Pinoresinol Diglucoside Relieves Osteoporosis Through Enhancing Osteogenic Differentiation via Activating Phosphatidylinositol-3-Kinase/Protein Kinase B Signaling Pathway

Lipopolysaccharide (LPS) has been confirmed to be the main inhibitor in osteogenic differentiation, posing a clinical challenge to bone healing, particularly for trauma followed by endotoxinemia/sepsis. However, the molecular mechanism remains ambiguous. miR-23b, which regulates multiple signaling pathways in inflammation, has been shown to be deregulated by LPS. In this study, we examined the LPS-mediated regulation on the expression of miR-23b and Smad 3 in preosteoblast MC3T3-E1 cells. Then we determined the regulation of miR-23b overexpression on the Smad 3 expression and on the LPS-mediated inhibition of bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. Our results demonstrated that LPS significantly downregulated the expression of miR-23b, while upregulating Smad 3 in MC3T3-E1 cells. However, the transfection with miR-23b mimics markedly downregulated the Smad 3 in both mRNA and protein levels, via the specific binding to the 3'-untranslated region (UTR) of Smad 3. Moreover, though LPS markedly downregulated the BMP-2-induced osteogenic differentiation of MC3T3-E1 cells by inhibiting the expression of alkaline phosphatase (ALP), Osteocalcin (OCN), Osteopontin (OPN) and Runt-related transcription factor 2 (RUNX2). The upregulated miR-23b reversed such downregulation of ALP, OCN, OPN and RUNX2 in the MC3T3-E1 cells which were treated both with LPS and BMP-2. In conclusion, our data indicates that miR-23b ameliorates the LPS-mediated inhibition of BMP-2-induced osteogenic differentiation in MC3T3-E1 cells, implying the protective role of miR-23b in the LPS-mediated inhibition of osteogenic differentiation and bone formation.

To study the effects of morroniside (MOR) on the proliferation and osteogenic differentiation of mouse MC3T3-E1 cells. The 4th generation MC3T3-E1 cells were randomly divided into 6 groups: control group (group A), MOR low dose group (10 μmol/L, group B), MOR medium-low dose group (20 μmol/L, group C), MOR medium dose group (40 μmol/L, group D), MOR medium-high dose group (80 μmol/L, group E), and MOR high dose group (100 μmol/L, group F). The proliferation activity of each group was detected by cell counting kit 8 (CCK-8) assay; the bone differentiation and mineralized nodule formation of each group were detected by alizarin red staining; real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect cyclin-dependent kinase inhibitor 1A (P21), recombinant Cyclin D1 (CCND1), proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP), collagen type Ⅰ (COL-1), bone morphogenetic protein 2 (BMP-2), and adenosine A2A receptor (A2AR) mRNA expressions; Western blot was used to detecte the expressions of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and adenosine A2AR protein. The CCK-8 assay showed that the absorbance ( A) values of groups B to F were significantly higher than that of group A at 24 hours of culture, with group C significantly higher than the rest of the groups ( P<0.05). The MOR concentration (20 μmol/L) of group C was selected for the subsequent CCK-8 assay; the results showed that the A values of group C were significantly higher than those of group A at 24, 48, and 72 hours of culture ( P<0.05). Alizarin red staining showed that orange-red mineralized nodules were visible in all groups and the number of mineralized nodules was significantly higher in groups B and C than in group A ( P<0.05). RT-qPCR showed that the relative expressions of P21, CCND1, and PCNA mRNAs were significantly higher in group C than in group A ( P<0.05). The expressions of ALP, BMP-2, COL-1, and adenosine A2AR mRNAs in groups B to E were significantly higher than those in group A, with the expressions of ALP, BMP-2, COL-1 mRNAs in group C significantly higher than the rest of the groups ( P<0.05). Compared with group A, the expressions of OPN and RUNX2 proteins in groups B and C were significantly increased, while those in group D and E were significantly inhibited ( P<0.05). There was no significant difference between groups B and C and between groups D and E ( P>0.05). The relative expression of adenosine A2AR protein in groups B to E was significantly higher than that in group A, with group C significantly higher than the rest of the groups ( P<0.05). MOR can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells; the mechanism of MOR may be achieved by interacting with adenosine A2AR.

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.

Glucocorticoid-induced osteoporosis is the third epidemic osteoporosis following postmenopausal and senileosteoporosis. According to one study, salidroside made ovariectomized rats' bones strong. Salidroside's potential for treating glucocorticoid-induced osteoporosis remains unproven. This study aimed to investigate the protective effect and mechanism of salidroside on dexamethasone-induced osteogenic differentiation and bone formation in MC3T3-E1 cells and zebrafish. The study proved that salindroside had no harmful impact on MC3T3E1 cells. Salidroside significantly relieved dexamethasone-induced inhibition of ALP (alkaline phosphatase) activity and mineralization in MC3T3-E1 cells, and promoted osteogenic differentiation of cells. Salidroside increased the expression of osteopontin (OPN), runt-related transcription factor 2 (Runx2), osterix (Osx), transforming growth factor-beta (TGF-β) proteins and promoted the phosphorylation of Smad2/3 in MC3T3-E1 cells treated with dexamethasone. In addition, the effect of salidroside in relieving dexamethasone-induced inhibition of osteogenic differentiation in MC3T3-E1 cells can be blocked by TGF-β receptor type I/II inhibitor (LY2109761). At the same time, we found that salidroside significantly alleviated the inhibition of dexamethasone-induced bone formation in zebrafish and promoted the mineralization of zebrafish skulls. LY2109761 reversed the protective impact of salidroside on dexamethasone-mediated bone impairment in zebrafish. These findings suggested that salidroside alleviated dexamethasone-induced inhibition of osteogenic differentiation and bone formation via TGF-β/Smad2/3 signaling pathway.

Statement of Retraction Qiang Wang, Xiaoxing Xie, Dehua Zhang, Feng Mao, Shaobo Wang, Yi Liao- 2022, Saxagliptin enhances osteogenic differentiation in MC3T3-E1 cells, dependent on the activation of AMP-activated protein kinase α (AMPKα)/runt-related transcription factor-2 (Runx-2), Bioengineered 13(1), doi: 10.1080/21655979.2021.2008667 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Introduction Tripartite motif 65 (TRIM65) is a crucial regulator of cell differentiation, proliferation, migration, invasion, and carcinogenesis. However, its role in osteoporosis (OP) remains unclear. In this study, we evaluated the role of TRIM65 in regulating osteoblast differentiation and calcification. Materials and Methods The role of TRIM65 during the osteogenic differentiation of MC3T3-E1 cells was evaluated. The expression of COL1A1, RUNX2, and OCN was examined using western blot analysis and immunofluorescence staining. The formation of calcium nodules was evaluated using alizarin red staining. Alkaline phosphatase activity was evaluated using ALP staining. Results TRIM65 expression was significantly elevated during the osteogenic differentiation of bone marrow mesenchymal stem and MC3T3-E1 cells. We demonstrated that TRIM65 overexpression enhanced osteogenic differentiation and promoted bone formation in the MC3T3-E1 cells. Conversely, TRIM65 inhibited the osteogenic differentiation and bone formation of the MC3T3-E1 cells. Mechanistically, we found that TRIM65 knockdown in MC3T3-E1 cells up-regulated the phosphorylated protein expression of PI3K and AKT, which was contrary to the results of the TRIM65-overexpression group. Conclusion Our research suggests that TRIM65 is an important osteogenic differentiation and bone formation regulator and offers a therapeutic application for OP.

Differential circRNA expression profiles during the BMP2-induced osteogenic differentiation of MC3T3-E1 cells

The effects of gold nanoparticles on the proliferation, differentiation, and mineralization function of MC3T3-E1 cells in vitro

Osteogenic differentiation is a crucial process of new bone formation. This study aimed to explore the roles and mechanism of SRY-Box Transcription Factor 2 (SOX2) on proliferation and osteogenic differentiation of MC3T3-E1 cells. Bone morphogenetic protein 2 (BMP2) was used to induce the osteogenic differentiation of MC3T3-E1 cells. The expression of SOX2 was determined by quantitative real-time PCR (RT-PCR) at different time points after induction. The SOX2 overexpression plasmids were constructed and transfected into MC3T3-E1 cells. Osteogenic differentiation was evaluated by Alizarin Red S staining and alkaline phosphatase (ALP) assay. The expressions of osteogenic differentiation markers including runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) were detected by western blot assay. Luciferase reporter and CHIP assays were used to confirm that SOX2 regulated the transcriptional activation of leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4). We found that SOX2 was down-regulated upon BMP2-induced osteogenic differentiation in MC3T3-E1 cells. Overexpression of SOX2 effectively inhibited osteogenic differentiation with decreased ALP activity, calcification, and osteogenic differentiation markers' expression including Runx2, OPN, and OCN. LGR4 was identified as a target of SOX2, and the inhibitory effect of SOX2 on osteogenic differentiation was reversed by knockdown of LGR4. The present study confirmed that SOX2 suppressed osteogenic differentiation of MC3T3-E1 cells through targeting LGR4, which possesses a therapeutic strategy for bone formation and generation.

Chromobox protein homolog 7 (CBX7) deficiency inhibits osteoblast ferroptosis by activating the Nrf2 function in type 2 diabetic osteoporosis.

Concentrate Growth Factors Regulate Osteogenic Dysfunction of MC3T3-E1 Cells Induced by High Glucose Through PI3K/Akt Signaling Pathway.

The E3 ligase RNF185 negatively regulates osteogenic differentiation by targeting Dvl2 for degradation

Diabetes is a worldwide health problem with increasing prevalence. Some reports indicate the interplay between bone and glucose metabolism. The imbalance between bone resorption and formation resulted in the structural integrity and strength of bone. Glucagon-like peptide-1 (GLP-1) and its agonists (Liraglutide) have an anabolic action on bone remodeling by stimulating osteoblast differentiation as well as increasing osteoblast longevity. However, the underlying mechanisms remain elusive. We detected the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells via immunocytochemistry assay. Alkaline phosphatase activity assay, alizarin red stain, quantitative real-time polymerase chain reaction, and western blot were employed to detect the effect of Liraglutide on osteogenic differentiation. Liraglutide promoted the expression of GLP-1R in a dosage- and time-dependent manner, and it enhanced the osteogenic differentiation in MC3T3-E1 cells. Liraglutide application improved the levels of Smad2/3 and p-Smad2/3; however, the silencing of Smad2/3 blocked the osteogenic differentiation induced by Liraglutide. What is more, the application of PI3K and Wnt inhibitors inhibited the upregulation of Akt, p-Akt, β-catenin, Smad2/3, and p-Smad2/3 induced by Liraglutide. Liraglutide facilitated the osteogenic differentiation via the regulation of Smad2/3 via PI3K/AKT and Wnt/β-catenin pathways. These data revealed a new mechanism of Liraglutide inducing osteogenic differentiation and provided theory evidence to maintain normal bone metabolism during diabetes therapy.

Background Mechanical stress plays an important role in the maintenance of bone homeostasis. Current hypotheses suggest that interstitial fluid flow is an important component of the system by which tissue level strains are amplified in bone. This study aimed to test the hypothesis that the short-term and appropriate fluid shear stress (FSS) is expected to promote the terminal differentiation of pre-osteoblasts and detect the expression profile of microRNAs in the FSS-induced osteogenic differentiation in MC3T3-E1 cells. Methods MC3T3-E1 cells were subjected to 1 hour of FSS at 12 dyn/cm2 using a parallel plate flow system. After FSS treatment, cytoskeleton immunohistochemical staining and microRNAs (miRNAs) were detected immediately. Osteogenic gene expression and immunohistochemical staining for collagen type I were tested at the 24th hour after treatment, alkaline phosphatase (ALP) activity assay was performed at 24th, 48th, and 72th hours after FSS treatment, and Alizarin Red Staining was checked at day 12. Results One hour of FSS at 12 dyn/cm2 induced actin stress fiber formation and rearrangement, up-regulated osteogenic gene expression, increased ALP activity, promoted synthesis and secretion of type I collagen, enhanced nodule formation, and promoted terminal differentiation in MC3T3-E1 cells. During osteogenic differentiation, expression levels of miR-20a, -21, -19b, -34a, -34c, -140, and -200b in FSS-induced cells were significantly down-regulated. Conclusion The short-term and appropriate FSS is sufficient to promote terminal differentiation of pre-osteoblasts and a group of miRNAs may be invovled in FSS-induced pre-osteoblast differentiation.

Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in osteoblastic differentiation. However, the exact role of STAT3 in osteogenic differentiation of the pre-osteoblastic cell line MC3T3-E1 is still controversial. In this study, we demonstrated that eradication of STAT3 signaling by the inhibitors cryptotanshinone (CPT, a STAT3-specific inhibitor) or STAT3 siRNA both suppressed osteogenic differentiation of MC3T3-E1 cells, with a decrease in alkaline phosphatase (ALP) activity, protein expressions of the osteogenic differentiation markers Collagen I (ColI), ALP, and osteocalcin (OCN), and reduced matrix mineralization capacity at the terminal stage of osteogenic differentiation. However, the inhibition of STAT3 by CPT did not affect MC3T3-E1 cell proliferation. To further clarify the effect of STAT3 on osteogenic differentiation of MC3T3-E1 cells, we forced STAT3 expression and found that this ameliorated osteogenic differentiation. Thus, our results confirmed that STAT3 is a likely positive regulator of osteogenic differentiation in MC3T3-E1 cells. These findings may provide a basis for the development of more efficient and controllable protocols for osteoblastic differentiation and facilitate their use in regenerative medicine. In addition, our results provide novel insights into the effect of the STAT3 antagonist CPT on modulation of osteogenesis.