The present study investigated the synergistic effect of co‑administering fibroblast growth factor‑2 (FGF‑2) and bone morphogenetic protein‑2 (BMP‑2) on osteoblastic differentiation in C2C12 cells and in rats. C2C12 murine myoblast cells represent a well‑accepted in vitro model system to study the ability of BMP‑2 to alter cell lineage from the myogenic to the osteogenic phenotype. The osteoblastic differentiation potency was determined by alkaline phosphatase (ALP) and Alizarin red S staining. ALP activity and calcium concentrations were colorimetrically measured. Simultaneous administration of 4 µg/ml recombinant human BMP‑2 with 2 ng/ml FGF‑2 markedly enhanced ALP activity (an early marker of osteogenesis) of C2C12 cells. This combination also increased extracellular signal‑regulated kinase1/2 mitogen activated protein kinase signaling that is involved in the promoting effect of FGF‑2 on BMP‑2‑induced osteoblastic differentiation in C2C12 cells. Calcium deposition (a late marker of osteogenesis) and the expression of CD34 (a marker of new vessels) were promoted optimally by simultaneous local sustained administration of FGF‑2 and BMP‑2 using collagen and chitosan‑coated antigen‑extracted porcine cancellous implants in a rat ectopic implantation model. The synergistic effects of a combination of BMP‑2 and FGF‑2 may have potential for bone regenerative therapeutics.

Fracture healing involves the coordinated actions of multiple cytokines. Bone morphogenetic protein 9 (BMP9) is an important factor in bone formation. The present study aimed to investigate the osteogenic potential of bone marrow stem cells (BMSCs) in response to adenoviral (Ad)BMP9, and the early fracture repair properties of AdBMP9 in surgically‑created fractures in osteoporotic rats. Alkaline phosphatase (ALP) activity was assayed and matrix mineralization was examined by Alizarin Red S staining. mRNA and protein expression levels of BMP9, runt‑related transcription factor 2 (RUNX2) and type 1 collagen (COL‑1) were detected in vitro and in vivo. Femoral bone mineral density was assessed for osteoporosis in ovariectomized rats. An open femora fracture was subsequently created, and gelatin sponges containing AdBMP9 were implanted. The femora were harvested for radiographical, micro‑computed tomography, biomechanical and histological analysis 4 weeks later. BMP9 successfully increased ALP activity and induced mineralized nodule formation in BMSCs. BMP9 in gelatin sponges demonstrated marked effects on microstructural parameters and the biomechanical strength of bone callus. In addition, it upregulated the expression levels of RUNX2 and COL‑1. AdBMP9 in gelatin sponges significantly mediated callus formation, and increased bone mass and strength in osteoporotic rats with femora fractures. The results of the present study suggested that BMP9 enhanced callus formation and maintained early mechanical stability during fracture healing in osteoporotic rats, implicating it as a potential novel therapeutic target for fracture healing.

Bone mesenchymal stem cells (BMSCs) have been an area of interest in biomedical research and tissue engineering due to their diverse differentiation abilities. In osteogenesis, bone morphogenetic proteins (BMPs), particularly BMP‑2, are important. However, the effect of BMP‑2 on the osteogenetic capacity of BMSCs remains to be fully elucidated. In the present study, primary rat BMSCs were infected with a recombinant lentivirus carrying the BMP‑2 gene (Lenti‑BMP‑2), and the effects of BMP‑2 on the activity of alkaline phosphatase (ALP) on days 3, 7, 14 and 21, and on mineralization on day 21 were evaluated. In addition, the adhesive ability of BMP‑2‑overexpressed BMSCs was detected using an adhesion assay. Following forced expression of BMP‑2 in the BMSCs, the levels of osteogenic genes, including osteopontin (OPN), osteocalcin (OC) and collagen type I (Col‑Ⅰ), were detected and the nuclear accumulation of Runt‑related transcription factor (Runx)‑2 and phosphorylated small mothers against decapentaplegic (p‑Smad) 1/5/8 was also evaluated. The results demonstrated that the rat BMSCs had been isolated, cultured and passaged from Sprague‑Dawley rat bone marrow successfully, and the third‑generation BMSCs were identified using flow cytometry with CD29 staining. The osteogenetic phenotype of the BMSCs, expressing ALP and osteocalcin, was significantly induced by BMP‑2, and the proliferation of the BMSCs was enhanced by BMP‑2. Furthermore, the adhesive potential of the BMP‑2‑overexpressed BMSCs was increased, the expression levels of OPN, OCN and Col‑Ⅰe osteogenetic factors were upregulated and the nuclear accumulation of Runx‑2 and p‑Smads1/5/8 were increased significantly. These data suggested that BMP‑2 may facilitate the osteogenetic differentiation of rat BMSCs and provide a favorable cell resource for tissue engineering.

Bone morphogenetic protein 9 (BMP9) belongs to the transforming growth factor‑β (TGF‑β) superfamily, and has been reported to promote cancer cell proliferation and epithelial‑mesenchymal transition (EMT). Cisplatin (DDP) is the first line treatment for ovarian cancer. However, initiation of EMT confers insensitivity to chemotherapy. The present study aimed to verify and examine the mechanisms underlying the effects of BMP9 on treatment with DDP for ovarian cancer. Prior to treatment with DDP, ovarian cancer cells were exposed to BMP9 for 3 days. Following this, cell viability, apoptosis rate and the extent of DNA damage were evaluated to compare the effects of DDP on BMP9‑pretreated and non‑pretreated ovarian cancer cells. In addition, EMT marker expression was evaluated by western blotting and immunofluorescence. The results demonstrated that BMP9 pretreatment inhibited the cytotoxicity of DDP on ovarian cancer cells. Additionally, BMP9‑pretreated ovarian cancer cells had downregulated expression of the epithelial marker E‑cadherin, which was accompanied by an upregulation of the mesenchymal markers N‑cadherin, Snail, Slug, and Twist. Taken together, the findings of the present study indicated that BMP9 conferred resistance to DDP in ovarian cancer cells by inducing EMT. The present study provided valuable insight into the mechanisms of chemotherapy in ovarian cancer and highlighted the potential of BMP9 as a novel therapeutic target for improving cisplatin chemosensitivity.

Mesenchymal stem cells (MSCs) are used to investigate regeneration and differentiation. MicroRNA‑204 (miR‑204) in involved in the Runt‑related transcription factor 2/alkaline phosphatase/bone morphogenic protein 2 (Runx2/ALP/BMP2) signaling pathway that regulates bone marrow mesenchymal stem cell (BMSC) differentiation; however, the mechanisms underlying the effects of miR‑204 are yet to be determined. The aim of the present study was to investigate the effects of miR‑204 on BMSC differentiation. BMSCs were derived from rat bone marrow. The expression levels of Runx2, ALP and BMP2 were measured via reverse transcription‑quantitative polymerase chain reaction and western blot analyses following transfection of BMSCs with miR‑204 agomir or BMP2 expression vector. The ability of the miR‑204 gene to directly bind BMP2 mRNA was assessed using dual‑luciferase assays. Ossification was measured via alizarin red stain assays. It was observed that the expression levels of Runx2 and ALP increased over time, whereas those of miR‑204 decreased; additionally, miR‑204 agomir upregulation inhibited the expression of Runx2, ALP and BMP2 in BMSCs. It was revealed that miR‑204 directly interacted with BMP2 mRNA, and that transfection with miR‑204 agomir suppressed ossification in BMSCs by targeting the BMP2/Runx2/ALP signaling pathway.

The present study explored whether bone morphogenetic proteins (BMPs) and Wnt/β‑catenin signaling pathways were involved in the 1,25(OH)2D3‑induced inhibition of osteogenic differentiation in bone marrow‑derived mesenchymal stem cells (BMSCs). To evaluate the osteogenic differentiation of BMSCs, the expression levels of ossification markers, including BMP2, Runt‑related transcription factor 2 (Runx2), Msh homeobox 2 (Msx2), osteopontin (OPN) and osteocalcin (OCN), and the activity of alkaline phosphatase (ALP), as well as the calcified area observed by Alizarin red‑S staining, were investigated. Chromatin immunoprecipitation (ChIP) assay was used to detect the effect of 1,25(OH)2D3 on the DNA methylation and histone modification of BMP2, while an immunoprecipitation (IP) assay was performed to assess the crosstalk between Smad1 and disheveled‑1 (Dvl‑1) proteins. It was observed that 1,25(OH)2D3 significantly decreased the expression levels of BMP2, Runx2, Msx2, OPN and OCN, and reduced ALP activity and the calcified area in BMSCs, whereas these effects were rescued by BMP2 overexpression. ChIP assay revealed that BMSCs treated with 1,25(OH)2D3 exhibited a significant increase in H3K9me2 level and a decrease in the acetylation of histone H3 at the same BMP2 promoter region. In addition, 1,25(OH)2D3 treatment promoted the nuclear accumulation of β‑catenin by downregulating BMP2. Furthermore, the β‑catenin signaling inhibitor XAV‑939 weakened the inhibitory effect of 1,25(OH)2D3 on osteogenic differentiation. Additionally, knockdown of β‑catenin rescued the attenuation in Dvl‑1 and Smad1 interaction caused by 1,25(OH)2D3. Overexpression of Smad1 also reversed the inhibitory effect of 1,25(OH)2D3 on osteogenic differentiation. Taken together, the current study demonstrated that 1,25(OH)2D3 inhibited the differentiation of BMSCs into osteoblast‑like cells by inactivating BMP2 and activating Wnt/β‑catenin signaling.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor superfamily and have been implicated in chondrogenesis and neuronal differentiation. In order to examine the function of bone morphogenetic protein 2 (BMP‑2) on the differentiation of nitrergic enteric neurons in slow transit constipation (STC), the expression of BMP‑2 and neuronal nitric oxide synthase (nNOS) was investigated in the myenteric nerve plexus in STC and control tissues by immunohistochemical assays. The present study demonstrated that BMP‑2 and nNOS were expressed in the myenteric nerve plexus and their levels were differentially altered in the STC group and control group. In addition, the effect of BMP‑2 on primary myenteric neurons was investigated by measuring the neurite length. The results demonstrated that BMP‑2 regulated the differentiation of primary enteric neurons and increased the length of neurites compared with the control group. In addition, the effect of BMP‑2 on the expression of nNOS was also investigated in primary enteric neurons and the Smad1 signal transduction pathway by western blot analysis, reverse transcription quantitative polymerase chain reaction and immunofluorescence assay. The results suggested that BMP‑2 promoted the expression of nNOS in primary myenteric neurons and induced phosphorylation of Smad1. These data indicate a new role for BMP‑2 as an important transcriptional cofactor that regulates the differentiation of nitrergic enteric neurons through the Smad1 pathway. Intervention of BMP‑2 may be useful for the treatment of STC.

Cytokines and their intercellular signals regulate the multipotency of mesenchymal stem cells (MSCs). The present study established the MSC lines SG‑2, ‑3, and ‑5 from the bone marrow of green fluorescent protein (GFP)‑transgenic mice. These cell lines clearly expressed mouse MSC markers Sca‑1 and CD44, and SG‑2 and ‑5 cells retained the potential for osteogenic and adipogenic differentiation in the absence of members of the transforming growth factor (TGF)‑β superfamily. By contrast, SG‑3 cells only retained adipogenic differentiation potential. Analysis of cytokine and cytokine receptor expression in these SG cell lines showed that bone morphogenetic protein (BMP) receptor 1B was most highly expressed in the SG‑3 cells, which underwent osteogenesis in response to BMP, while TGF‑β receptor II was most highly expressed in SG‑3 and ‑5 cells. However, it was unexpectedly noted that phosphorylation of Smad 2, a major transcription factor, was induced by TGF‑β1 in SG‑2 cells but not in SG‑3 or ‑5 cells. Furthermore, TGF‑β1 clearly induced the expression of Smad‑interacting transcription factor CCAAT/enhancer binding protein‑β in SG‑2 but not in SG‑3 or ‑5 cells. These results demonstrated the establishment of TGF‑β‑responsive SG‑2 MSCs, BMP‑responsive SG‑3 MSCs and TGF‑β/BMP‑unresponsive SG‑5 MSCs, each of which was able to be traced by GFP fluorescence after transplantation into in vivo experimental models. In conclusion, the present study suggested that these cell lines may be used to explore how the TGF‑β superfamily affects the proliferation and differentiation status of MSCs in vivo.

The aim of the present study was to investigate the expression and function mechanism of bone morphogenetic protein 9 (BMP9) in cerebral ischemia‑reperfusion (I/R) injuries in vivo and in vitro. A total of 40 Sprague‑Dawley rats were randomly divided into four groups (n=10): i) Normal control; ii) sham surgery group, the procedure without occlusion; iii) I/R group, right middle cerebral artery occlusion (MCAO) followed by reperfusion; and iv) adenoviral vector (Ad)‑BMP9 + I/R group, Ad‑BMP9 intracerebroventricular injection was performed 2 days prior to MCAO. Neurological deficit score and infarct volume were measured at 24 h following reperfusion. To further test the mechanism of BMP9, astrocytes were isolated and treated with Ad‑BMP9, Ad‑BMP9 + extracellular signal‑regulated kinase (ERK) inhibitor PD098059, Ad‑BMP9 + c‑Jun N‑terminal kinase inhibitor SP600125 and Ad‑BMP9 + p38 inhibitor SB203580 for 24 h, followed by undergoing oxygen‑glucose deprivation and reoxygenation (OGD/R) treatment. Cell viability and death were assessed by 3‑(4,5‑dimethylthiazol‑2yl)‑5‑(3‑carboxymethoxyphenyl)‑(4‑sulfophenyl)‑2H‑tetrazolium and lactate dehydrogenase release, respectively. Gene expression was determined by quantitative polymerase chain reaction and western blotting. BMP9 was identified to be upregulated at mRNA and protein levels in cerebral I/R animal and cell models. BMP9 pretreatment significantly reduced the neurological score and infarct volume compared with I/R rats. In astrocytes, overexpression of BMP9 significantly decreased cell death and improved cell viability, an effect which may be mediated by the ERK signaling pathway, as ERK was activated by BMP9 and the use of PD098059 partially reversed the protective effect of BMP9. Pretreatment with BMP‑9 may be a promising treatment option for prevention of cerebral I/R injuries.

At present, the molecular mechanism underlying the protective effect of Ginsenoside Rb1 remains unclear. The present study was designed to investigate whether Ginsenoside Rb1 weakened the steroid‑induced avascular necrosis of the femoral head (SANFH) and to explore the possible mechanisms of the above effects. As a result, it was revealed that Ginsenoside Rb1 was protective against steroid‑induced avascular necrosis and inhibited serum osteocalcin in a rat model of SANFH. Ginsenoside Rb1 reduced inflammation, oxidative stress and bone cell apoptosis in a rat model of SANFH. Furthermore, Ginsenoside Rb1 attenuated trabecula parameters, total cholesterol and low density lipoprotein/high density lipoprotein in SANFH rat. Additionally, Ginsenoside Rb1 significantly reversed alkaline phosphatase and osteocalcin activities, vascular endothelial growth factor (VEGF) receptor, VEGF, Runt related transcription factor 2 (Runx2) and bone morphogenetic protein (BMP)‑2 protein expression in SANFH rat. Collectively, the present study demonstrated that Ginsenoside Rb1 attenuated SANFH through the VEGF/RUNX2/BMP‑2 signaling pathway.

It has been confirmed that bone morphogenetic protein-9 (BMP-9) promotes the differentiation of osteoblasts. However, the ways in which BMP‑9 exerts its effects on the differentiation of osteoclasts and bone resorption remain to be elucidated. The present study was designed to investigate the roles and the molecular mechanism of BMP‑9 on the proliferation and differentiation of osteoclast precursors in vitro. Mouse spleen macrophages (RAW 264.7 cells) were cultured in the presence of receptor activator for nuclear factor‑κb ligand (RANKL) in vitro. Following treatment with different concentrations of BMP‑9, a number of parameters were quantitatively monitored. Cell proliferation was determined using an MTT assay. The expression levels of cell BMP receptor‑IA (BMPR‑IA), BMPR‑IB, BMPR‑II and anaplastic lymphoma kinase 1 (ALK1) receptor were detected by ELISA, the small mothers against decapentaplegic pathway, extracellular signal‑regulated kinase (ERK)1/2 pathways and markers of osteoclast differentiation were detected by western blotting. The results showed that treatment with BMP‑9 alone promoted mouse spleen macrophage proliferation, and the differentiation into osteoclasts occurred only in the presence of RANK. The promoting effect of BMP‑9 on cell proliferation and osteoclast differentiation occurred in dose‑dependent manner. In addition, BMP‑9 significantly upregulated the expression of the ALK1 receptor and inhibited the ERK1/2 pathway. The inhibition of the ERK1/2 pathways was ameliorated by transfection with small interfering (si)RNA ALK1. The effect of BMP‑9 on osteoclast differentiation was reduced by transfection with siRNA ALK1, however, the effect was enhanced by the ERK1/2 pathway inhibitor, U0126. The results of the present study demonstrated that BMP‑9 promoted the osteoclast differentiation of osteoclast precursors via binding to the ALK1 receptor on the cell surface, and inhibiting the ERK1/2 signaling pathways in the cell.

Scarring is the inevitable consequence of wound repair, which may cause significant physical and mental pain to patients when excessive. Bone morphogenetic protein‑7 (BMP‑7) has been proved to inhibit TGF‑β‑induced fibrosis in various tissues including dermal papilla cells. However, the effect of BMP‑7 on hypertrophic scarring, a common proliferative disorder of dermal fibroblasts, has not been determined. To overcome this problem, the present study established a mouse model of thermal injury to investigate the inhibitory effects of BMP‑7 on scar formation. The histological analysis of scar tissues was performed by H&E and Masson's trichrome staining. Western blot assay was used to determine the level changes of related proteins and TUNEL assay was performed to assess the apoptosis of scar tissues. The results demonstrated that BMP‑7 promoted wound healing and inhibited scar formation when compared with untreated mice. Collagen deposition and the expression of fibrotic proteins were suppressed in the scar tissues of mice treated with BMP‑7. In addition, BMP‑7 induced fibroblast apoptosis in scar tissues. Furthermore, activation of the BMP‑7/Smad1/5/8 signaling pathway may have been involved in the inhibitory effects of BMP‑7 on scar formation. In conclusion, the results of the present study indicate that BMP‑7 may inhibit excessive scar formation via activation of the BMP‑7/Smad1/5/8 signaling pathway. The results present a potential alternative therapeutic strategy for the treatment of hypertrophic scarring.

Estradiol (E2) is a first‑line drug for osteoporosis (OP) treatment via promotion of osteoblastic proliferation and differentiation. However, a long‑term use of E2 would produce side effects thus, it is imperative to discover safer and more effective drugs. Pinoresinol (PINO) has a similar chemical structure to E2. The present study aimed to investigate whether PINO could promote osteoblastic proliferation and differentiation and the potential mechanisms. After treatment with 0.1 µg/l PINO for 2 days, MC3T3‑E1 cell migration was assessed by wound healing assay. Estrogen (E2) treatment served as a positive control. RT‑qPCR and western blotting were used for mRNA and protein expression analyses. Alkaline phosphatase (ALP) activity assay and Alizarin red staining were performed to investigate the calcification and mineralization, and the cyclic AMP (cAMP) level was detected by enzyme‑linked immunosorbent assay (ELISA). H89, an inhibitor of protein kinase A (PKA), was introduced to verify the role of cAMP/PKA in the effect of PINO on MC3T3‑E1 cells. Cell viability was the highest under 48 h of 0.1 µg/l PINO treatment. After treatment with PINO, a significant increase was observed in the migration rate and the expression of collagen type I (Col‑I), ALP, osteopontin (OPN), runt‑related transcription factor 2 (Runx2) and bone morphogenetic protein‑2 (BMP‑2) (P<0.01). The ALP activity and Alizarin red size in PINO and E2 groups were notably increased. The increased cAMP, PKA and phosphorylated cAMP response element‑binding protein (CREB) levels were also observed in the PINO group. Furthermore, H89 co‑treatment abolished the positive effects of PINO on cell viability and migration. PINO had similar effects to E2 on the osteoblastic proliferation and differentiation, and these positive effects may be attributed to the regulation of the cAMP/PKA signaling pathway.

Reports of the ameliorative effect of angelicin on sex hormone deficiency‑induced osteoporosis have highlighted this compound as a candidate for the treatment of osteoporosis. However, the molecular mechanisms of action of angelicin on osteoblast differentiation have not been thoroughly researched. The aim of the present study was to evaluate the effect of angelicin on the proliferation, differentiation and mineralization of rat calvarial osteoblasts using a Cell Counting Kit‑8, alkaline phosphatase activity and the expression of osteogenic genes and proteins. Treatment with angelicin promoted the proliferation, matrix mineralization and upregulation of osteogenic marker genes including collagen type I α 1 and bone γ‑carboxyglutamate in fetal rat calvarial osteoblasts. Furthermore, angelicin promoted the expression of β‑catenin and runt related transcription factor 2, which serve a vital role in the Wnt/β‑catenin signaling pathway. Consistently, the osteogenic effect of angelicin was attenuated by the use of a Wnt inhibitor. Moreover, angelicin increased the expression of estrogen receptor α (ERα), which also serves a key role in osteoblast differentiation. Taken together, these results demonstrated that angelicin may promote osteoblast differentiation through activation of ERα and the Wnt/β‑catenin signaling pathway.

Aucubin represents an iridoid glucoside separated from multiple Chinese herbs, which has been demonstrated to possess numerous pharmacological activities. In the present study, the aim was to investigate the roles and mechanisms of aucubin in the suppression of mouse MC3T3‑E1 osteoblast apoptosis induced by Titanium particles and the promotion of bone formation. MTT assay and flow cytometry were performed to analyze cell viability and apoptosis, respectively. ELISA and para‑nitrophenyl phosphate colorimetry were carried out to evaluate the oxidative stress markers and alkaline phosphatase (ALP). Western blotting and reverse transcription‑quantitative polymerase chain reaction assays were used to evaluate the associated mRNA and protein expression. The results revealed that aucubin enhanced the cell activity of MC3T3‑E1 cells treated with Ti particles. Aucubin suppressed the apoptosis of Ti particles‑induced MC3T3‑E1 cells and facilitated osteogenesis by affecting the B‑cell lymphoma‑2 (Bcl‑2), Bcl‑2 associated X protein, ALP and associated osteogenic factors expression. Aucubin reduced the oxidative stress in Ti particles‑induced MC3T3‑E1 cells. In addition, aucubin upregulated the bone morphogenetic protein 2 (BMP2)/Smads/runt related transcription factor 2 (RunX2) pathway in Ti particles‑induced MC3T3‑E1 cells. In conclusion, the present study confirmed that aucubin suppressed the Ti particles‑mediated apoptosis of MC3T3‑E1 cells and facilitated osteogenesis by affecting the BMP2/Smads/RunX2 signaling pathway.

The differentiation of pluripotent mesenchymal stem cells to mature osteoblasts is crucial for the maintenance of the adult skeleton. In rheumatic arthritis, osteoblast differentiation is impaired by the overproduction of cytokine tumor necrosis factor (TNF)‑α. It has been demonstrated that TNF-α is able to inhibit osteoblast differentiation through the activation of nuclear factor (NF)-κB signaling. As a result of the critical role of TNF-α and NF-κB in the pathogenesis of bone-loss associated diseases, these factors are regarded as key targets for the development of therapeutic agents. In the current study, the role of the NF-κB inhibitor gliotoxin (GTX) in the regulation of osteoblast differentiation was evaluated. The non-toxic GTX doses were determined to be ≤ 3 µg/ml. It was revealed that GTX was able to block TNF-α-induced inhibition of osteoblast differentiation, as indicated by alkaline phosphatase (ALP) activity and ALP staining assays, as well as the expression levels of osteoblast-associated genes Col I, Ocn, Bsp, Runx2, Osx and ATF4. Additionally, it was identified that gliotoxin directly promoted bone morphogenetic protein-2-induced osteoblast differentiation. GTX was found to inhibit the accumulation of NF-κB protein p65 in the nucleus and reduce NF-κB transcriptional activity, suggesting that GTX potentiated osteoblast differentiation via the suppression of NF-κB signaling.

Lonicera japonica has been used in traditional Chinese medicine as an important medicinal plant, with the ability to inhibit osteoclast development and bone loss. However, it is not clear which active ingredient exerts these effects. (R)‑dehydroxyabscisic alcohol β‑D‑apiofuranosyl‑(1ˮ→6')‑β‑D‑glucopyranoside (DAG) is an active constituent isolated from Lonicera japonica. In the present study, the ST2 bone marrow stromal cell line was treated by DAG at different concentrations and the osteoblastic differentiation was explored by ELISA assay, Von Kossa staining, Alizarin Red S staining, reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The results revealed that DAG promoted osteoblastic differentiation, as evidenced by increasing mineralization and alkaline phosphatase (ALP) activity, as well as the expression of genes encoding bone differentiation markers, including Alp, osteopontin (Opn) and osteocalcin (Ocn). In addition, DAG upregulated the gene expression of bone morphogenetic protein (Bmp)‑2, Bmp4, Wnt family member (Wnt)‑1, Wnt3 and runt‑related transcription factor 2 (Runx2), as well as the protein expression of phosphorylated‑mothers against decapentaplegic homolog (Smad) 1, Smad5 Smad8, β‑catenin and Runx2 in ST2 cells. The osteogenic effects induced by DAG were attenuated by the BMP antagonist Noggin and the WNT signaling pathway inhibitor Dickkopf related protein‑1. The data indicated that DAG promoted the osteoblastic differentiation of ST2 cells, at least partially through regulating the BMP/WNT signaling pathways. This provides scientific rationale for the development of DAG as a treatment for bone loss‑associated diseases, such as osteoporosis.

Osteoporosis is a bone‑related disease that results from impaired bone formation and excessive bone resorption. The potential value of adipokines has been investigated previously, due to their influence on osteogenesis. However, the osteogenic effects induced by omentin‑1 remain unclear. The aim of the present study was to determine the regulatory effects of omentin‑1 on osteoblast viability and differentiation, as well as to explore the underlying molecular mechanism. The present study investigated the effects of omentin‑1 on the viability and differentiation of mouse pre‑osteoblast cells (MC3T3‑E1) using quantitative and qualitative measures. A Cell Counting Kit‑8 assay was used to assess the viability of MC3T3‑E1 cells following treatment with different doses of omentin‑1. Omentin‑1 and bone morphogenetic protein (BMP) inhibitor were added to osteogenic induction mediums in different ways to assess their effect. The alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining of MC3T3‑E1 cells treated with omentin‑1 and/or BMP inhibitor were used to examine the effects of omentin‑1 on differentiation and mineralization. Western blotting was used to further explore its potential mechanism, and to study the role of omentin‑1 on the viability and differentiation of osteoblasts. The results showed that omentin‑1 altered the viability of MC3T3‑E1 cells in a dose‑dependent manner. Omentin‑1 treatment significantly increased the expression of members of the TGF‑β/Smad signaling pathway. In the omentin‑1 group, the ALP activity of the MC3T3‑E1 cells was increased, and the ARS staining area was also increased. The mRNA and protein expression levels of BMP2, Runt‑related transcription factor 2, collagen1, osteopontin, osteocalcin and osterix in the omentin‑1 group were also significantly upregulated. All these effects were reversed following treatment with SIS3 HCl. These results demonstrated that omentin‑1 can significantly promote osteoblast viability and differentiation via the TGF‑β/Smad signaling pathway, thereby promoting bone formation and preventing osteoporosis.

Our previous study demonstrated that febuxostat, a xanthine oxidase inhibitor, can alleviate kidney dysfunction and ameliorate renal tubulointerstitial fibrosis in a rat unilateral ureteral obstruction (UUO) model; however, the underlying mechanisms remain unknown. Increasing evidence has revealed that epithelial‑mesenchymal transition (EMT) is one of the key mechanisms mediating the progression of renal tubulointerstitial fibrosis in chronic kidney disease (CKD). Uterine sensitization‑associated gene‑1 (USAG‑1), a kidney‑specific bone morphogenetic protein antagonist, is involved in the development of numerous types of CKDs. The present study aimed to investigate the role of febuxostat in the process of EMT in Madin‑Darby canine kidney (MDCK) cells in vitro. Western blotting, reverse transcription‑semiquantitative polymerase chain reaction analysis and immunofluorescence staining were used to evaluate the expression levels of bone morphogenetic protein 7, USAG‑1, α‑smooth muscle actin (α‑SMA) and E‑cadherin, respectively. The results demonstrated that the expression of USAG‑1 and α‑SMA increased, and that of E‑cadherin decreased significantly in MDCK cells following treatment with transforming growth factor‑β1 (TGF‑β1). The application of small interfering RNA‑USAG‑1 potently inhibited TGF‑β1‑induced EMT. Subsequently, the effects of febuxostat on TGF‑β1‑induced EMT was investigated. The results demonstrated that febuxostat downregulated the expression of USAG‑1, and reversed TGF‑β1‑induced EMT in MDCK cells. Furthermore, pretreatment with febuxostat significantly restored the decreased expression levels of phosphorylated Smad1/5/8 induced by TGF‑β1 in MDCK cells. The results of the present study suggested that USAG‑1 may be involved in the EMT process of MDCK cells induced by TGF‑β1, and febuxostat inhibited EMT by activating the Smad1/5/8 signaling pathway via downregulating the expression of USAG‑1 in MDCK cells.

Osteoporosis is an aging process of skeletal tissues with characteristics of reductions in bone mass and microarchitectural deterioration of bone tissue. The present study aimed to investigate the effects of glucocorticoid‑induced osteoporosis on osteoblasts and to examine the roles of β‑ecdysterone (β‑Ecd) involved. In the present study, an in vivo model of osteoporosis was established through the subcutaneous implantation of prednisolone (PRED) into Sprague‑Dawley rats, with or without a subcutaneous injection of β‑Ecd (5 or 10 mg/kg body weight). Expression of Beclin‑1 and microtubule‑associated protein 1A/1B‑light chain 3I/II and apoptosis in lumbar vertebrae tissues was measured by immunofluorescence and TUNEL assays, respectively. Serum concentration of calcium and phosphorus, and the activity of tartrate‑resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) were measured by biochemical assay. Reverse transcription‑quantitative polymerase chain reaction and western blotting was used for detect the expression of related genes and proteins. PRED treatment inhibited bone formation by decreasing bone mineral density, and suppressing the expression of Runt‑related transcription factor 2 and bone morphogenetic protein 2, while enhancing the activity of alkaline phosphatase, upregulating the expression of receptor activator of nuclear factor-κB ligand, and increasing the serum content of calcium, phosphorus and tartrate‑resistant acid phosphatase in rats. Additionally, PRED was revealed to inhibit autophagy through the downregulation of Beclin‑1, autophagy protein 5 and microtubule‑associated protein 1A/1B‑light chain 3I/II expression, whereas it induced the apoptosis, through the activation of caspase‑3 and the suppression of apoptosis regulator BCL2 expression. Notably, the PRED‑induced alterations in bone formation, autophagy and apoptosis were revealed to be attenuated by β‑Ecd administration. In conclusion, the findings of the present study suggested that β‑Ecd may be a promising candidate for the development of therapeutic strategies for the treatment of osteoporosis, through the induction of autophagy and the inhibition of apoptosis in vivo.