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Bone metastatic cancer-secreted extracellular factors are capable of modifying the bone microenvironment through interacting with bone cells, including osteoblasts. Reticulum ribosome-binding protein 1 (RRBP1) is substantially expressed in certain bone metastatic cancer cells. This study was undertaken to determine whether RRBP1 from bone metastatic cancer cells affects the osteoblastic phenotype expression. Breast and prostate cancer cells, MDA-MB-231 and PC3, were cultured, respectively, followed by collecting conditioned mediums (CMs) and identifying the abundance of RRBP1 in CMs using LC-MS/MS. MC3T3-E1 cells were cultured with a mixed medium (including CMs from sh RRBP1 -transduced two-type cancer cells) with or without endoplasmic reticulum (ER) stress inhibitor 4-PBA, followed by measuring the levels of osteoblastic phenotype expression and biomarkers of ER stress using western blotting, qPCR, and ARS staining, respectively. Similar experiments were performed in sh Rrbp1 -transduced MC3T3-E1 cells cultured with a mixed medium (including CMs from the two-type cancer cells). Bone formation parameters were measured in the tibia of nude mice injected with sh RRBP1 -transduced two-type cancer cells using micro-CT analysis. These results showed that RRBP1 is the sole shared high-abundance protein in CMs from the two-type cancer cells, involving osteoblast differentiation. CMs from sh RRBP1 -transduced two-type cells boosted the osteoblastic phenotype expression partially through increasing ER stress. CMs from the two-type cancer cells partially offset the similar alterations induced by sh Rrbp1 in MC3T3-E1 cells. Injection with sh RRBP1 -transduced two-type cells ameliorated the bone lesions in nude mice. Therefore, RRBP1 depletion of bone metastatic cancer enhanced the osteoblastic phenotype expression, suggesting a role of RRBP1 in the bone microenvironment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Wang, Xu, He, Li, Xia and Zhang.)

Oxidative stress represents a major cause of cellular damage and death in pathological conditions including osteoporosis, in which oxidative stress is associated with increased bone resorption and low bone mass. And grape seed proanthocyanidins are a group of polyphenolic bioflavonoids which are known to possess broad pharmacological activity and therapeutic potential, exerting a protective role against oxidant injury. The aim of our study was to investigate whether proanthocyanidins exert an anti-apoptosis effect in osteoblastic MC3T3-E1 cells, via their antioxidant activity. Firstly, we determined the anti-apoptosis effect of proanthocyanidins in osteoblastic MC3T3-E1 cells, which were subject to H2O2 treatment, then we determined the association of the antioxidant activity exerted by proanthocyanidins with their anti-apoptosis effect. Results demonstrated that proanthocyanidins inhibit H2O2-promoted apoptosis in MC3T3-E1 cells, via ameliorating the viability of MC3T3-E1 cells post H2O2treatment and reducing the apoptotic cell numbers. And the proanthocyanidins treatment also ameliorates the H2O2-induced mitochondrial dysfunction via promoting the mitochondrial membrane potential (MMP) and respiratory chain complex IV, and reducing the mitochondrial free radical production, ROS and mitochondrial superoxide. Moreover, the proanthocyanidins inhibit H2O2-induced apoptosis signaling which is mediated by p53. This study implied a possible anti-osteoporosis effect of proanthocyanidins via their antioxidant and anti-apoptosis activity. [ABSTRACT FROM AUTHOR]

Phosphate has been shown to work as a signaling molecule in several cells including endothelial cells and chondrocytes. However, it is largely unknown how phosphate affects osteoblastic cells. In the present study, we investigated the effects of phosphate on reactive oxygen species (ROS) production and osteoblastic differentiation in murine osteoblastic MC3T3-E1 cells. Phosphate increased production of ROS in MC3T3-E1 cells and the inhibitors of sodium-phosphate cotransporter and NADPH oxidase suppressed ROS production by phosphate. Silencing Nox1 and Nox4 also inhibited the increase of ROS by phosphate. Phosphate also decreased alkaline phosphatase activity induced by bone morphogenetic protein 2 and this inhibition was abrogated by an inhibitor of NADPH oxidase. Furthermore, phosphate decreased the expression of osteoblastic marker genes in MC3T3-E1 cells. These results indicate that phosphate suppresses osteoblastic differentiation at least in part by enhancing ROS production in MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

Abstract: Bioactive amorphous phase/TiO2 nanocrystal (APTN) composite coatings were fabricated by microarc oxidation (MAO) on Ti. The APTN coatings are composed of much amorphous phase with Si, Na, Ca, Ti and O elements and a few TiO2 nanocrystals. With increasing applied voltage, the micropore density of the APTN coating decreases and the micropore size of the APTN coating increases. The results indicate that less MC3T3-E1 cells attach on the APTN coatings as compared to Ti. However, the APTN coatings greatly enhance the cell proliferation ability and the activity of alkaline phosphatase. The amorphous phase and the concentrations of the released Ca and Si from the APTN coatings during cell culture have significant effects on the cell response. [Copyright &y& Elsevier]

We investigated the protective effect of liquiritigenin, one of the flavonoids present in Glycyrrhizae radix, against antimycin A-induced mitochondrial dysfunction in MC3T3-E1 osteoblast cells. Osteoblastic MC3T3-E1 cells were pre-incubated with liquiritigenin before treatment with antimycin A, and markers of mitochondrial function and oxidative damage were examined. In addition, the effects of liquiritigenin on the activation of phosphoinositide 3-kinase (PI3K) were examined in MC3T3-E1 cells. Liquiritigenin protected MC3T3-E1 cells from antimycin A-induced cell death. However, the PI3K inhibitor, LY294002, significantly attenuated liquiritigenin-mediated cell survival, indicating the involvement of PI3K in the cytoprotective effect of liquiritigenin. Pretreatment with liquiritigenin prior to antimycin A exposure significantly reduced antimycin A-induced PI3K inactivation, mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Liquiritigenin also reduced mitochondrial superoxide generation, nitrotyrosine production, and cardiolipin peroxidation during mitochondrial complex inhibition with antimycin A. Taken together, the results of this study show that modulation of PI3K, antioxidant effects, and the attenuation of mitochondrial dysfunction by liquiritigenin represent an important mechanism for its protection of osteoblasts against cytotoxicity resulting from mitochondrial oxidative stress. [ABSTRACT FROM AUTHOR]

Actein, isolated from black cohosh, was subjected to in vitro experiments to investigate its functional bioactivities in osteoblastic MC3T3-E1 cells. Actein caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, mineralization, and glutathione content in the cells, suggesting that actein has a stimulatory effect on osteoblastic bone formation or has potential activity against osteoporosis. We investigated the protective effects of actein on mitochondrial electron transport inhibitor, antimycin A induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant decrease in cell viability and mineralization. However, pretreatment with actein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, cardiolipin oxidation, ROS release, and nitrotyrosine increase, suggesting that actein may be useful for protecting mitochondria against a burst of oxidative stress. In addition, actein increased the phosphorylation of CREB (cAMP-response element-binding protein) inhibited by antimycin A and decreased the production of TNF-α induced by antimycin A. These findings suggest that actein could prevent oxidative damage to osteoblasts in osteoporotic patients. [ABSTRACT FROM AUTHOR]

Recent experiments have explored the impact of Wnt/β-catenin signaling and Substance P (SP) on the regulation of osteogenesis. However, the molecular regulatory mechanisms of SP on the formation of osteoblasts is still unknown. In this study, we investigated the impact of SP on the differentiation of MC3T3-E1 cells. The osteogenic effect of SP was observed at different SP concentrations (ranging from 10-10 to 10-8 M). To unravel the underlying mechanism, the MC3T3-E1 cells were treated with SP after the pretreatment by neurokinin-1 (NK1) antagonists and Dickkopf-1 (DKK1) and gene expression levels of Wnt/β-catenin signaling pathway components, as well as osteoblast differentiation markers (collagen type I, alkaline phosphatase, osteocalcin, and Runx2), were measured using quantitative polymerase chain reaction (PCR). Furthermore, protein levels of Wnt/β-catenin signaling pathway were detected using Western blotting and the effects of SP, NK1 antagonist, and DKK1 on β-catenin activation were investigated by immunofluorescence staining. Our data indicated that SP (10-9 to 10-8 M) significantly up-regulated the expressions of osteoblastic genes. SP (10-8 M) also elevated the mRNA level of c-myc, cyclin D1, and lymphocyte enhancer factor-1 (Lef1), as well as c-myc and β-catenin protein levels, but decreased the expression of Tcf7 mRNA. Moreover, SP (10-8 M) promoted the transfer of β-catenin into nucleus. The effects of SP treatment were inhibited by the NK1 antagonist and DKK1. These findings suggest that SP may enhance differentiation of MC3T3-E1 cells via regulation of the Wnt/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

The conformation change of bovine serum albumin (BSA) induced by bioactive titanium surfaces, including acid-alkali-treated titanium (AA-Ti) and alkali-heat-treated titanium (AH-Ti), was studied, and its effects on the activity of MC3T3-E1 cell were evaluated. Pure titanium metal (P-Ti) was used as control. The AA-Ti could adsorb more BSA on its surface than AH-Ti and P-Ti. The α-helix part of the protein adsorbed on P-Ti has weakly decreased compared with native BSA, and it dramatically decreased on AA-Ti and AH-Ti. The β-sheet segment of proteins adsorbed on P-Ti and AH-Ti had obviously increased. Much more tryptophan residues were exposed after the protein conformation changed when it interacted with AH-Ti, and some tryptophan residues were enveloped after it interacted with AA-Ti and P-Ti. AA-Ti has more tryptophan residues enveloped than P-Ti. All titanium surfaces induced tyrosine residues exposed, especially for the P-Ti. The higher ratio of COO−/NH3+ for the proteins on P-Ti and AA-Ti indicated an orientation of proteins on P-Ti and AA-Ti, which makes more COO− exposed. The lower ratio of COO−/NH3+ on AH-Ti indicates that more NH3+ is exposed on its surface. The cell proliferation ability on different treated titanium surfaces coated with BSA followed by the order: P-Ti > AA-Ti > AH-Ti, which indicated that the protein conformation change on different bioactive titanium surfaces has great effect on the cell activity. Our results showed that the different biological response of bioactive titanium metals might depend on the protein conformation change induced by the surface structure. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1053-1062, 2014. [ABSTRACT FROM AUTHOR]

Cp-Ti is the most common material used for dental implants, but its elastic modulus is around five times higher than that of bone. Recently, promising alloys that add Nb, Ta, Zr and Mo to Ti have been developed. The mechanical properties of these alloys are directly related to its microstructure and the presence of interstitial elements, such as oxygen, carbon, nitrogen and hydrogen. In this study, the in vitro cytotoxicity of Ti-35Nb-7Zr-5Ta (TNZT) alloys was analyzed in the as-received condition and after being doped with several small quantities of oxygen on a cultured osteogenic cell. The cell's morphology was also examined by scanning electron microscopy (SEM). The TNZT alloy presented no cytotoxic effects on osteoblastic cells in the studied conditions. [ABSTRACT FROM AUTHOR]

Bone morphogenetic protein-2 (BMP-2), a glycosylated protein, has been demonstrated to play a key role in osteoblast differentiation. However, the function of its glycosylation is incompletely understood. In this study, we examined the role that N-linked glycans (NLG) play in the secretion of BMP-2. Blocking the addition of NLGs or inhibiting initial glycan processing prevented the secretion of BMP-2. To identify the specific glycosylation sites, we abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in BMP-2 by mutating the Asn residues to Gln individually or in combination, expressed the BMP-2 mutants in Chinese hamster ovary (CHO) and human embryonic kidney 293T (HEK293T) cells and determined their glycosylation state by using peptide:N-glycosidase F and endoglycosidase H digestion. We found that human BMP-2 contains three NLG on N135, N200 and N338. Elimination of N-glycosylation by mutation of N135 (N135Q) abolished the BMP-2 secretion from CHO cells. Overexpression of the BMP-2 mutant N135Q elicited endoplasmic reticulum (ER) stress and retention within the ER in CHO cells, indicating that N-glycosylation is required for folding of human BMP-2. Furthermore, we demonstrated that glycosylation at N135 was necessary for BMP-2-induced osteoblast differentiation in MC3T3-E1 cells. Taken together, these data provide further evidence of the critical role that individual NLG may play an important role in determining BMP-2 folding, secretion and function. [ABSTRACT FROM AUTHOR]

Ti6Al4V discs with a thickness of 2.5 mm and dimensions of 15 × 15 mm2 were fabricated. The titanium nitride (TiN) surface was formed via Nd:YAG laser-nitriding. A sandblast acid-etched (SA) surface was used as a control. Scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), and surface roughness tests were conducted to study the surface and cross-section morphologies as well as the properties of TiN and SA surfaces. MC3T3-E1 osteoblast-like cells were cultured on the TiN and SA surfaces to evaluate the effect of TiN surface on cellular behaviors, including attachment, proliferation and differentiation. Morphological testing results revealed that the cross-section of TiN exhibited dendritic crystallization without cracking. The proliferation and differentiation of MC3T3-E1 cells on the laser-nitriding TiN surface were significantly increased compared to those cultured on SA surface. These findings suggested that the TiN surface generated from Nd:YAG laser-nitriding were favorable for the proliferation and differentiation of MC3T3-E1 cells, which is significant for implant surface modification. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Velvet antlers (VA) have been claimed for centuries to have numerous medical benefits including strengthen bones. To investigate and compare the anti-osteoporotic activities from different sections of VA. Materials and methods: Fresh VA prepared from farmed sika deers (Cervus nippon) was divided into upper (VAU), middle (VAM), and basal (VAB) sections. The chemical constituents and anti-osteoporotic effect of different sections from VA were evaluated using ovariectomized rats. Results: Levels of water-soluble extracts, diluted alcoholic extract, amino acids, testosterone, insulin-like growth factor (IGF)-1and testosterone plus estradiol significantly differed among the different sections. Levels of these constituents were significantly higher in the upper section than in the basal section. Moreover, levels of testosterone and IGF-1 of the VAM were also significantly higher than those of the VAB. Calcium level increased downward from the tip with statistical significance. The strength of vertebrae increased in all VA-treated groups compared to the control, but only treatment with VAU and VAM increased the strength of the femur and the microarchitecure of the trabecular bone. Alkaline phosphatase levels of VAU- and VAM-treated groups significantly decreased, but osteocalcin did not significantly change. Moreover, VAU and VAM dose-dependently increased proliferation and mineralization of MC3T3-E1 cells. Conclusion: Our study provides strong evidence for the regional differences in the effectiveness of velvet antler in treating osteoporosis. However, further studies are needed to elucidate the bioactive chemical constituents associated with the anti-osteoporotic effects of velvet antler. [ABSTRACT FROM AUTHOR]

Induction of some specific cell signaling molecules in peptide scaffold play a critical role in developing of functional substitutes for damaged tissue. In this study, a new peptide ELK8 (Ac-LELELKLK-CONH2) was designed by replacing Ala in EAK16 (Ac-AEAEAKAKAEAEAKAK-CONH2) with more hydrophobic residue Leu in order to enhance the strength of peptide. The functionalized peptides were obtained through directly coupling three functional motifs to C-terminal of ELK8, such as osteogenic growth peptide, osteopontin cell adhesion motif, and 2-unit RGD binding sequence. Then, the new self-assembling peptide scaffolds were fabricated by mixing three functionalized peptides severally with pure ELK8 at the ratio of 1:1 (v:v) and the nanostructures of self-assembling peptides were investigated with AFM and DLS. The osteogenic efficacy of designed peptides on mouse pre-osteoblast MC3T3-E1 were examined after cells were incubated in scaffolds for 14 d. The results showed that these functionalized peptides, ALK mix particularly, promoted the attachment, proliferation, and osteogenic differentiation of MC3T3-E1 cells by increasing in cell numbers, ALP activities and osteocalcin concentrations. [ABSTRACT FROM AUTHOR]

Abstract: 1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) regulates osteoblasts through genomic and rapid membrane-mediated responses. Here we examined the interaction of protein disulfide isomerase family A, member 3 (Pdia3) and the traditional vitamin D receptor (VDR) in plasma membrane-associated responses to 1α,25(OH)2D3. We found that Pdia3 co-localized with VDR and the caveolae scaffolding protein, caveolin-1 on the surface of MC3T3-E1 osteoblasts. Immunoprecipitation showed that both Pdia3 and VDR interacted with caveolin-1. Pdia3 further interacted with phospholipase A2 activating protein (PLAA), whereas VDR interacted with c-Src. 1α,25(OH)2D3 changed the interactions and transport of the two receptors and rapidly activated phospholipase A2 (PLA2) and c-Src. Silencing either receptor or caveolin-1 inhibited both PLA2 and c-Src, indicating that the two receptors function interdependently. These two receptor dependent rapid responses to 1α,25(OH)2D3 regulated gene expression, proliferation and apoptosis of MC3T3-E1 cells. These data demonstrate the importance of both receptors and caveolin-1 in mediating membrane responses to 1α,25(OH)2D3 and subsequently regulating osteoblast biology. [Copyright &y& Elsevier]

Fibronectin (FN) containing a heparin-binding domain (HBD) and an Arg-Gly-Asp (RGD) domain can promote cell adhesion and proliferation compared to FN that contained only RGD. Here, we have engineered recombinant human osteocalcin (rhOC) with FN type III9-14 (rhOC-FNIII9-14) containing RGD and HBD to promote the cellular activity of MC3T3-E1 cells, including adhesion, proliferation, and differentiation. RhOC-FNIII9-14 significantly increased cell adhesion and proliferation of MC3T3-E1 cells compared to rhOC-FNIII9-10 ( P < 0.05). Moreover, rhOC-FNIII9-14 showed osteogenic differentiation of MC3T3-E1 cells in mineralization activity and osteogenic gene expression. [ABSTRACT FROM AUTHOR]

Background: Deer antler is a traditional Chinese medicine with the function of tonifying kidney and strengthening bone, which is often used to treat orthopedic diseases., Methods: Eight-week-old C57BL/6 mice were used as the fixation model of open tibial fracture with intramedullary nail. The mice were treated with deer antler extract (DAE) or PBS by oral gavage once daily. The tibial fracture samples were collected and performed to the tissue analysis, including X-ray, micro-CT, histology, qRT-PCR, immunohistochemistry. MC3T3-E1 cells were used to detect the effect of deer antler extract on ability of cell proliferation and migration by CCK-8 assay and cell scratch test., Results: Imaging and micro-CT showed that DAE could promote the healing of tibial fracture in mice, and histological analysis showed that DAE could promote the transformation of cartilage callus to bone callus in fracture area. The results of qRT-PCR and immunohistochemistry showed that DAE could promote intrachondral ossification in fracture zone and the mechanism of promoting fracture healing may be related to the activation of BMP-2/SMAD4 signaling pathway. In the cytological experiment of DAE, it can be found that DAE promoted the proliferation of MC3T3-E1 cells and the migration of MC3T3-E1 cells at a certain concentration, which is also related to the promotion of fracture healing by DAE., Conclusion: DAE can promote fracture healing by activating BMP-2/SMAD4 signaling pathway. DAE has the potential to be used in clinic as an important means of promoting fracture healing., (© 2022. The Author(s).)

Periodontitis is an excessive inflammatory event in tooth-supporting tissues and can cause tooth loss. We used erythropoietin (EPO), which has been reported to play an important role in bone healing and modulation of angiogenesis, as a therapeutic agent in vivo and in vitro experimental models to analyze its effect on periodontitis. First , EPO was applied to in vitro MC3T3-E1 cells and human periodontal ligament fibroblast (hPDLF) cells to examine its function in altered cellular events and gene expression patterns. In vitro cultivation of MC3T3-E1 and hPDLF cells with 10 IU/ml EPO at 24 and 48 h showed an obvious increase in cell proliferation. Interestingly, EPO treatment altered the expression of osteogenesis-related molecules, including alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OC) in MC3T3-E1 cells but not in hPDLF cells. In particular, MC3T3-E1 cells showed increased expression of ALP, BMP-2, and OC on day 5, while hPDLF cells showed increased expression of BMP-2, and OC on day 14. Based on the in vitro examination, we evaluated the effect of EPO on bone formation using an experimentally-induced animal periodontitis model. After the induction of periodontitis in the maxillary left second M, 10 IU/ml of EPO was locally applied to the extraction tooth sockets. Histomorphological examination using Masson's trichrome (MTC) staining showed facilitated bone formation in the EPO-treated groups after 14 days. Similarly, stronger positive reactions against vascular endothelial growth factor (VEGF), cluster of differentiation 31 (CD31), runt-related transcription factor 2 (RUNX2), and osteocalcin (OC) were detected in the EPO-treated group compared to the control. Meanwhile, myeloperoxidase, an inflammatory marker, was decreased in the EPO-treated group on days 1 and 5. Overall, EPO facilitates bone healing and regeneration through altered signaling regulation and modulation of inflammation in the osteoblast cell lineage and to a lesser extent in hPDLF cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bae, Hwang, Aryal, Kim, Sohn, An, Kim, An, Lee, Kim, Park, Lee, Kim and Suh.)

Cyanidin-3-O-glucoside (C3G) is the most widely distributed anthocyanin and it can reportedly reduce the risk of osteoporosis, but the molecular mechanism by which C3G promotes bone formation is poorly understood. In the current study, RNA sequencing (RNA-seq) was used to investigate the mechanism of action of C3G in osteogenesis. MC3T3-E1 mouse osteoblasts were divided into a C3G (100 μmol/L)-treated group and a vehicle-treated control group, and differentially expressed genes (DEGs) in groups were evaluated via RNA-seq analysis. The functions of the DEGs were evaluated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and the genes were validated by quantitative real-time PCR. The RNA-seq analysis identified 34 genes that were upregulated in C3G-treated cells compared to vehicle-treated cells, and 17 that were downregulated GO and KEGG pathway analyses indicated that these genes were highly enriched in functions related to lysosomes and glycolipid biosynthesis, among others. The differential expression of ATPase H+-transporting V0 subunit C ( Atp6v0c ), chemokine (C-X3-C motif) ligand 1 ( Cx3cl1 ), and lymphocyte antigen 6 complex, locus A ( Ly6a ) genes was validated by quantitative real-time-PCR. Because these genes have been previously implicated in osteoporosis, they are potential target genes of C3G action in MC3T3-E1 cells. These results provide molecular level evidence for the therapeutic potential of C3G in the treatment of osteoporosis and other disorders of bone metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Hu, Wang, Chen and Zhou.)

Osteoporosis is a serious threat to human life. Guben Zenggu Granule is an empirical prescription for clinical treatment of osteoporosis. MC3T3-E1 cells are mouse osteogenic precursor cells with osteogenic differentiation, and are classic cells for studying bone metabolism and osteogenic mechanism, as well as mechanical stimulation sensitive cells. Therefore, it can be inferred that Guben Zenggu granule can repair MC3T3-E1 cells under continuous static pressure overload. This study aims to through the network of pharmacology and gene sequencing method, reveal thrift increase bone particles under the condition of continuous static pressure overload on osteogenesis mechanism of MC3T3-E1 cells. In the process of analysis, from a variety of 98 compounds was predicted in the database, a collection of 474 goals, a total of 29,164 difference between two groups of genes. Then, construction of composite targets between cells and predict targets and protein - protein interaction networks, and through the cluster analysis to further explore the relationship between the target. In addition, linkages between target proteins and cells were further identified using Gene Ontology (GO) and Pathways (KEGG Pathway). Finally, the repair effect of Guben Zenggu granule on MC3T3-E1 cells under continuous static pressure overload was verified through experiments, so as to accurately explain the pharmacodynamic mechanism of Traditional Chinese medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Song, Zhang, Jiang, Zhao, Pei, Ning, Chen, Pan, Gong, Song and Wang.)

Diabetic osteoporosis (DOP) is a complication of diabetes mellitus (DM) and occurs due to alterations in bone metabolism under hyperglycemic condition. ELAV-like RNA binding protein 1 (ELAVL1) is abnormally up-regulated in diabetes-related diseases. Bioinformatics prediction indicates that divalent metal transporter 1 (DMT1) is a potential target of ELAVL1. To explore the role of ELAVL1 and the involvement of ELAVL1/DMT1 axis in DOP, we established a mouse model of DM by administration of high-fat diet and intraperitoneal injection with streptozotocin (STZ). The expression of ELAVL1 and DMT1 was increased in the bone tissues of DM mice. Knockdown of ELAVL1 reduced iron level and oxidative stress, promoted osteogensis, and prevented bone mass loss, thus mitigating DOP in DM mice. In vitro , mouse pre-osteoblast MC3T3-E1 cells were treated with high glucose (25 mM) and ferric ammonium citrate (FAC, 200 μM). The inhibitory effects of ELAVL1 knockdown on iron accumulation and oxidative stress were evidenced in MC3T3-E1 cells. Knockdown of ELAVL1 enhanced osteoblast viability, differentiation and mineralization. Notably, the expression of DMT1 was positively correlated with that of ELAVL1 in vivo and in vitro. Overexpression of DMT1 abolished the effect of ELAVL1 knockdown on the behaviors of MC3T3-E1 cells, suggesting that ELAVL1 might function through regulating DMT1. In conclusion, knockdown of ELAVL1 likely alleviated DOP by inhibiting iron overload and oxidative stress and promoting osteogenesis, and DMT1 might be involved in this process. These findings provide insights into the pathogenesis of DOP and suggest a potential therapeutic target for DOP treatment. • The expression of ELAVL1 and DMT1 was increased in bone tissues of DM mice. • Kncokdown of ELAVL1 inhibited iron overload and oxidative stress in bone tissues of DM mice. • Knockdown of ELAVL1 promoted osteogenesis and prevented bone mass loss in DM mice. • The expression of DMT1 was positively correlated with that of ELAVL1. • ELAVL1 functioned by targeting DMT1 in high glucose-treated MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

The mature fruit of Psoralea corylifolia L. is a common traditional Chinese medicine used to tonify the kidney and yang, and as well as to treat osteoporosis. Systematic phytochemical investigations have established the most comprehensive constituent library to date, covering over 180 compounds. In this study, 109 chemical constituents containing 37 undescribed compounds were reported and incorrect structures of four known coumarins were corrected. The structures of these undescribed compounds were elucidated using spectroscopic methods, single-crystal X-ray diffraction, Rh 2 (OCOCF 3) 4 and Mo 2 (OAc) 4 -induced circular dichroism spectra. To identify potentially active compounds and investigate their structure-activity relationship (SAR), 89 constituents in the library were evaluated for their osteogenic differentiation and mineralisation activities in MC3T3-E1 cells. We found that coumarins, isoflavones, flavonones, and meroterpenoids were the material basis for Psoralea corylifolia -based treatment of osteoporosis, with some compounds exhibiting excellent activities. These compounds function via the estrogen receptor (ER) pathway and were natural phytoestrogen. Further SAR analysis showed that compounds with an intact isopentenyl replacement possessed superior activities, which was explained by their improved affinity with the ER. [Display omitted] • We established the most comprehensive constituent library to date. • 37 undescribed compounds were reported and wrong structures of 4 coumarins were corrected. • Activities on promoting osteogenesis of 89 constituents were evaluated. • Effective substances were initially identified and excellent compounds were found. • Their SAR were discussed, further explained by SPR analysis. [ABSTRACT FROM AUTHOR]

Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 μg/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01∼1 μg/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 μg/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells ( P < 0.05). Moreover, pretreatment with ZnO NPs (0.01∼1 μg/ml) significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Measurement of reactive oxygen species (ROS) indicated decrease in ROS level upon exposure to ZnO nanoparticles (0.01 μg/ml). Hence, our study indicated that ZnO nanoparticles can have protective effects on osteoblasts at low concentrations where there are little or no observable cytotoxic effects. [ABSTRACT FROM AUTHOR]

Highlights: [•] Sciadopitysin increased the differentiation in osteoblastic MC3T3-E1 cells. [•] Sciadopitysin increased glutathione content in the cells. [•] Sciadopitysin decreased the production of ROS induced by antimycin A. [•] Sciadopitysin increased mitochondrial function. [ABSTRACT FROM AUTHOR]

Gold nanoparticles have shown promising biological applications due to their unique properties. Understanding the interaction mechanisms between nanomaterials and biological cells is important for the control and manipulation of these interactions for biomedical applications. In the present study, we investigated the effects of gold nanoparticles on the differentiation of osteoblastic MC3T3-E1 cells and antimycin A-induced mitochondrial dysfunction. The results showed that gold nanoparticles (5, 10, and 20 nm) caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, and osteocalcin content in the cells ( P < 0.05). Moreover, pretreatment with gold nanoparticles prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, cardiolipin peroxidation, and reactive oxygen species generation. Taken together, our study indicated that gold nanoparticles may improve the differentiation and have protective effects on mitochondrial dysfunction of osteoblastic cells. [ABSTRACT FROM AUTHOR]

This study examined the effect of pinacidil, a nonselective adenosine triphosphate-sensitive potassium channel opener, on the function of osteoblastic MC3T3-E1 cells. Pinacidil caused a significant elevation of collagen synthesis, alkaline phosphatase activity, osteocalcin synthesis and mineralization in the cells ( p < 0.05). Pinacidil significantly decreased the production of osteoclast differentiation inducing factors such as TNF-α, IL-6 and receptor activator of nuclear factor-κB ligand in the presence of antimycin A, which inhibits mitochondrial electron transport. Moreover, pinacidil prevented antimycin A-induced reactive oxygen species and nitrotyrosine production. These results demonstrate that pinacidil may have positive effects on skeletal structure. [ABSTRACT FROM AUTHOR]

ABSTRACT Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca2+]i) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca2+]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

In this study, we investigated whether fluid shear stress and melatonin in combination stimulate the anabolic proteins through the phosphorylation of extracellular signal-regulated kinase (p-ERK) in MC3T3- E1 osteoblast cells. First, we researched why fluid shear stress and melatonin in combination influence cell survival. Fluid shear stress (1 hr) and melatonin (1 mM) in combination reduced autophagic marker LC3-II compared with fluid shear stress (1 hr) and/or melatonin (0.1 mM). Under the same conditions for fluid shear stress, markers of cell survival signaling pathway p-ERK, phosphorylation of serine-threonine protein kinase (p-Akt), phosphorylation of mammalian target of rapamycin (p-mTOR), and p85-S6K were investigated. p-Akt, p-mTOR (Ser 2481) expressions increased with the addition of 1 mM melatonin prior to 0.1 mM melatonin treatment. However, p-S6K expression did not change significantly. Next, mitochondria activity including Bcl-2, Bax, catalase, and Mn-superoxide dismutase (Mn-SOD) were studied. Expressions of Bcl-2, Bax, and catalase proteins were low under fluid shear stress plus 1 mM melatonin compared with only fluid shear stress alone, whereas Mn-SOD expression was high compared with conditions of no fluid shear stress. Finally, the anabolic proteins of bone, osteoprotegerin, type I collagen (collagen I), and bone sialoprotein II (BSP II) were checked. These proteins increased with combined fluid shear stress (1, 4 hr) and melatonin (0.1, 1 mM). Together, these results suggest that fluid shear stress and melatonin in combination may increase the expression of anabolic proteins through the p-ERK in MC3T3-E1 osteoblast cells. Therefore, fluid shear stress in combination with melatonin may promote the anabolic response of osteoblasts. [ABSTRACT FROM AUTHOR]

Nowadays there is a short-term need of investigating in orthopedic implants with a greater functionality, including an improved osseointegration and also antibacterial properties. The coating of metallic implants with hydroxyapatite (HA) remains to be the main proposal, but superior quality HA coatings with compositions closer to natural bone apatites, including carbonates, trace elements are required. Selenium is an essential nutrient in biological tissues and, at the same time, it also presents antibacterial properties. A pioneering study on the fabrication of selenium-doped carbonated hydroxyapatite ( iHA:Se) coatings by Pulsed Laser Deposition (PLD) is presented. Different proportions of selenium were incorporated to obtain the iHA:Se coatings. Their physicochemical characterization, performed by SEM/EDS, FTIR, FT-Raman, Interferometric Profilometry and XPS, revealed typical columnar growth of HA in globular aggregates and the efficient incorporation of selenium into the HA coatings by the, most probably, substitution of SeO [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia is a risk factor for osteoporotic fractures. Homocysteine (Hcys) inhibits collagen cross-linking and consequently decreases bone extracellular matrix (ECM) quality. Serum amyloid A (A-SAA), an acute-phase protein family, plays an important role in chronic and inflammatory diseases and up-regulates MMP13, which plays an important role in bone development and remodeling. Here, we investigate the effect of Hcys on expression of SAA3, a member of the A-SAA gene family, in osteoblasts characterizing underlying mechanisms and possible consequences on bone metabolism. MC3T3-E1 osteoblast-like cells were cultured up to 21 d with Hcys (low millimolar range) or reseeded onto ECM resulting from untreated or Hcys-treated MC3T3-E1 cells. Fourier-transformed infrared spectroscopy and a discriminative antibody were used to characterize the resulting ECM. Gene expression and signaling pathways were analyzed by gene chip, quantitative RT-PCR, and immunoblotting. Transcriptional regulation of Saa3 was studied by promoter transfection assays, chromatin immunoprecipitation, and immunofluorescence microscopy. Hcys treatment resulted in reduced collagen cross-linking, uncovering of RGD-motifs, and activation of the PTK2-PXN-CTNNB1 pathway followed by RELA activation. These signaling events led to increased SAA3 expression followed by the production of MMP13 and several chemoldnes, including Ccl5, Cd2, Cxcl10, and 116. Our data suggest Saa3 as link between hyperhomocysteinemia and development of osteoporosis. [ABSTRACT FROM AUTHOR]

Abstract: Two new chromone glycosides, drynachromoside A (1), drynachromoside B (2), along with three known flavanones, 5,7,3′,5′-tetrahydroxy-flavanone (3), 5,7,3′,5′-tetrahydroxy-flavanone-7-O-β-d-glucopyranoside (4), and 5,7,3′,5′-tetrahydroxy-flavanone-7-O-neohesperidoside (5), were isolated from the dry rhizomes of Drynaria fortunei by means of bio-active screening. The two former compounds were elucidated on the basis of physico-chemical property and spectroscopic data. The osteoblastic proliferation activities of these flavonoids were evaluated by the method of MTT. The results showed that compound 1 exhibited the biochemical effects on the proliferation of MC3T3-E1 cells, while Compound 2 showed inhibitory effects against MC3T3-E1 cells. [Copyright &y& Elsevier]