Inhibition Of Osteogenic Differentiation Research Articles

Local Application of Tanshinone IIA protects mesenchymal stem cells from apoptosis and promotes fracture healing in ovariectomized mice

BackgroundElderly patients suffering from osteoporotic fractures are more susceptible to delayed union or nonunion, and their bodies then are in a state of low-grade chronic inflammation with decreased antioxidant capacity. Tanshinone IIA is widely used in treating cardiovascular and cerebrovascular diseases in China and has anti-inflammatory and antioxidant effects. We aimed to observe the antioxidant effects of Tanshinone IIA on mesenchymal stem cells (MSCs), which play important roles in bone repair, and the effects of local application of Tanshinone IIA using an injectable biodegradable hydrogel on osteoporotic fracture healing.MethodsMSCs were pretreated with or without different concentrations of Tanshinone IIA followed by H2O2 treatment. Ovariectomized (OVX) C57BL/6 mice received a mid-shaft transverse osteotomy fracture on the left tibia, and Tanshinone IIA was applied to the fracture site using an injectable hydrogel.ResultsTanshinone IIA pretreatment promoted the expression of nuclear factor erythroid 2-related factor 2 and antioxidant enzymes, and inhibited H2O2-induced reactive oxygen species accumulation in MSCs. Furthermore, Tanshinone IIA reversed H2O2-induced apoptosis and decrease in osteogenic differentiation in MSCs. After 4 weeks of treatment with Tanshinone IIA in OVX mice, the bone mineral density of the callus was significantly increased and the biomechanical properties of the healed tibias were improved. Cell apoptosis was decreased and Nrf2 expression was increased in the early stage of callus formation.ConclusionsTaken together, these results indicate that Tanshinone IIA can activate antioxidant enzymes to protect MSCs from H2O2-induced cell apoptosis and osteogenic differentiation inhibition. Local application of Tanshinone IIA accelerates fracture healing in ovariectomized mice.

MicroRNA-18a regulates the Pyroptosis, Apoptosis, and Necroptosis (PANoptosis) of osteoblasts induced by tumor necrosis factor-α via hypoxia-inducible factor-1α

BackgroundTumor necrosis factor-α (TNF-α) is involved in inflammatory responses and promotes cell death and the inhibition of osteogenic differentiation. MicroRNA (miRNA) plays a crucial role in the infected bone diseases, however, the biological role of miRNAs in inflammation-induced impaired osteogenic differentiation remains unclear. This study aimed to explore the role of miRNA-18a-5p (miR-18a) in regulating PANoptosis and osteogenic differentiation in an inflammatory environment via hypoxia-inducible factor-1α (HIF1-α). MethodsThe expression of miR-18a in MC3T3-E1 cells was analyzed using quantitative reverse transcription-polymerase chain reaction in an inflammatory environment induced by TNF-α. The expression of HIF1-α and NLRP3 in LV-miR-18a or sh-miR-18a cells was analyzed using western blotting. Fluorescence imaging for cell death, flow cytometry, and alkaline phosphatase activity analysis were used to analyze the role of miR-18a in TNF-α-induced PANoptosis and the inhibition of osteogenic differentiation. An animal model of infectious bone defect was established to validate the regulatory role of miR-18a in an inflammatory environment. ResultsThe expression of miRNA-18a in the MC3T3-E1 cell line was significantly lower under TNF-α stimulation than in the normal environment. miR-18a significantly inhibited the expression of HIF1-α and NLRP3, and inhibition of HIF1-α expression further inhibited NLRP3 expression. Furthermore, inhibition of miR-18a expression promoted the TNF-α-induced PANoptosis and inhibition of osteogenic differentiation, whereas miR-18a overexpression and the inhibition of both HIF1-α and NLRP3 reduced the effects of TNF-α. These findings are consistent with those of the animal experiments. ConclusionmiRNA-18a negatively affects HIF1-α/NLRP3 expression, inhibits inflammation-induced PANoptosis, and impairs osteogenic differentiation. Thus, it is a potential therapeutic candidate for developing anti-inflammatory strategies for infected bone diseases.

Effect of Adefovir Dipivoxil on the Inhibition of Osteogenic Differentiation of Mesenchymal Stem Cells and Osteoblasts

Effect of Adefovir Dipivoxil on the Inhibition of Osteogenic Differentiation of Mesenchymal Stem Cells and Osteoblasts

PCAT-1' s role in wound healing impairment: Mitochondrial dysfunction and bone marrow stem cell differentiation inhibition via PKM2/β-catenin pathway and its impact on implant osseo-integration.

This study focused on unravelling the role of PCAT-1 in wound-healing process, particularly its impact on regenerative and osteogenic abilities of mesenchymal stem cells (MSCs). We delved into how PCAT-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) and interacts with pivotal molecular pathways, especially β-catenin and PKM2, using human bone marrow-derived MSCs. MSCs were cultured under specific conditions and PCAT-1 expression was modified through transfection. We thoroughly assessed several critical parameters: MSC proliferation, mitochondrial functionality, ATP production and expression of wound healing and osteogenic differentiation markers. Further, we evaluated alkaline phosphatase (ALP) activity and mineral deposition, essential for bone healing. Our findings revealed that overexpressing PCAT-1 significantly reduced MSC proliferation, hampered mitochondrial performance and lowered ATP levels, suggesting the clear inhibitory effect of PCAT-1 on these vital wound-healing processes. Additionally, PCAT-1 overexpression notably decreased ALP activity and calcium accumulation in MSCs, crucial for effective bone regeneration. This overexpression also led to the reduction in osteogenic marker expression, indicating suppression of osteogenic differentiation, essential in wound-healing scenarios. Moreover, our study uncovered a direct interaction between PCAT-1 and the PKM2/β-catenin pathway, where PCAT-1 overexpression intensified PKM2 activity while inhibiting β-catenin, thereby adversely affecting osteogenesis. This research thus highlights PCAT-1's significant role in impairing wound healing, offering insights into the molecular mechanisms that may guide future therapeutic strategies for enhancing wound repair and bone regeneration.

The Effect of Two Years of Secukinumab Treatment on Bone Metabolism in Patients with Radiographic Axial Spondyloarthritis: Results from Daily Clinical Practice.

Our objective was to explore bone-related outcome and bone turnover markers (BTM) during 2 years of secukinumab treatment in patients with radiographic axial spondyloarthritis (r-axSpA) in daily clinical practice. Included were consecutive r-axSpA outpatients from the Groningen Leeuwarden axSpA (GLAS) cohort treated with secukinumab for 2 years. At baseline and 2 years, spinal radiographic damage was assessed using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS; 0-72), cervical facet joint involvement according the "de Vlam" scoring method (0-15) and radiographic vertebral fractures (VF) using the "Genant" method (grade 0-3). At all visits, BTM reflecting collagen resorption (serum type I collagen C-telopeptide; sCTX), collagen formation (procollagen type 1 N-terminal peptide; PINP) and bone mineralization (bone-specific alkaline phosphatase; BALP) were measured and expressed in Z-scores to correct for the normal influence of age and gender. 17 r-axSpA patients were included; 53% male, mean age was 47±15 years, mean Ankylosing Spondylitis Disease Activity Score (ASDAS) 3.9±1.2, and 53% was biological naïve. The median 2-year progression rates were 1.1 for mSASSS and 0.5 for facet joints, which was less than the smallest detectable change. One traumatic VF (grade 3) occurred. Serum levels of sCTX and PINP remained stable during secukinumab treatment and BALP decreased significantly after 2 years, with median 0-2 year change in Z-scores of +0.1, -0.4, and -1.2, respectively. This explorative study of r-axSpA patients treated with secukinumab in daily clinical practice showed low radiographic spinal progression during 2 years of follow-up. Collagen resorption and formation markers remained stable, whereas mineralization marker BALP decreased significantly after 2 years. Our results are in line with the results of in vitro studies demonstrating that inhibition of IL17-A resulted in suppression of osteogenic differentiation with significant decrease in mineralization.

HuR-mediated nucleocytoplasmic translocation of HOTAIR relieves its inhibition of osteogenic differentiation and promotes bone formation

Bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoblast function play critical roles in bone formation, which is a highly regulated process. Long noncoding RNAs (lncRNAs) perform diverse functions in a variety of biological processes, including BMSC osteogenic differentiation. Although several studies have reported that HOX transcript antisense RNA (HOTAIR) is involved in BMSC osteogenic differentiation, its effect on bone formation in vivo remains unclear. Here, by constructing transgenic mice with BMSC (Prx1-HOTAIR)- and osteoblast (Bglap-HOTAIR)-specific overexpression of HOTAIR, we found that Prx1-HOTAIR and Bglap-HOTAIR transgenic mice show different bone phenotypes in vivo. Specifically, Prx1-HOTAIR mice showed delayed bone formation, while Bglap-HOTAIR mice showed increased bone formation. HOTAIR inhibits BMSC osteogenic differentiation but promotes osteoblast function in vitro. Furthermore, we identified that HOTAIR is mainly located in the nucleus of BMSCs and in the cytoplasm of osteoblasts. HOTAIR displays a nucleocytoplasmic translocation pattern during BMSC osteogenic differentiation. We first identified that the RNA-binding protein human antigen R (HuR) is responsible for HOTAIR nucleocytoplasmic translocation. HOTAIR is essential for osteoblast function, and cytoplasmic HOTAIR binds to miR-214 and acts as a ceRNA to increase Atf4 protein levels and osteoblast function. Bglap-HOTAIR mice, but not Prx1-HOTAIR mice, showed alleviation of bone loss induced by unloading. This study reveals the importance of temporal and spatial regulation of HOTAIR in BMSC osteogenic differentiation and bone formation, which provides new insights into precise regulation as a target for bone loss.

Pmepa1 knockdown alleviates SpA-induced pyroptosis and osteogenic differentiation inhibition of hBMSCs via p38MAPK/NLRP3 axis

BackgroundOsteomyelitis is a refractory bone infectious disease, which usually results in progressive bone destruction and bone loss. The invasion of pathogens and subsequent inflammatory response could damage bone marrow mesenchymal stem cells (BMSCs) and inhibit osteogenic differentiation, and finally aggravate uncontrolled bone remodeling in osteomyelitis by affecting bone formation. Exploring the mechanisms of BMSCs injury and osteogenic differentiation inhibition may would help us to find potential therapeutic targets. MethodFirstly, staphylococcal protein A (SpA)-treated human bone marrow mesenchymal stem cells (hBMSCs) were used to construct cell models of osteomyelitis. Secondly, transcriptome sequencing was performed to screen differentially expressed genes and then verified the expression of target genes. Next, in vitro experiments were conducted to explore the functions and mechanisms of prostate transmembrane protein androgen induced 1 (Pmepa1) in SpA-treated hBMSCs. Finally, the rat model of osteomyelitis was established to provide an auxiliary validation of the in vitro experimental results. ResultsWe found that SpA treatment induced inflammatory injury and inhibited osteogenic differentiation in hBMSCs, then the transcriptome sequencing and further detection results showed that Pmepa1 was significantly upregulated in this process. Functionally, Pmepa1 knockdown alleviated inflammatory injury and promoted osteogenic differentiation in SpA-treated hBMSCs. Among them, it was demonstrated that Pmepa1 knockdown exerted cytoprotective effects by alleviating pyroptosis of SpA-infected hBMSCs. Furthermore, recovery experiments revealed that Pmepa1 knockdown reversed SpA-mediated adverse effects by downregulating the p38MAPK/NLRP3 axis. Finally, the detection results of rat femoral osteomyelitis showed that the expression of Pmepa1 was up-regulated, and the expression trends of other indicators including p38MAPK, NLRP3, and caspase-1 were also consistent with the in vitro model. ConclusionPmepa1 knockdown alleviates SpA-induced pyroptosis and inhibition of osteogenic differentiation in hBMSCs by downregulating p38MAPK/NLRP3 signaling axis. Modulating the expression of Pmepa1 may be a potential strategy to ameliorate osteomyelitis.

Lipocalin 2 induces self-limited inhibition of osteoblast differentiation of mesenchymal stem cells

To explore the role of lipocalin 2 (Lcn2) in bone development and senile osteoporosis. Chromatin immunoprecipitation with H3K27AC and H3K9Me3 antibodies coupled with massively parallel sequencing (ChIP-Seq) was performed to analyze the changes in binding of modified histones at the Lcn2 gene locus in C3H10T1/2 mesenchymal stem cells (MSCs) induced with osteogenic induction medium for 3 days. ITRAQ-MS/MS and ELISA were used to compare Lcn2 protein expressions in the cancellous bone and the serum between 16- and 3-month-old male mice. The effect of treatment with recombinant Lcn2 protein on osteogenic differentiation of C3H10T1/2 cells was evaluated by detecting changes in ALP expression, and Western blotting was performed to examine the changes in OSX and OCN expression. The expression of Lcn2 protein in the cancellous bone and its serum levels were significantly higher in 16-month-old than in 3-month-old male mice (P < 0.01). In C3H10T1/2 cells, ALP expression level decreased significantly after treatment with recombinant Lcn2 protein (P < 0.05) accompanied by lowered protein expressions of OSX and OCN (P < 0.05). Analysis with ROSE software revealed a super enhancer in the Lcn2 gene region of C3H10T1/2 cells after osteogenesis induction. RNA-seq results confirmed that Lcn2 was among the top 4 up-regulated genes in C3H10T1/2 cells following osteogenic induction, and a super enhancer was also detected in Zbtb16 gene, which showed the highest up- regulation after osteogenic induction. Real- time quantitative PCR further confirmed that the mRNA level of Lcn2 increased sharply in C3H10T1/2 cells after osteogenic induction (P < 0.001). Lcn2 is essential for normal osteogenic differentiation of MSCs, but its overexpression and excessive secretion in aging induce self-limited inhibition of osteogenic differentiation of MSCs.

Melatonin promotes osteogenesis of bone marrow mesenchymal stem cells by improving the inflammatory state in ovariectomized rats

To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.

Thioredoxin-1 promotes the restoration of alveolar bone in periodontitis with diabetes

Treatment of periodontitis in people with diabetes remains challenging. The present study aimed to investigate the therapeutic potential of thioredoxin-1 (TRX1) in periodontitis with diabetes, as well as its role in modulating osteogenic differentiation. Our findings indicated that the production of reactive oxygen species (ROS) was elevated, while the expression of TRX1 was significantly reduced in the periodontal tissues of periodontitis mice with diabetes. Furthermore, knockdown of TRX1 in periodontal ligament stem cells (PDLSCs) resulted in the inhibition of osteogenic differentiation through disrupting Wnt/β-catenin signaling. However, this inhibition was restored upon administration of recombinant human TRX1 (rhTRX1). Importantly, rhTRX1 treatment decreased ROS generation, activated Wnt/β-catenin signal pathway and considerably promoted the alveolar bone repair of periodontitis mice with diabetes. These findings highlighted the crucial protective role of TRX1 in periodontitis with diabetes and suggested that it may serve as a potential therapeutic target for refractory periodontitis associated with oxidative stress.

Hmga1-overexpressing lentivirus protects against osteoporosis by activating the Wnt/β-catenin pathway in the osteogenic differentiation of BMSCs.

Postmenopausal osteoporosis is associated with bone formation inhibition mediated by the impaired osteogenic differentiation potential of bone marrow mesenchymal stem cells (BMSCs). However, identifying and confirming the essential genes in the osteogenic differentiation of BMSCs and osteoporosis remain challenging. The study aimed at revealing the key gene that regulated osteogenic differentiation of BMSCs and led to osteoporosis, thus exploring its therapeutic effect in osteoporosis. In the present study, six essential genes related to the osteogenic differentiation of BMSCs and osteoporosis were identified, namely, fibrillin 2 (Fbn2), leucine-rich repeat-containing 17 (Lrrc17), heat shock protein b7 (Hspb7), high mobility group AT-hook 1 (Hmga1), nexilin F-actin-binding protein (Nexn), and endothelial cell-specific molecule 1 (Esm1). Furthermore, the in vivo and in vitro experiments showed that Hmga1 expression was increased during the osteogenic differentiation of rat BMSCs, while Hmga1 expression was decreased in the bone tissue of ovariectomized (OVX) rats. Moreover, the expression of osteogenic differentiation-related genes, the activity of alkaline phosphatase (ALP), and the number of mineralized nodules were increased after Hmga1 overexpression, which was partially reversed by a Wnt signaling inhibitor (DKK1). In addition, after injecting Hmga1-overexpressing lentivirus into the bone marrow cavity of OVX rats, the bone loss, and osteogenic differentiation inhibition of BMSCs in OVX rats were partially reversed, while osteoclast differentiation promotion of BMSCs in OVX rats was unaffected. Taken together, the present study confirms that Hmga1 prevents OVX-induced bone loss by the Wnt signaling pathway and reveals that Hmga1 is a potential gene therapeutic target for postmenopausal osteoporosis.

Quercetin promotes autophagy to alleviate cigarette smoke-related periodontitis.

Cigarette smoking has been reported as an independent risk factor for periodontitis. Tobacco toxins affect periodontal tissue not only locally but also systemically, leading to the deterioration and recurrence of periodontitis. However, the mechanism of cigarette smoke-related periodontitis (CSRP) is unclear and thus lacks targeted treatment strategies. Quercetin, a plant-derived polyphenolic flavonoid, has been reported to have therapeutic effects on periodontitis due to its documented antioxidant activity. This study aimed to evaluate the effects of quercetin on CSRP and elucidated the underlying mechanism. The cigarette smoke-related ligature-induced periodontitis mouse model was established by intraperitoneal injection of cigarette smoke extract (CSE) and silk ligation of bilateral maxillary second molars. Quercetin was adopted by gavage as a therapeutic strategy. Micro-computed tomography was used to evaluate the alveolar bone resorption. Immunohistochemistry detected the oxidative stress and autophagy markers in vivo. Cell viability was determined by Cell Counting Kit-8, and oxidative stress levels were tested by 2,7-dichlorodihydrofluorescein diacetate probe and lipid peroxidation malondialdehyde assay kit. Alkaline phosphatase and alizarin red staining were used to determine osteogenic differentiation. Network pharmacology analysis, molecular docking, and western blot were utilized to elucidate the underlying molecular mechanism. Alveolar bone resorption was exacerbated and oxidative stress products were accumulated during CSE exposure in vivo. Oxidative stress damage induced by CSE caused inhibition of osteogenic differentiation in vitro. Quercetin effectively protected the osteogenic differentiation of human periodontal ligament cells (hPDLCs) and periodontal tissue by upregulating the expression of Beclin-1 thus to promote autophagy and reduce oxidative stress damage. Our results established a role of oxidative stress damage and autophagy dysfunction in the mechanism of CSE-induced destruction of periodontal tissue and hPDLCs, and provided a potential application value of quercetin to ameliorate CSRP.

Titanium particles inhibit bone marrow mesenchymal stem cell osteogenic differentiation through the MAPK signaling pathway.

Metallic implants have great application in clinical orthopedics. Implants wear out in vivo due to long-term mechanical loading. The formation of wear debris is one of the long-term complications of prosthesis. In the case of artificial joint replacement in particular, aseptic loosening is the most common reason for secondary revision surgery. Previous studies suggested that wear debris caused aseptic loosening mainly by promoting osteolysis around the prosthesis. In this study, titanium particles, the most commonly used particles in clinical practice, were selected to simulate wear debris and explore the influence of titanium particles on osteogenic differentiation of mesenchymal stem cells. Our results show that titanium particles can significantly inhibit osteogenic differentiation in a dose-dependent manner. While engaged in preliminary exploration of the underlying mechanisms, we found that titanium particles significantly affect phosphorylation of ERK1/2, a key component of MAPK signaling. This suggests that the MAPK signaling pathway is involved in the inhibition of osteogenic differentiation by titanium particles.

Alleviation of Dexamethasone-induced Inhibition of Bone Formation by Salidroside

Alleviation of Dexamethasone-induced Inhibition of Bone Formation by Salidroside

Retracted: miR-141-3p Regulates EZH2 to Attenuate Porphyromonas gingivalis Lipopolysaccharide-Caused Inflammation and Inhibition of Osteogenic Differentiation in Human Periodontal Ligament Stem Cells.

[This retracts the article DOI: 10.1155/2022/4634925.].

MiR-126 mitigates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting the ERK1/2 and Bcl-2 pathways.

Human bone marrow mesenchymal stem cells (hBMMSCs) are a promising cell source for bone engineering owing to their high potential to differentiate into osteoblasts. The objective of the present study is to assess microRNA-126 (miR-126) and examine its effects on the osteogenic differentiation of hBMMSCs. In this study, we investigate the role of miR-126 in the progression of osteogenic differentiation (OD) as well as the apoptosis and inflammation of hBMMSCs during OD induction. OD is induced in hBMMSCs, and matrix mineralization along with other OD-associated markers are evaluated by Alizarin Red S (AR) staining and quantitative PCR (qPCR). Gain- and loss-of-function studies are performed to demonstrate the role of miR-126 in the OD of hBMMSCs. Flow cytometry and qPCR-based cytokine expression studies are performed to investigate the effect of miR-126 on the apoptosis and inflammation of hBMMSCs. The results indicate that miR-126 expression is downregulated during the OD of hBMMSCs. Gain- and loss-of function assays reveal that miR-126 upregulation inhibits the differentiation of hBMMSCs into osteoblasts, whereas the downregulation of miR-126 promotes hBMMSC differentiation, as assessed by the determination of osteogenic genes and alkaline phosphatase activity. Furthermore, the miR-126 level is positively correlated with the production of inflammatory cytokines and apoptotic cell death. Additionally, our results suggest that miR-126 negatively regulates not only B-cell lymphoma 2 (Bcl-2) expression but also the phosphorylation of extracellular signal‑regulated protein kinase (ERK) 1/2. Moreover, restoring ERK1/2 activity and upregulating Bcl-2 expression counteract the miR-126-mediated suppression of OD in hBMMSCs by promoting inflammation and apoptosis, respectively. Overall, our findings suggest a novel molecular mechanism relevant to the differentiation of hBMMSCs into osteoblasts, which can potentially facilitate bone formation by counteracting miR-126-mediated suppression of ERK1/2 activity and Bcl-2 expression.

Macrophage migration inhibitory factor (MIF) inhibitor iSO-1 promotes staphylococcal protein A-induced osteogenic differentiation by inhibiting NF-κB signaling pathway.

Osteomyelitis is among the most difficult to treat diseases in the field of orthopedics, and there is a lack of effective treatment modalities. Exploring the mechanisms of its development is beneficial for finding molecular targets for treatment. Increasing evidence suggests that macrophage migration inhibitory factor (MIF), as a proinflammatory mediator, is not only involved in various pathophysiological processes of inflammation but also plays an important role in osteogenic differentiation, while its specific regulatory mechanism in osteomyelitis remains unclear. In the present study, staphylococcal protein A (SPA)-treated rat bone marrow mesenchymal stem cells (rBMSCs) were used to construct cell models of osteomyelitis. Rat and cell models of osteomyelitis were used to validate the expression levels of MIF, and to further explore the regulatory mechanisms of the MIF inhibitor methyl ester of (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (iSO-1) and MIF knockdown on cell model of osteomyelitis toward osteogenic differentiation. We found that the expression level of MIF was upregulated in rat and cell models of osteomyelitis and subsequently demonstrated by the GSE30119 dataset that the expression level of MIF was also significantly upregulated in patients with osteomyelitis. Furthermore, SPA promotes MIF expression in rBMSCs while inhibiting the expression of osteogenic-related genes such as Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN) and collagen type-1 (COL-1) through activation of the nuclear factor kappa-B (NF-κB) pathway. In vivo, we further demonstrated that local injection of iSO-1 significantly increased the osteogenic activity in rat model of osteomyelitis. Importantly, we also demonstrated that MIF knockdown and the MIF inhibitor iSO-1 reversed the SPA-mediated inhibition of osteogenic differentiation of rBMSCs by inhibiting the activation of the NF-κB pathway, as evidenced by the upregulation of osteogenic-related gene expression and enhanced bone mineralization. ISO-1 and MIF knockdown can reverse the SPA-mediated inhibition of osteogenic differentiation in the rBMSCs model of osteomyelitis by inhibiting the NF-κB signaling pathway, providing a potential target for the treatment of osteomyelitis.

MicroRNA-29b-3p/sirtuin-1/peroxisome proliferator-activated receptor γ suppress osteogenic differentiation.

Osteoporosis is described as an age-associated impairment of bone formation. microRNA (miR)-29b-3p was thought to be linked to osteoblast differentiation; however, the underlying molecular pathways are yet unknown. The study's goal was to look into the involvement of miR-29b-3p in osteoporosis and the pathophysiological mechanisms. A murine model of estrogen deficiency-induced bone loss was established to simulate postmenopausal osteoporosis. Reverse transcription-quantitative PCR (RT-qPCR) was performed to assess the level of miR-29b-3p of bone tissue. Additionally, miR-29b-3p/sirtuin-1 (SIRT1)/peroxisome proliferator-activated receptor γ (PPARγ) axis in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was examined. Osteogenesis-related markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2), were assessed at protein and molecular levels. ALP staining and Alizarin Red staining were used to detect ALP activity and calcium deposition. The ovariectomy group was shown to express miR-29b-3p at higher levels in vitro, and miR-29b-3p mimics suppressed osteogenic differentiation and protein/mRNA expression levels of osteogenesis-related markers in vivo. SIRT1 was identified as a target of miR-29b-3p using luciferase reporter assays. Overexpression of SIRT1 reduced the inhibition of osteogenic differentiation by miR-29b-3p. Rosiglitazone, an activator of PPARγ signaling, was able to reverse the downregulation of the osteogenic differentiation of BMSCs and the protein expression of PPARγ caused by miR-29b-3p inhibitors. The results revealed that osteogenesis was suppressed by miR-29b-3p, which blocks the SIRT1/PPARγ axis. These results suggested that postmenopausal osteoporosis could be treated by targeting miR-29b-3p SIRT1/PPARγ.

Effect of PKM2 on Osteogenic and Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Myeloma Bone Disease

To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs. BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs. Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2. The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.

Study on the treatment of postmenopausal osteoporosis with quercetin in Liuwei Dihuang Pill based on network pharmacology

BackgroundLiuwei Dihuang Pill (LP) was verified to alleviate postmenopausal osteoporosis (PMOP) development. Nevertheless, the major constituent of LP and the related network pharmacology study remain unexplored.MethodsProtein–protein interaction was established to identify the downstream target of LP in PMOP, and the related signaling pathway was investigated by bioinformatics analysis. MC3T3-E1 cells were added to ferric ammonium citrate (FAC) to mimic osteoporosis in vitro. The osteoblasts were identified by Alizarin red staining. Western blot was applied to evaluate protein levels. In addition, Cell Counting Kit-8 (CCK8) assay was applied to assess cell viability, and cell apoptosis was assessed by flow cytometry.ResultsQuercetin was the major constituent of LP. In addition, quercetin significantly reversed FAC-induced inhibition of osteogenic differentiation in MC3T3-E1 cells. In addition, quercetin notably abolished the FAC-induced upregulation of Bax, Caspase-3, FOS, JUN, TGFB1 and PPARD. In contrast, Bcl-2, p-mTOR/mTOR, p-AKT/AKT and p-PI3K/PI3K levels in MC3T3-E1 cells were reduced by FAC, which was restored by quercetin. Meanwhile, FAC notably inhibited the viability of MC3T3-E1 cells via inducing apoptosis, but this impact was abolished by quercetin. Furthermore, quercetin could reverse pcDNA3.1-FOS-mediated growth of FAC-treated osteoblasts by mediating PI3K/AKT/mTOR signaling.ConclusionQuercetin alleviated the progression of PMOP via activation of PI3K/AKT/mTOR signaling. Hence, this study would shed novel insights into discovering new methods against PMOP.