ObjectiveTo elucidate the molecular mechanisms underlying thrombin-induced suppression of osteoblast differentiation, and to identify the MEK inhibitor PD0325901 (PD03) as a potential therapeutic candidate.MethodsFollowing treatment of primary rat osteoblasts with thrombin (20 U/mL) and PD03 (0.1 μM) during osteogenic induction, the cells were harvested and subjected to RNA sequencing to identify differentially expressed genes (DEGs). The combination of network pharmacology and RNA sequencing was used to predict the targets of PD03 in thrombin-induced osteoblasts. Alkaline phosphatase (ALP) activity was assessed through staining and quantitative analysis; the expression of osteogenic genes was measured by quantitative PCR (qPCR) and western blot; mineralized nodule formation was evaluated by Alizarin Red S staining; the expression of signaling pathway-related proteins (p-Erk1/2, p-Stat3, p-p65, MMP-9, COX-2) and proliferation-related proteins (PCNA and MCM2) were examined by western blot; nuclear localization of NF-κB was observed by immunofluorescence (IF); intracellular calcium levels were quantified using a calcium assay and probe labeling; osteoblast proliferation was evaluated by EdU staining; IL-1β secretion in cell supernatants was detected by ELISA; the expression of IL-1RA was measured by western blot; the effects of MMP-9 knockdown and COX-2 overexpression on osteogenic differentiation were investigated.ResultsThrombin promoted osteoblast proliferation and inhibited osteogenic differentiation by upregulating inflammatory factors and activating inflammatory signaling pathways, including MEK-Erk1/2 and NF-κB, which in turn reduced ALP activity, calcium ion influx, expression of osteogenic markers (e.g., Col1α1, Runx2, OCN), and mineralized nodule formation. PD03 reverses these effects by suppressing thrombin-induced activation of IL-1β-dependent signaling pathway, in which the downstream gene MMP-9 plays a critical role.ConclusionPD03 inhibits thrombin-induced activation of the IL-1β-mediated feedback loop between the MEK-Erk1/2 and NF-κB pathways, thereby restoring bone formation and offering a promising therapeutic approach for mitigating bone loss in patients with elevated thrombin levels.

Transplantation therapy combining scaffolds and cells can be used for extensive bone regeneration (e.g., to manage severe alveolar bone defects). Gelatin methacryloyl (GelMA) may be a suitable scaffold for cell transplantation. We developed a novel GelMA that polymerizes under visible light, using riboflavin as a photoinitiator (GelMA-RF). Here we investigated the efficacy of bone-regenerative therapy combining GelMA-RF and immature osteoblasts in rats. Rat alveolar bone immature osteoblasts (RAOBs) from 70-week-old rats were encapsulated in GelMA-RF (aged RAOBs+GelMA-RF) and transplanted into palatal (control, RAOBs alone, and RAOBs+GelMA-RF groups; n=8, respectively) or femoral (control, GelMA-RF alone, RAOBs alone, and RAOBs+GelMA-RF groups; n=8, respectively) bone defects. Macroscopic assessment, micro-computed tomography, and histological analyses were performed, and mineralization (aged and young RAOBs with or without osteogenic differentiation (OD) groups; n=8, respectively) and the calcium/phosphorus ratio (aged and young RAOBs in OD+ or OD- groups; n=8, respectively) for RAOBs under 3D-culture conditions were evaluated. Bone differentiation-related gene expression (aged and young RAOBs in OD+ or OD- groups; n=8, respectively) was analyzed by quantitative polymerase chain reaction. Transplantation of RAOBs encapsulated in GelMA-RF into palatal or femoral defects resulted in significantly earlier bone-like tissue formation compared with controls. RAOBs showed sufficient mineralization in a 3D-culture environment. Bone differentiation-related gene expression was significantly increased in 3D cultures. Taken together, GelMA-RF, particularly when combined with RAOBs, provides a supportive scaffold for bone differentiation and may represent a novel bone-regeneration therapy for complex/extensive bone defects, even when combined with immature alveolar-bone osteoblasts from aged rats. Regenerative medicine combining scaffold materials and cell transplantation shows promise for widespread bone regeneration. This study focused on gelatin methacryloyl (GelMA) as a scaffold material. We developed GelMA (GelMA-RF) that gelated under visible light, avoiding the disadvantage of UV irradiation in conventional methods, and used it as a scaffold for cell transplantation. Furthermore, considering the increasing need for regenerative medicine in elderly patients, this study focused on using immature-osteoblasts derived from aged individuals as transplant cells to verify the bone regenerative capacity. In this study, immature-osteoblasts isolated from the alveolar bone of aged rats were encapsulated in GelMA-RF and transplanted into bone defects, confirming early bone regeneration sufficiently. Furthermore, in vitro analysis confirmed bone formation-related gene expression and calcification capacity, demonstrating that GelMA-RF is a suitable material for bone regeneration. These findings suggest that even alveolar bone immature-osteoblasts derived from aged individuals, when combined with GelMA-RF, hold promise as a novel therapeutic approach for regenerating complex and extensive bone defects.

This article is being retracted from Bioscience Reports at the request of the Editor-in-Chief and the Editorial Board. This follows the receipt of a notification from a reader, alerting the Editorial Board to irregularities in the flow cytometry graphs, some of which suggest that the graphs could have been hand-drawn. The authors were contacted regarding the concerns and the retraction but have not yet responded or provided requested raw data. Given the extent of the issues raised, the Editorial Board stand by the decision to retract the article.

This study aims to explore the mechanism of carvacrol (CV) in regulating alveolar bone repair in periodontal disease. A rat model of periodontal disease (P group) was created by ligating the first molar and injecting lipopolysaccharide (LPS). A control model (C group) was also created. The treatment models received low (L group), medium (M group), and high (H group) doses of CV hydrogel. Hematoxylin-eosin (HE) staining was used to observe the pathological changes in periodontal tissues. Immunohistochemical staining was employed to analyze the expression of collagen typeⅠ(COL1) and Runt-related transcription factor 2 (Runx2). In vitro, the rat osteoblast cells were divided into C, P, L, M, H, CV and CV+LY294002 (CV+LY) groups. Western blot analysis detected the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase 3 beta (GSK-3β) pathway-related and osteoblastic proteins. Quantitative reverse-transcription po-lymerase chain reaction was used to measure the expression of inflammatory factors and osteoblastic proteins. The alkaline phosphatase (ALP) colorimetric kit and alizarin red S staining kit were utilized to assess osteogenic ability. Immunofluorescence (IF) was used to detect COL1 expression in osteoblasts. Transmission electron microscopy was applied to detect cell apoptosis. CV hydrogel alleviated periodontal symptoms, upregulated PI3K/AKT/GSK-3β pathway-related and osteoblastic proteins, and increased the expression of ALP and the number of calcified nodules. However, it decreased cell apoptosis and inflammatory factors. LY294002 inhibited the PI3K/AKT pathway and decreased osteoblastic protein expression, ALP coloration, and calcified nodule quantity. CV hydrogel promotes the proliferation and differentiation of alveolar bone osteoblasts by activating the PI3K pathway and inhibiting inflammation-induced bone resorption. This study emphasizes the potential of CV for the treatment of periodontal diseases.

Objectives: We evaluated the effect of diacerein, an anti-inflammatory drug, on the activity and survival of alveolar bone osteoblasts in rats with periodontitis. Methods: The rats with periodontitis received diacerein (PDG) or saline solution (PSG) for 7, 15 and 30 days. In gingiva samples, Nfkb1 and Bmp2 gene expressions were evaluated, and maxillae were processed for light and transmission electron microscopy. Results: In PDG, the tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) immunoexpression decreased in parallel with the increase in alkaline phosphatase (ALP) and bone area over time. At 15 and 30 days, Nfkb1 expression decreased in PDG compared to PSG, whereas at 30 days, the Bmp2 expression was greater in PDG than in PSG. Immunofluorescence for IL-10, an anti-inflammatory cytokine, was greater in PDG than in PSG at 15 and 30 days. In PSG, the significant increase in the number of TUNEL-positive osteoblasts was accompanied by the presence of osteoblasts with condensed chromatin nuclei or caspase-3-immunolabelled osteoblasts. In contrast, the number of TUNEL-positive osteoblasts was significantly lower in PDG than in PSG specimens at all time points. Conclusions: Therefore, the diacerein-induced TNF-α and IL-1β inhibitory effect caused Nfkb1 downregulation and, hence, prevented apoptosis in osteoblasts. The increased ALP activity and IL-10 in PDG indicate that diacerein mitigates periodontitis impact on alveolar bone in rat molars.

ABSTRACTObjectiveGlucocorticoid‐induced osteonecrosis of the femoral head (GC‐ONFH) represents a devastating complication of steroid therapy, primarily driven by osteoblast apoptosis and impaired osteogenesis. Although selenium (Se) is renowned for its potent bone‐protective properties, its therapeutic potential, and specific mechanisms in GC‐ONFH remain largely unexplored and thus require further investigation.MethodsTo assess the therapeutic effectiveness of oral selenium supplementation in GC‐ONFH, a rat model of GC‐ONFH was utilized. The rats were randomly allocated into three groups (n = 6 per group): (1) Control group, (2) Methylprednisolone sodium succinate (MPS) group, and (3) Se group. The intervention was carried out for 4 weeks. In vitro experiments utilized primary rat osteoblasts and MC3T3‐E1 cells to elucidate the mechanisms through which selenium mitigates dexamethasone (DEX)‐induced alterations in cell proliferation, apoptosis, and osteogenic differentiation. The assessments were conducted using micro‐CT and histomorphometry, CCK‐8 assays and flow cytometry, as well as RT–qPCR, Western blotting, and immunofluorescence.ResultsSelenium supplementation effectively prevented trabecular collapse and significantly reduced the number of empty lacunae in rats with GC‐ONFH. Specifically, an optimal dose of 10 μmol Se successfully reversed the damage induced by DEX, including the restoration of cell proliferation, suppression of apoptosis, and rescue of osteogenic activity. Mechanistically, Se counteracts the DEX‐induced suppression of phosphorylated phosphatidylinositol 3‐kinase (p‐PI3K), phosphorylated protein kinase B (p‐AKT), and phosphorylated glycogen synthase kinase 3β (GSK3β) (p‐GSK3β), thereby activating the PI3K/AKT/GSK3β signaling pathway, which promotes cell proliferation, inhibits apoptosis, and enhances osteogenesis in osteoblasts.ConclusionSelenium can activate the PI3K/AKT/GSK3β pathway, reverse DEX‐induced hypoproliferation and apoptosis, restore osteogenic capacity, prevent trabecular collapse, and attenuate GC‐ONFH in rat models. Our findings demonstrate that selenium supplementation can be regarded as a clinically applicable strategy for impeding the progression of GC‐ONFH in at‐risk patients.

ABSTRACTOsteoblast dysfunction plays a central role in osteoporosis. CXC chemokine ligand 1 (CXCL1), a known inflammatory mediator, is increasingly recognised for its role in bone homeostasis. However, its influence on osteoblast survival mechanisms such as ferroptosis and autophagy remains unclear. This study explores the role of CXCL1 in promoting osteoblast differentiation and activity by inhibiting ferroptosis and enhancing autophagy via the TGF‐β/Smad signalling pathway. Primary rat osteoblasts were treated with recombinant CXCL1, shRNA constructs and pathway modulators, such as Galunisertib, Fer‐1 and Chloroquine (CQ). Osteoblast differentiation, autophagy, ferroptosis and TGF‐β/Smad pathway activity were evaluated using qPCR, western blotting, staining and densitometric analysis. CXCL1 knockdown impaired osteoblast proliferation and differentiation, while increasing intracellular iron and ROS and enhancing ACSL4 expression, indicative of ferroptosis. These effects were partially reversed by Fer‐1. Besides, CXCL1 activated the TGF‐β/Smad signalling cascade, and Galunisertib inhibited this signalling and partially suppressed CXCL1‐induced effects. Furthermore, CXCL1 promoted autophagy via increased Beclin‐1 and LC3B and reduced p62, which mitigated ferroptosis and supported osteogenesis. Our findings suggest that CXCL1 promotes osteoblast differentiation by inhibiting ferroptosis and enhancing autophagy through activation of the TGF‐β/Smad signalling pathway. Collectively, our results highlight CXCL1 as a promising therapeutic target for osteoporosis.

Oral biofilms contribute to apical periodontitis, a major cause of tooth loss. This study evaluated the potential of an on-demand aqueous chlorine dioxide solution, “matching transformation system” (MA-T), as a safer alternative to conventional root canal irrigants. The antibacterial activity of MA-T was assessed against mono-species biofilms (Enterococcus faecalis, Parvimonas micra, Fusobacterium nucleatum), polymicrobial biofilms derived from human supragingival plaque, and an in vitro infected root canal model using E. faecalis. Antibacterial efficacy was evaluated using viable cell counting and confocal laser scanning microscopy (CLSM) to quantify the reduction in bacterial load. Biofilm mass was assessed by crystal violet (CV) staining. Cytotoxicity was evaluated by treating rat osteoblasts and human periodontal ligament fibroblasts with MA-T or NaOCl, followed by assessment of ATP levels and morphological integrity. MA-T eradicated planktonic bacteria, inhibited biofilm formation, and significantly reduced viable bacteria in established biofilms without affecting the total amount of biofilm biomass time-dependently. The infected root canal model showed a substantial decrease in bacterial load. Compared with NaOCl, MA-T-treated cells retained higher ATP levels and normal morphology. MA-T could be a promising root canal irrigant offering effective disinfection and superior biocompatibility, making it a potential alternative to traditional root canal irrigants as NaOCl.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-20131-5.

Bioscience Reports has been made aware of potential issues surrounding the scientific validity of this paper and hence is issuing an Expression of Concern to notify readers whilst the Editorial Office investigates. Irregularities have been noted in the flow cytometry graphs – the authors were contacted for comment but have not yet responded or provided requested raw data.

Expression of Concern: Extracellular regulated kinase 5 mediates osteoporosis through modulating viability and apoptosis of osteoblasts in ovariectomized rats.

Chronic exposure to cadmium (Cd) has been linked to bone dysfunction, including osteomalacia and osteoporosis. Such dysfunction may result from indirect disturbances in calcium (Ca) and phosphorus metabolism due to Cd-induced nephrotoxicity, as well as from direct Cd accumulation in bone tissue. However, the mechanisms by which Cd is taken up by bone-derived cells such as osteoblasts remain unclear. Cd may enter cells via metal transporters, such as ZIP8, ZIP14, and DMT1, as well as Ca2+ channels, including voltage-gated Ca2+ channels, TRPV6, and TRPM7. In this study, we systematically analyzed Cd transport efficiency and the expression levels of candidate pathways using rat osteosarcoma-derived UMR-106 cells, rat liver-derived TRL1215 cells, and rat basophilic leukemia RBL-2H3 cells. Cd accumulation in UMR-106 cells was similar to that in TRL1215 cells but lower than that in RBL-2H3 cells, which exhibited the highest expression of ZIP8 and ZIP14. The uptake of Cd2+ and Mn2+ in UMR-106 cells was competitively inhibited by Mn2+ and Cd2+, respectively, a pattern characteristic of ZIP8- and ZIP14-mediated transport. Treatment with verapamil, an L-type Ca2+ channel inhibitor, significantly reduced Cd uptake in UMR-106 cells. Suppression of ZIP8, ZIP14, DMT1, CaV1.3 (a component of L-type Ca2+ channels), TRPV6, and TRPM7 expression via siRNA transfection also significantly reduced Cd uptake. However, the degree of reduction ranged from 15 to 35%, indicating that no single pathway makes a predominant contribution. These findings suggest that diverse pathways, encompassing metal transporters and Ca2+ channels, contribute to Cd uptake in UMR-106 cells, although no single pathway predominates.

Objective Osteoporosis (OP) is a systemic skeletal disease that increases the risk of fractures by weaking bone. Hypoxia-inducible factor-1α (HIF-1α) plays a crucial role in osteogenesis and osteoblastic differentiation. The purpose of this study is to examine the underlying mechanism of HIF-1α overexpression and its impact on osteoblast development. Methods First, we used the autophagy inhibitor 3-MA in conjunction with either a control lentivirus or an HIF-1α overexpression lentivirus to transfect rat osteoblasts in osteogenic induction media for 3, 7, 14, and 21 days. The effects of HIF-1α overexpression on osteogenic differentiation were evaluated using CCK-8, alkaline phosphatase (ALP) staining, and Alizarin Red staining. Furthermore, we investigated the mechanism by which HIF-1α overexpression mediates autophagy to regulate osteogenic differentiation through immunofluorescence, western blot, and transmission electron microscopy. Ovariectomy (OVX) was performed to establish an osteoporotic rat model. The impact of HIF-1α overexpression on autophagy and bone metabolism was evaluated by locally injecting HIF-1α overexpression lentivirus or control lentivirus, in combination with HE staining, micro-CT, immunohistochemistry, ELISA, western blot, and transmission electron microscopy. Results Overexpression of HIF-1a promotes osteoblast proliferation and enhances ALP staining as well as calcium nodule formation. In addition, the overexpression of HIF-1a significantly increases the relative protein expression levels of osteocalcin (OCN), osteoprotegerin (OPG), HIF-1a, BNIP3, Beclin1, ATG5, and LC3 II/I. This indicates that HIF-1a may facilitate osteoblast differentiation by promoting autophagy. These findings were further corroborated by in vivo experiments, which demonstrated improved pathological morphology in rat femurs, alongside increased bone mineral density (BMD), trabecular thickness (Tb. Th), bone volume/total volume ratio (BV/TV), and trabecular number (Tb. N). Additionally, there was a decrease in trabecular separation (Tb. Sp) and structural model index (SMI), along with upregulated expression of OCN, OPG, HIF-1α, BNIP3, Beclin1, ATG5, and LC3 II. Conclusion HIF-1a overexpression can promote osteogenic differentiation and ameliorate osteoporosis through the induction of autophagy. These insights provide a valuable reference for its potential application in targeted therapy. Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-16046-w.

Cellular damage induced by oxidative stress contributes to systemic bone disorders leading to osteoporosis. Bone homeostasis regulates the balance between the functions of osteoblasts and osteoclasts. Osteoblast cells are responsible for bone formation and are very sensitive to oxidative stress. Polyphenolic compounds possess the ability to scavenge free radicals, thus reducing intracellular oxidative stress. Natural compounds such as salvianolic acid A (SAL-A) exhibit prominent antioxidant properties. However, its antioxidant role in bone homeostasis is poorly defined. In this study, we aimed to elucidate the potential role of SAL-A in protecting the osteoblasts from H2O2-induced oxidative stress. Rat osteoblast cells were treated with or without 500μM H2O2 in the presence or absence of 5μM and 10μM of SAL-A. A series of assays such as cell viability by CCK-8 kit, detection of reactive oxygen species by dichlorodihydrofluorescein diacetate (DCFH-DA), mitochondrial membrane potential by JC-1 fluorescence, level of bone mineralization proteins osteocalcin, bone sialoprotein, and alkaline phosphatase by immunocytochemistry studies, were conducted. SAL-A protected the rat osteoblast cells from H2O2-induced cytotoxicity by significantly attenuating free radical generation, thus improving cell viability. SAL-A treatment also significantly restored bone mineralization proteins, including osteocalcin, bone sialoprotein, and alkaline phosphatase, which were aggravated by H2O2-induced oxidative stress. The study results provide the role of SAL-A in protecting the osteoblasts from H2O2-induced oxidative stress in rat osteoblast cells by scavenging the free radicals, increasing the cell viability, mineralization, and differentiation of osteoblasts.

Background:Compelling evidence has implicated osteoblast ferroptosis as a critical contributor to the pathogenesis of glucocorticoid-induced osteoporosis (GIOP). However, the underlying regulatory mechanisms remain poorly understood.Methods:In the glucocorticoids (GCs)-induced GIOP rat model, both osteoblast dysfunction and ferroptosis markers were assessed. Ferroptosis was inhibited with deferoxamine (DFO). Transcriptomic profiling was performed to analyze the correlation between mitophagy and SIRT3 levels. In MC3T3-E1 cells exposed to GCs, we examined key mitophagy markers PINK1 and PARKIN, mitochondrial function, and SIRT3 expression. Treatments included DFO, mitophagy inhibitors (Mdivi-1), and SIRT3 agonists (Nicotinamide riboside chloride).Results:In the GIOP rat model, significant osteoblast dysfunction and elevated ferroptosis markers were observed. Although DFO treatment inhibited ferroptosis, it failed to restore osteogenesis, suggesting the involvement of additional regulatory mechanisms in osteogenic function regulation. Transcriptomic profiling highlighted a robust correlation between mitophagy and SIRT3 levels in GIOP. In GC-exposed MC3T3-E1 cells, key mitophagy markers PINK1 and PARKIN were upregulated, mitochondrial function was impaired, and SIRT3 expression was significantly reduced. Notably, while DFO treatment did not restore mitochondrial homeostasis, the application of Mdivi-1 (mitophagy inhibitor) and Nicotinamide riboside chloride (SIRT3 agonists) effectively alleviated ferroptosis and restored mitochondrial function.Conclusions:SIRT3 regulates ferroptosis by inhibiting excessive mitophagy in osteoblasts, providing a novel mechanistic pathway for mitigating GIOP. These findings suggest that SIRT3 represents a critical regulator of mitophagy-dependent ferroptosis and a potential therapeutic target for GIOP.

Objectives: Osteoarthritis (OA) is a prevalent chronic degenerative joint disorder marked by cartilage degradation, subchondral bone remodeling, and synovial inflammation. Despite its widespread occurrence, effective pharmacological interventions to halt or reverse OA progression remain elusive. Thus, an in-depth understanding of its pathogenesis is imperative for developing novel therapeutic strategies. Methods: Sixty-four male Sprague-Dawley rats (8 weeks old, weighing 180-220 g) were randomly assigned to two groups: the anterior cruciate ligament transection (ACLT) group and the sham-operated group. Primary osteoblasts were isolated from the subchondral bone at 0, 4, 8, and 12 weeks after ACLT. Nuclear magnetic resonance (NMR)-based metabolomics was used to elucidate metabolic changes and the underlying mechanisms in osteoblasts. Results: A total of 26 metabolites were identified from the NMR spectra of osteoblasts. Distinct metabolic profiles were observed in the ACLT group at 0, 4, 8, and 12 weeks after surgery. In particular, several differential metabolites were identified, including glucose, lactate, NADP+, phosphocreatine, and alanine, as well as eight perturbed pathways, such as alanine, aspartate, and glutamate metabolism, phenylalanine metabolism, and taurine metabolism. Conclusions: Key energy-related metabolites, including glucose, lactate, creatine phosphate, and creatine, were identified as key markers of osteoblast dysfunction in OA, underscoring the profound metabolic perturbations induced by ACL injury. These disturbances in energy homeostasis are strongly implicated in the progression of OA. In addition, branched-chain amino acids emerged as potential biomarkers, further highlighting the metabolic dysregulation associated with the disease. Taken together, the metabolic changes observed in rat osteoblasts following ACL injury reveal a complex interplay between energy and amino acid metabolism, providing critical insights into the pathogenesis of post-traumatic OA and highlighting potential therapeutic targets.

BackgroundParthenolide (PTL) is a natural sesquiterpene lactone that possesses significant effects on stimulating osteoblast differentiation. The present study focused on the potential of PTL in the treatment of glucocorticoid-induced osteoporosis (GIOP).MethodsMC3T3-E1 cells were treated with dexamethasone (DEX; 10 µM) or/and PTL (5, 10, and 20 µM). The changes in osteogenic differentiation were analyzed by conducting ALP and Alizarin Red staining and assessing the levels of osteogenic markers (Runx2, Osx, and OPN). PTL (3 and 10 mg/kg/day) was injected into rat models of GIOP induced by DEX. Bone formation was analyzed by assessing the levels of bone turnover markers (ALP, TRAP, OCN, and CTx) in the serum and osteoblast differentiation markers (BMP2 and Runx2) in the femurs. The pathological changes of the femurs were determined by H&E staining. Bone mass and osteoblast numbers in the femurs were measured. Western blotting evaluated ERK phosphorylation in vitro and in vivo.ResultsPTL promoted osteogenic differentiation and enhanced the levels of Runx2, Osx, OPN, and ERK phosphorylation in DEX-treated MC3T3-E1 cells. ERK inhibitor U0126 reversed the promoting effect of PTL on osteogenesis in DEX-treated MC3T3-E1 cells. After the administration of PTL in rat models of GIOP, the levels of ALP, TRAP, OCN, and CTx in the serum and the levels of BMP2, Runx2, and ERK phosphorylation in the femurs were restored. PTL increased trabecular bone number, reduced trabecular separation, and increased the number of osteoblasts in GIOP rat model.ConclusionOverall, PTL alleviates osteoporosis by promoting osteogenic differentiation via activation of ERK signaling.

ObjectivesOxymatrine (OMT), a quinolizidine alkaloid derived from Sophora flavescens Ait., has demonstrated therapeutic potential in type 2 diabetes mellitus (T2DM). This study aimed to investigate its effects on diabetic osteoporosis (DOP) and explore the underlying mechanisms involving gut microbiota and osteogenic regulation.MethodsIn a rat model of T2DM, intragastric Oxymatrine was used to study trabecular bone repair through bone microstructure and histopathology analyses. Changes in gut microbiota, especially Gram-negative bacteria releasing lipopolysaccharides (LPS), were assessed via 16S rRNA sequencing. miRNA sequencing on LPS-induced rat osteoblasts, with and without Oxymatrine, explored osteoblast proliferation, mineralization, and the miR-539-5p/OGN/Runx2 pathway.ResultsThe administration of OMT resulted in an enhancement of diabetic osteopathy by reversing trabecular bone loss and modifying the composition of gut microbiota, specifically affecting Gram-negative bacteria that release LPS into the bloodstream. miRNA sequencing revealed that miR-539-5p, which was upregulated in LPS-induced ROBs, was downregulated following OMT treatment. Furthermore, OMT was found to promote osteoblast proliferation and mineralization under conditions of LPS exposure and modulate the miR-539-5p/OGN/Runx2 signaling pathway.ConclusionsOMT improves diabetic osteoporosis by altering gut microbiota, decreasing LPS release, and enhancing osteoblast growth and differentiation through the miR-539-5p/OGN/Runx2 pathway, suggesting its potential as a treatment.

PurposeThis study aims to evaluate the effects of miR-374-5p on osteogenesis in rat osteoblasts, validate its target on PTEN, and explore its role in the PTEN/PI3K/AKT signaling pathway during osteoblast differentiation.MethodsWe transfected 293T cells with miR-374-5p mimics and inhibitors, followed by Western blot and qRT-PCR analyses to assess protein and mRNA expression levels. A dual-luciferase assay was performed to confirm direct targeting. Markers of osteoblast function, such as Runx2, OSX, and OCN, were examined in osteoblasts from rats by qRT-PCR and Western blot. Additionally, we developed a lentiviral vector to overexpress miR-374-5p, which successfully infected rat osteoblast progenitors. Bone formation was subsequently assessed using Alizarin Red staining and ALP activity assays. Finally, rescue experiments were conducted to validate the involvement of the miR-374-5p/PTEN/PI3K/AKT signaling pathway.ResultsOur results demonstrate that miR-374-5p significantly downregulates both the protein and mRNA levels of its target gene PTEN, as confirmed by dual luciferase assays. qRT-PCR and Western blot analyses revealed that osteoblastic markers-including Runx2, OSX, and OCN—were markedly reduced in the miR-374-5p mimic group, whereas an opposite trend was observed in the inhibitor group. In vitro, overexpression of miR-374-5p suppressed osteoblast differentiation, as evidenced by decreased calcium nodule formation and reduced ALP activity compared to controls. Furthermore, co-transfection of miR-374-5p mimics with the PI3K/AKT pathway inhibitor LY294002 in osteoblasts led to significantly lower expression of PI3K/AKT pathway-related genes, and notably, the inhibitory effect of miR-374-5p on osteoblast differentiation was reversed by LY294002 treatment.ConclusionOur findings indicate that miR-374-5p inhibits osteogenesis in rat osteoblasts by targeting PTEN and modulating the PI3K/AKT signaling pathway.

This study focuses on exploring the impact of Astragaloside IV [AS-IV] on osteogenic differentiation. Osteogenic differentiation was induced in rat osteoblasts, following which treatment with AS-IV at varied doses was performed. Using Alizarin red staining and alkaline phosphatase (ALP) detection assay, the osteogenic differentiation of the cells was investigated. The expressions of osteogenic differentiation-related genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathwayassociated protein expressions were examined using Western blot. After osteoblasts were transfected with protein tyrosine phosphatase non-receptor type 2 (PTPN2) overexpression plasmid, the impact of PTPN2 on osteoblasts treated with AS-IV was examined. AS-IV treatment enhanced osteogenic differentiation and up-regulated the expression of osteogenic differentiation-related genes, as well as the levels of p- PI3K/PI3K and p-AKT/AKT, while reducing phosphatase and tensin homolog (PTEN) protein production in osteoblasts. Overexpression of PTEN inhibited osteogenic differentiation, and PTPN2 overexpression counteracted the effects of AS-IV on osteogenic differentiation. AS-IV contributing to osteogenic differentiation may be related to the PTPN2-mediated PTEN/PI3K/Akt pathway.

Alleviating osteoarthritis-induced damage through extracellular vesicles derived from inflammatory chondrocytes.