OBJECTIVE: To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). METHODS: Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ). RESULTS: Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area. CONCLUSION After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.
Female ; Mice ; Animals ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; PPAR gamma/metabolism* ; Steroid 12-alpha-Hydroxylase/metabolism* ; Mice, Inbred C57BL ; Cell Differentiation ; Osteogenesis ; Mesenchymal Stem Cells ; Bile Acids and Salts/pharmacology* ; Bone Marrow Cells ; Cells, Cultured ; Azo Compounds

This study aimed to investigate the effects of Erxian Decoction(EXD)-containing serum on the proliferation and osteogenic differentiation of MC3T3-E1 cells under oxidative stress through BK channels. The oxidative stress model was induced in MC3T3-E1 cells by H_2O_2, and 3 mmol·L~(-1) tetraethylammonium(TEA) chloride was used to block the BK channels in MC3T3-E1 cells. MC3T3-E1 cells were divided into a control group, a model group, an EXD group, a TEA group, and a TEA+EXD group. After MC3T3-E1 cells were treated with corresponding drugs for 2 days, 700 μmol·L~(-1) H_2O_2 was added for treatment for another 2 hours. CCK-8 assay was used to detect cell proliferation activity. The alkaline phosphatase(ALP) assay kit was used to detect the ALP activity of cells. Western blot and real-time fluorescence-based quantitative PCR(RT-qPCR) were used to detect protein and mRNA expression, respectively. Alizarin red staining was used to detect the mineralization area of osteoblasts. The results showed that compared with the control group, the model group showed significantly blunted cell proliferation activity and ALP activity, reduced expression of BK channel α subunit(BKα), collagen Ⅰ(COL1), bone morphogenetic protein 2(BMP2), osteoprotegerin(OPG), and phosphorylated Akt, decreased mRNA expression levels of Runt-related transcription factor 2(RUNX2), BMP2, and OPG, and declining area of calcium nodules. EXD-containing serum could significantly potentiate the cell proliferation activity and ALP activity, up-regulate the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt, and forkhead box protein O1(FoxO1), promote the mRNA expression of RUNX2, BMP2, and OPG, and enlarge the area of calcium nodules. However, BK channel blockage by TEA reversed the effects of EXD-containing serum in promoting the protein expression of BKα, COL1, BMP2, OPG, and phosphorylated Akt and FoxO1, increasing the mRNA expression of RUNX2, BMP2, and OPG, and enlarging the area of calcium nodules. EXD-containing serum could improve the proliferation activity, osteogenic differentiation, and mineralization ability of MC3T3-E1 cells under oxidative stress, which might be related to the regulation of BK channels and downstream Akt/FoxO1 signaling pathway.
Osteogenesis ; Core Binding Factor Alpha 1 Subunit/pharmacology* ; Large-Conductance Calcium-Activated Potassium Channels/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Calcium/metabolism* ; Cell Differentiation ; RNA, Messenger/metabolism* ; Cell Proliferation ; Osteoblasts

OBJECTIVES: To investigate the effect of recombinant human fibroblast growth factor 21 (rhFGF21) on the proliferation and mineralization of cementoblasts and its mechanism. METHODS: Hematoxylin eosin, immunohistochemical staining, and immunofluorescence were used to detect the expression and distribution of fibroblast growth factor 21 (FGF21) in rat periodontal tissues and cementoblasts (OCCM-30), separately. Cell Counting Kit-8 was used to detect the proliferation of OCCM-30 under treatment with rhFGF21. Alkaline phosphatase staining and Alizarin Red staining were used to detect the mineralization state of OCCM-30 after 3 and 7 days of mineralization induction. The transcription and protein expression of the osteogenic-related genes Runx2 and Osterix were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot analysis. The expression levels of genes of transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling pathway in OCCM-30 were detected through PCR array analysis. RESULTS: FGF21 was expressed in rat periodontal tissues and OCCM-30. Although rhFGF21 had no significant effect on the proliferation of OCCM-30, treatment with 50 ng/mL rhFGF21 could promote the mineralization of OCCM-30 cells after 7 days of mineralization induction. The transcriptional levels of Runx2 and Osterix increased significantly at 3 days of mineralization induction and decreased at 5 days of mineralization induction. Western blot analysis showed that the protein expression levels of Runx2 and Osterix increased during mineralization induction. rhFGF21 up-regulated Bmpr1b protein expression in cells. CONCLUSIONS rhFGF21 can promote the mineralization ability of OCCM-30. This effect is related to the activation of the TGFβ/BMP signaling pathway.
Humans ; Rats ; Animals ; Dental Cementum ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cell Differentiation ; Bone Morphogenetic Proteins/metabolism* ; Transforming Growth Factor beta/pharmacology*

OBJECTIVE: To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). METHODS: The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. RESULTS: The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05). CONCLUSION Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.
Animals ; Female ; Mice ; Cell Differentiation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cyclic AMP Response Element-Binding Protein/metabolism* ; Mesenchymal Stem Cells ; Mice, Inbred C57BL ; MicroRNAs/metabolism* ; Osteocalcin/metabolism* ; Osteogenesis/genetics* ; RNA, Messenger/genetics*

OBJECTIVE: To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect. METHODS: Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining. RESULTS: The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05). CONCLUSION Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.
Animals ; Calcium Carbonate/metabolism* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Exosomes/metabolism* ; Humans ; Male ; Osteogenesis/genetics* ; RNA, Messenger/metabolism* ; Rabbits

OBJECTIVE: To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC). METHODS: Mouse models of VC were established in ApoE-deficient (ApoE^-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with N^ε-carboxymethyl-lysine for 16 weeks. ApoE^-/- mice (control group), ApoE^-/- diabetic mice (VC group), ApoE^-/- diabetic mice with BI-1 overexpression (VC + BI-1^TG group), and ApoE^-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1^TG+OPA1^-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L β-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining. RESULTS: ApoE^-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007). CONCLUSION BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.
Animals ; Apolipoproteins E/metabolism* ; Calcium/metabolism* ; Caspase 3/metabolism* ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Diabetes Mellitus, Experimental/pathology* ; GTP Phosphohydrolases/metabolism* ; Membrane Proteins/metabolism* ; Mice ; Mice, Knockout ; Muscle, Smooth, Vascular/pathology* ; Myocytes, Smooth Muscle/pathology* ; Optic Atrophy, Autosomal Dominant/pathology* ; Osteogenesis ; Vascular Calcification/pathology* ; bcl-2-Associated X Protein/metabolism*

In this study, the secondary osteoporosis model was induced by oral administration of retinoic acid for two weeks in SD male rats. The efficacy and mechanism of LG on secondary osteoporosis in rats were explored through the bone morphogenetic protein 2(BMP-2)/Runt-related transcription factor 2(Runx2)/Osterix signaling pathway. With Xianling Gubao Capsules(XLGB) as the positive control, three dose groups of low glycoside from Epimedii Folium flavonoids(LG), i.e., low-dose group(LG-L), medium-dose group(LG-M), and high-dose group(LG-H), were set up. After modeling, the rats in each group were treated correspondingly by gavage for eight weeks. The action target of LG in the treatment of secondary osteoporosis in rats was analyzed by measuring the body weight and the organ indexes of rats including heart index and testis index. The efficacy of LG was characterized by the pathological changes of the femur, the microstructural parameters of the trabecular bone, and the biomechanical properties of femoral tissues in rats. The mechanism of LG was explored by measuring the relevant biochemical indexes and the changes in BMP-2, Runx2, and Osterix content in rats with secondary osteoporosis. The results showed that the action target of LG in the treatment of secondary osteoporosis in rats was the testis. LG can improve the bone loss of the femur, increase the number and thickness of the trabecular bone, reduce the porosity and separation of the trabecular bone, potentiate the resistance of bone to deformation and destruction, up-regulate the serum content of Ca, P, aminoterminal propeptide of type Ⅰ procollagen(PINP), and osteocalcin(OC), promote bone matrix calcification and the expression of BMP-2, Runx2, and Osterix proteins, and accelerate bone formation, thereby reducing the risk of fractures, and ultimately exerting anti-secondary osteoporosis efficacy.
Animals ; Bone Density ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Drugs, Chinese Herbal ; Flavonoids/therapeutic use* ; Glycosides/therapeutic use* ; Male ; Osteoporosis/metabolism* ; Rats ; Rats, Sprague-Dawley ; Tretinoin/adverse effects*

This study compared the effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus healthy obesity due to pre-hibernation fattening in a hibernator (ground squirrels). Kunming mice were fed a high-fat diet to provide a model of pathological obesity (OB group). Daurian ground squirrels fattened naturally in their pre-hibernation season (PRE group) were used as a healthy obesity model. Micro-computed tomography (micro-CT) and three-point bending tests were used to determine the microstructure and mechanical properties of bone. Western blots were used to analyze protein expression levels related to bone metabolism (Runt-related transcription factor 2 (RunX2), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor-‍κB ligand (RANKL), cathepsin K, matrix metallopeptidase 9 (MMP9), patched protein homolog 1 (Ptch1), phosphorylated β‍-‍catenin (P‍-‍β‍-‍catenin), and glycogen synthase kinase-3β (GSK-3β)). Compared with controls, there was no obvious bone loss in the OB mice, and the stiffness of the femur was increased significantly. Compared with summer active squirrels, bone formation was enhanced but the mechanical properties did not change in the PRE group squirrels. In OB mice, western blots showed significantly increased expression levels of all proteins except RunX2, OPG, and Ptch1. PRE ground squirrels showed significantly increased expression of most proteins except OCN and Ptch1, which decreased significantly, and P‍-‍β‍-‍catenin and OPG, which did not change. In conclusion, for non-hibernating mice, moderate obesity had a certain protective effect on bones, demonstrating two-way regulation, increasing both bone loss and bone formation. For pre-hibernating ground squirrels, the healthy obesity acquired before hibernation had a positive effect on the microstructure of bones, and also enhanced the expression levels of proteins related to bone formation, bone resorption, and Wnt signaling.
Mice ; Animals ; Hibernation ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Diet, High-Fat ; X-Ray Microtomography ; Sciuridae/metabolism* ; Obesity

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*

Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.
Animals ; Cell Differentiation ; physiology ; Core Binding Factor Alpha 1 Subunit ; Homeodomain Proteins ; metabolism ; Mesenchymal Stem Cells ; metabolism ; Mice ; Mice, Nude ; Osteoblasts ; metabolism ; Osteocalcin ; drug effects ; Osteogenesis ; physiology ; Transcription Factors ; metabolism ; Up-Regulation