoneal injected 100 μl (8 μM) of Wnt pathway inhibitor IWR-1-endo (IWR-1) before molding at 1 day. At 1 day after molding, 100 μl (100 μM) of TOB was intraperitoneally injected into group C and D at once a day for 7 days. At 7 weeks after modling, fracture healing of group B, C and D were observed by X-ray, Western blotting was appilied to detect alkaline phosphatase(ALP) and Runt related transcription factor 2 (RUNX2) and β-catenin of Wnt passway. RESULTS: X-ray results showed fracture line disappeared, callus formation and fracture healing well in group C compared with begning of molding; while a little fracture line, callus formation and fracture malunion in group B and d could be seen. Western blotting results showed ALP, RUNX2 and expression of β-catenin in group B, C and D were higher than that of group A ( CONCLUSION Tobramycin could promote osteoblast differentiation and fracture healing by stimulating Wnt / β-catenin signaling pathway, up regulating expression of ALP and RUNX2.
Alkaline Phosphatase ; Animals ; Cell Differentiation ; Core Binding Factor Alpha 1 Subunit/genetics* ; Femoral Fractures ; Fracture Healing ; Male ; Osteogenesis ; Rats ; Tobramycin ; Wnt Signaling Pathway ; beta Catenin/metabolism*

OBJECTIVEPrevious studies have clarified that calcitonin gene-related peptide (CGRP) can promote the biologi- cal activity of osteoblasts. To further reveal the role of CGRP in bone repair, we studied its influence on osteogenic differentia- tion of mouse bone marrow stromal cells (BMSCs) and initially explored the effect of the Hippo signaling pathway with this process.

METHODSBMSCs were induced to osteogenic differentiate osteoblasts by different concentrations of CGRP for a screening of the optimal concentration. CGRP was added in BMSCs, then the activity of alkaline phosphatase (ALP) and the number of mineralized nodules were examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days, respectively. The protein expression of p-Mst1/2 was measured by Western blot. Verteporfin was used to block the downstream Yap signaling. The mRNA expression of collagen type I (Col I) and runt-related transcription factor 2 (Runx2) were detected by reverse transcription-polymerase chain reaction.

RESULTSCompared to the blank group, different concentrations of CGRP (10⁻⁹, 10⁻⁸, 10⁻⁷ mol · L⁻¹), especially 10⁻⁸ mol · L⁻¹, significantly increased the ALP activity of BMSCs (P < 0.05). Alizarin red staining also showed more mineralized nodules in 10⁻⁸ mol · L⁻¹ group. The expression of p-Mst1/2 increased in the CGRP group (P < 0.05). Verteporfin treatment effectively decreased the mRNA expression of Runx2 and Col I (P < 0.05).

CONCLUSIONThe Hippo signaling pathway plays a role in CGRP-induced osteogenic differentiation in mouse BMSCs.

Alkaline Phosphatase ; Animals ; Calcitonin ; genetics ; metabolism ; Calcitonin Gene-Related Peptide ; metabolism ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; Core Binding Factor Alpha 1 Subunit ; Mesenchymal Stromal Cells ; physiology ; Mice ; Osteoblasts ; Osteogenesis ; physiology ; Signal Transduction

To study the genic and non-genic regulation of 50 Hz 0.6 mT pulsed electromagnenic fields (PEMF) on rat calvarial osteoblasts (ROB) differentiation.ROBs were achieved by enzyme digestion, and treated with 50 Hz 0.6 mT PEMFs for 1.5 hours after subculture. The alkaline phosphatase (ALP) activity, mRNA transcription of ALP, Runx2 and OSX and protein expression of Runx2 and OSX were detected at 0, 3, 6, 9 and 12 hours after PEMF treatment.The ALP activity at 3 hours after treatment was significantly higher than that in the control(<0.01), while the mRNA transcription of ALP began to increase at 6 hours after treatment. The mRNA transcription of Runx2 increased immediately after treatment and regressed at 6 hours, then increased again. The protein expression of it corresponded but with a little lag. The mRNA transcription of OSX also raised instantly after treatment, then returned to the level of control at 6 hours, and lower than control at 12 hours significantly. The protein expression of it also corresponded but with a bit delay.There are genic regulation for the protein expression of Runx2 and OSX, and non-genic regulation for the ALP activity on the process of 50 Hz 0.6 mT PEMFs prompts ROBs differentiation.
Alkaline Phosphatase ; metabolism ; radiation effects ; Animals ; Cell Differentiation ; genetics ; radiation effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; radiation effects ; Electromagnetic Fields ; Osteoblasts ; chemistry ; radiation effects ; Osteogenesis ; genetics ; radiation effects ; Rats ; Transcription Factors ; metabolism ; radiation effects

There is an increasing interest in phytoestrogens due to their potential medical usage in hormone replacement therapy (HRT). The present study was designed to investigate the in vitro effects of estrogen-like activities of two widespread coumarins, osthole and imperatorin, using the MCF-7 cell proliferation assay and their alkaline phosphatase (ALP) activities in osteoblasts Saos-2 cells. The two compounds were found to strongly stimulate the proliferation of MCF-7 cells. The estrogen receptor-regulated ERα, progesterone receptor (PR) and PS2 mRNA levels were increased by treatment with osthole and imperatorin. All these effects were significantly inhibited by the specific estrogen receptor antagonist ICI182, 780. Cell cycle analysis revealed that their proliferation stimulatory effect was associated with a marked increase in the number of MCF-7 cells in S phase, which was similar to that observed with estradiol. It was also observed that they significantly increased ALP activity, which was reversed by ICI182,780. These results suggested that osthole and imperatorin could stimulate osteoblastic activity by displaying estrogenic properties or through the ER pathway. In conclusion, osthole and imperatorin may represent new pharmacological tools for the treatment of osteoporosis.
Alkaline Phosphatase ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cnidium ; chemistry ; Coumarins ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Furocoumarins ; pharmacology ; Humans ; MCF-7 Cells ; Osteoblasts ; cytology ; drug effects ; enzymology ; Phytoestrogens ; pharmacology ; Receptors, Estrogen ; genetics ; metabolism

OBJECTIVETo evaluate the effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to explore the possible application on the alveolar bone regeneration.

METHODSTo determine the optimum concentration, the effects of ginsenoside Rg-1 ranging from 10 to 100 μmol/L were evaluated by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, alkaline phosphatase activity and calcium deposition. Expressions of runt-related transcription factor 2, collagen alpha-2(I) chain, osteopontin, osteocalcin protein were examined using real-time polymerase chain reaction.

RESULTSCompared with the control group, a certain concentration (10 μmol/L) of the Rg-1 solution significantly enhanced the proliferation and osteogenic differentiation of hPDLSCs (P<0.05). However, concentrations that exceeds 100 μmol/L led to cytotoxicity whereas concentrations below 10 nmol/L showed no significant effect as compared with the control.

CONCLUSIONGinsenoside Rg-1 can enhance the proliferation and osteogenic differentiation of hPDLSCs at an optimal concentration of 10 μmol/L.

Adolescent ; Alkaline Phosphatase ; metabolism ; Biomarkers ; metabolism ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Separation ; Cell Shape ; drug effects ; Cells, Cultured ; Flow Cytometry ; Ginsenosides ; pharmacology ; Humans ; Osteoblasts ; drug effects ; metabolism ; Osteogenesis ; drug effects ; genetics ; Periodontal Ligament ; cytology ; Real-Time Polymerase Chain Reaction ; Stem Cells ; cytology ; drug effects ; enzymology ; Time Factors ; Young Adult