Overexpression of fucosyltransferase 8 reverses the inhibitory effect of high-dose dexamethasone on osteogenic response of MC3T3-E1 preosteoblasts

Background Core fucosylation catalyzed by FUT8 is essential for TGF-β binding to TGF-β receptors. Methods Indirect TGF-β1 binding assay was used to evaluate the ability of TGF-β1 to bind to TGFBRs, Alizarin red and alkaline phosphatase staining were used to detect osteogenic differentiation and mineralization ability , western blot and quantitative RT-PCR were used to measure the differential expression of osteogenesis-related proteins and genes. Plasmid-mediated gain-of-function study. The scale of core fucosylation modification was detected by Lectin-blot and LCA laser confocal. Results Our results showed that compared with vehicle treatment, high-dose (10−6 and 10−5 M) dexamethasone significantly inhibited cell proliferation, osteogenic differentiation, and FUT8 mRNA expression while promoting mRNA expression of adipogenesis-related genes in MC3T3-E1 cells, suggesting that downregulation of FUT8 is involved in the inhibitory effect of high-dose dexamethasone on osteogenesis. Overexpression of FUT8 significantly promoted osteogenic differentiation and activated TGF-β/Smad signaling in MC3T3-E1 cells in the presence of high-dose dexamethasone, suggesting that FUT8 reverses the inhibitory effect of high-dose dexamethasone on osteogenesis. In addition, lectin fluorescent staining and blotting showed that overexpression of FUT8 significantly reversed the inhibitory effects of high-dose dexamethasone on core fucosylation of TGFBR1 and TGFBR2. Furthermore, indirect TGF-β1 binding assay showed that overexpression of FUT8 remarkably promoted TGF-β1 binding to TGFBRs in MC3T3-E1 cells in the presence of high-dose dexamethasone. Conclusions Taken together, these results suggest that overexpression of FUT8 facilitates counteracting the inhibitory effect of dexamethasone on TGF-β signaling and osteogenesis.

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The inhibitory effects of vancomycin on rat bone marrow–derived mesenchymal stem cell differentiation

Journal of Neurosurgery Spine ◽

10.3171/2020.10.spine201511 ◽

2021 ◽

pp. 1-5

Author(s):

Kari Hanson ◽

Carly Isder ◽

Kristen Shogren ◽

Anthony L. Mikula ◽

Lichun Lu ◽

...

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

High Dose ◽

Inhibitory Effects ◽

Alizarin Red ◽

Osteogenic Factors

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

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Apigenin neutralizes the inhibitory effect of inflammation on the osteogenic differentiation of human mesenchymal stem cells

10.21203/rs.3.rs-283695/v1 ◽

2021 ◽

Author(s):

Azita Asadi ◽

Farjam Goudarzi ◽

Mustafa Ghanadian ◽

Adel Mohammadalipour

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Human Mesenchymal Stem Cells ◽

Inhibitory Effect ◽

Alizarin Red ◽

Inflammatory Conditions ◽

Cell Staining ◽

Degree Of Differentiation

Abstract Background: The stimulating effects of apigenin on mesenchymal stem cells (MSCs) osteogenesis, as well as the anti-inflammatory effect of this flavonoid, have been identified. In this study, osteogenic differentiation was investigated under inflammatory conditions and treatment with apigenin. Methods and Results: Along with osteogenic differentiation of MSCs, they became inflamed with LPS/PA, and treated simultaneously with apigenin. The degree of differentiation was assessed by alizarin red staining and alkaline phosphatase (ALP) activity. Also, gene expression of NLRP3 and RUNX2 was performed along with protein expression of IL-1β. Significant increase in NLRP3 and IL-1β were observed in MSCs when exposed to LPS/PA (p<0.01). Also, the osteogenesis was significantly decreased (p<0.01). Apigenin treatment induced significantly higher gene expression of RUNX2, the activity of ALP, and cell staining (p<0.01) which were also associated with reduced inflammation in these cells. Conclusions: The effectiveness of apigenin on osteogenesis under inflammatory conditions was cautiously observed.

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LncRNA DANCR and miR-320a suppressed osteogenic differentiation in osteoporosis by directly inhibiting the Wnt/β-catenin signaling pathway

Experimental & Molecular Medicine ◽

10.1038/s12276-020-0475-0 ◽

2020 ◽

Vol 52 (8) ◽

pp. 1310-1325

Author(s):

Cheng-Gong Wang ◽

Yi-He Hu ◽

Shi-Long Su ◽

Da Zhong

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathway ◽

Morphological Changes ◽

Osteoporosis Treatment ◽

Inhibitory Effect ◽

Luciferase Assay ◽

Blue Staining ◽

Alcian Blue Staining ◽

Alizarin Red

Abstract Our study aimed to determine how lncRNA DANCR, miR-320a, and CTNNB1 interact with each other and regulate osteogenic differentiation in osteoporosis. qRT-PCR and western blotting were performed to determine the expression of DANCR, miR-320a, CTNNB1, and the osteoporosis- or Wnt/β-catenin pathway-related markers T-cell factor 1 (TCF-1), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Interactions between CTNNB1, DANCR, and miR-320a were predicted by bioinformatics approaches and validated using a luciferase assay. Osteoblastic phenotypes were evaluated by ALP staining, ALP activity assay and Alizarin Red staining. The bilateral ovariectomy method was used to establish an in vivo osteoporosis model. Bone morphological changes were examined using hematoxylin and eosin (H&E) and Alcian Blue staining. The expression levels of DANCR and miR-320a in BMSCs derived from osteoporosis patients were upregulated, whereas CTNNB1 expression was downregulated compared with that in healthy controls. Importantly, we demonstrated that miR-320a and DANCR acted independently from each other and both inhibited CTNNB1 expression, whereas the inhibitory effect was additive when miR-320a and DANCR were cooverexpressed. Moreover, we found that DANCR overexpression largely abrogated the effect of the miR-320a inhibitor on CTNNB1 expression and the Wnt/β-catenin signaling pathway in BMSCs during osteogenic differentiation. We further confirmed the results above in BMSCs derived from an osteoporosis animal model. Taken together, our findings revealed that DANCR and miR-320a regulated the Wnt/β-catenin signaling pathway during osteogenic differentiation in osteoporosis through CTNNB1 inhibition. Our results highlight the potential value of DANCR and miR-320a as promising therapeutic targets for osteoporosis treatment.

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Influences of High-Dose Dexamethasone on Regulatory T Cells, Transforming Growth Factor-β1 and Interleukin-10 of Patients with Chronic ITP.

Blood ◽

10.1182/blood.v110.11.3920.3920 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3920-3920

Author(s):

Yun Ling ◽

Xiangshan Cao ◽

Ziqiang Yu ◽

Changgeng Ruan

Keyword(s):

T Cells ◽

Transforming Growth Factor ◽

Interleukin 10 ◽

Treg Cells ◽

Transforming Growth Factor Β1 ◽

High Dose ◽

Chronic Itp ◽

High Dose Dexamethasone ◽

Significant Difference ◽

Tgf Β1

Abstract Immune thrombocytopenic purpura (ITP) is an autoimmune disorder and high-dose dexamethasone (HD-DXM) has been used as a first-line therapy for patients with ITP. However, little is known about the role of CD4+CD25 + regulatory T (Treg) cells, interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1) in the pathogenesis of chronic ITP and the effects of HD-DXM on them contained Treg cells, IL-10 and TGF-β1. In this study, we investigated the expressions of Treg cells, IL-10 and TGF-β1 in 26 untreated adult patients with chronic ITP. All patients had thrombocytopenia (platelet count <50 × 109/L) for more than 6 months. We also observed short time changes of Treg cells, IL-10 and TGF-β1 after treatment with HD-DXM in these patients. The results showed that a good initial response to HD-DXM occurred in 24 of the 26 patients with chronic ITP (92.3%): the mean platelet count was (84.9±30.4)×109/L [range, (20∼150) ×109/L] one week after the initiation of treatment. The proportion of CD4+CD25+ T cells in the peripheral blood of patients with chronic ITP was significantly higher than that in normal controls(P<0.001); there was no significant difference in the percentage of CD4+CD25high T cells between patients and controls ( P=0.317); but the number of CD4+ FOXP3+ T cells in patients was significantly lower than that in controls (P<0.001). After 4-days treatment with HD-DXM, the numbers of CD4+ CD25+ T cells (P<0.001), CD4+CD25high T cells ( P<0.001), and CD4+FOXP3+ T cells ( P<0.001) in patients were all significantly increased. In the serum of chronic ITP patients, the expression level of TGF-β1 was lower than that of healthy controls (P<0.0001) and HD-DXM could significantly increase it; there was no significant difference in the expression level of IL-10 between patients and controls ( P>0.05) and there was no remarkable change of IL-10 in patients after HD-DXM treatment (P>0.05). The mRNA levels of Foxp3 and TGF-β1 gene in patients were lower than those of controls (P<0.05 and P<0.05); HD-DXM administration significantly increased the expressions of Foxp3 and TGF-β1 gene(P<0.05 and P<0.0001), which were even higher than those of controls(P<0.05 and P<0.05); There was a positive correlation between the Foxp3 mRNA expression and TGF-β1 after treatment with HD-DXM (r =0.403, P=0.041). These results suggest that Foxp3 and TGF-β1 gene are deficient in chronic ITP and the immunosuppressive therapy of glucocorticoids could improve the expression levels of these genes.

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Luteolin supports osteogenic differentiation of human periodontal ligament cells

BMC Oral Health ◽

10.1186/s12903-019-0926-y ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 2

Author(s):

He Quan ◽

Xiaopeng Dai ◽

Meiyan Liu ◽

Chuanjun Wu ◽

Dan Wang

Keyword(s):

Cyclin D1 ◽

Osteogenic Differentiation ◽

Periodontal Ligament ◽

Cell Activity ◽

Inhibitory Effect ◽

Bone Morphogenetic Protein 2 ◽

Periodontal Ligament Cells ◽

Alizarin Red ◽

Real Time Quantitative Pcr ◽

Related Transcription Factor

Abstract Background Previous research revealed that luteolin could improve the activation of alkaline phosphatase (ALP) and osteocalcin in mouse osteoblasts. We aimed to determine the effect of luteolin on osteogenic differentiation of periodontal ligament cells (PDLCs). Methods Cultured human PDLCs (HPDLCs) were treated by luteolin at 0.01, 0.1, 1, 10, 100 μmol/L, Wnt/β-catenin pathway inhibitor (XAV939, 5 μmol/L) alone or in combination with 1 μmol/L luteolin. Immunohistochemical staining was performed to ensure cells source. Cell activity and the ability of osteogenic differentiation in HPDLCs were determined by MTT, ALP and Alizarin Red S staining. Real-time Quantitative PCR Detecting System (qPCR) and Western blot were performed to measure the expressions of osteogenic differentiation-related genes such as bone morphogenetic protein 2 (BMP2), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), Osterix (OSX) and Wnt/β-catenin pathway proteins members cyclin D1 and β-catenin. Results Luteolin at concentrations of 0.01, 0.1, 1, 10, 100 μmol/L promoted cell viability, ALP activity and increased calcified nodules content in HPDLCs. The expressions of BMP2, OCN, OSX, RUNX2, β-catenin and cyclin D1 were increased by luteolin at concentrations of 0.01, 0.1, 1 μmol/L, noticeably, 1 μmol/L luteolin produced the strongest effects. In addition, XAV939 inhibited the expressions of calcification and osteogenic differentiation-related genes in HPDLCs, and 1 μmol/L luteolin availably decreased the inhibitory effect. Conclusion 1 μmol/L luteolin accelerated osteogenic differentiation of HPDLCs via activating the Wnt/β-catenin pathway, which could be clinically applied to treat periodontal disease.

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MiR-27a-3p promotes the osteogenic differentiation by activating CRY2/ERK1/2 axis

Molecular Medicine ◽

10.1186/s10020-021-00303-5 ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Li-Rong Ren ◽

Ru-Bin Yao ◽

Shi-Yong Wang ◽

Xiang-Dong Gong ◽

Ji-Tao Xu ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Osteoblast Differentiation ◽

Inhibitory Effect ◽

Luciferase Reporter ◽

Induction Medium ◽

Luciferase Reporter Gene ◽

Alizarin Red ◽

Protein Levels ◽

Phosphorylation Level ◽

Gene Assay

Abstract Background Osteoporosis seriously disturbs the life of people. Meanwhile, inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. It was reported that miR-27a-3p reduced the symptoms of osteoporosis. However, the mechanism by which miR-27a-3p in osteogenic differentiation remains largely unknown. Methods To induce the osteogenic differentiation in MC3T3-E1 cells, cells were treated with osteogenic induction medium (OIM). RT-qPCR was used to evaluate the mRNA expression of miR-27a-3p and CRY2 in cells. The protein levels of CRY2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and the phosphorylation level of extracellular regulated protein kinases (ERK) 1/2 in MC3T3-E1 cells were evaluated by western blotting. Meanwhile, calcium nodules and ALP activity were tested by alizarin red staining and ALP kit, respectively. Luciferase reporter gene assay was used to analyze the correlation between CRY2 and miR-27a-3p. Results The expression of miR-27a-3p and the phosphorylation level of ERK1/2 were increased by OIM in MC3T3-E1 cells, while CRY2 expression was decreased. In addition, OIM-induced increase of calcified nodules, ALP content and osteogenesis-related protein expression was significantly reversed by downregulation of miR-27a-3p and overexpression of CRY2. In addition, miR-27a-3p directly targeted CRY2 and negatively regulated CRY2. Meanwhile, the inhibitory effect of miR-27a-3p inhibitor on osteogenic differentiation was reversed by knockdown of CRY2 or using honokiol (ERK1/2 signal activator). Furthermore, miR-27a-3p significantly inhibited the apoptosis of MC3T3-E1 cells treated by OIM. Taken together, miR-27a-3p/CRY2/ERK axis plays an important role in osteoblast differentiation. Conclusions MiR-27a-3p promoted osteoblast differentiation via mediation of CRY2/ERK1/2 axis. Thereby, miR-27a-3p might serve as a new target for the treatment of osteoporosis.

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Role of 1,25-Dihydroxyvitamin D3 on Osteogenic Differentiation and Mineralization of Chicken Mesenchymal Stem Cells

Frontiers in Physiology ◽

10.3389/fphys.2021.479596 ◽

2021 ◽

Vol 12 ◽

Author(s):

Chongxiao Chen ◽

Roshan Adhikari ◽

Dima Lynn White ◽

Woo Kyun Kim

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Mrna Expression ◽

Inhibitory Effect ◽

Dependent Manner ◽

Cell Stage ◽

Dihydroxyvitamin D3

1,25-dihydroxyvitamin D3 (1,25OHD) has been suggested to play an important role in osteogenic differentiation and mineralization. However, limited data have been reported in avian species. In the present study, the direct role of 1,25OHD on osteogenic differentiation and mineralization in chicken mesenchymal stem cells (cMSCs) derived from day-old broiler bones was investigated. cMSCs were treated with control media (C), osteogenesis media (OM), OM with 1, 5, 10, and 50 nM 1,25OHD, respectively. The messenger RNA (mRNA) samples were obtained at 24 and 48 h and 3 and 7 days to examine mRNA expression of key osteogenic genes [runt related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen type I alpha 2 chain (COL1A2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and alkaline phosphatase (ALP)]. Cells were stained at 7, 14, and 21 days using Von Kossa (mineralization), Alizarin Red (AR; mineralization), and Alkaline Phosphatase (early marker) staining methods. From the mRNA expression results, we found a time-dependent manner of 1,25OHD on osteoblast differentiation and mineralization. In general, it showed an inhibitory effect on differentiation and mineralization during the early stage (24 and 48 h), and a stimulatory effect during the late cell stage (3 and 7 days). The staining showed 1,25OHD had an inhibitory effect on ALP enzyme activities and mineralization in a dosage-dependent manner up to 14 days. However, at 21 days, there was no difference between the treatments. This study provides a novel understanding of the effects of 1,25OHD on osteogenic differentiation and mineralization of cMSCs depending on cell stage and maturity.

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LINC00899 promotes osteogenic differentiation and alleviates osteoporosis via targeting miR-374a to regulate RUNX2

10.21203/rs.3.rs-31105/v2 ◽

2020 ◽

Author(s):

Xiaoya Gao ◽

Yun Xue ◽

Kechun Yang

Keyword(s):

Osteogenic Differentiation ◽

Molecular Mechanisms ◽

Rna Binding ◽

Quantitative Polymerase Chain Reaction ◽

Direct Interaction ◽

Inhibitory Effect ◽

Luciferase Reporter ◽

Bone Mesenchymal Stem Cells ◽

Alizarin Red ◽

Osteogenic Induction

Abstract Objective: This study aims to illustrate the underlying molecular mechanisms of long noncoding RNAs (LncRNAs) LINC00899 in osteoporosis.Methods: Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was used to examine the levels of LINC00899, miR-374a and RUNX2 in clinical tissues or human bone mesenchymal stem cells (hBMSCs). The interaction between miR-374a and LINC00899 or RUNX2 was predicted by starBase and verified by luciferase reporter assay and RNA binding protein immunoprecipitation (RIP) assay. Alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining were also used to evaluate the osteogenic ability of hBMSCs.Results: The expression levels of LINC00899 were gradually increased, but miR-374a expression was decreased with the prolongation of osteogenic induction. In addition, the expression of LINC00899 was lowly expressed in osteoporotic patients’ bone tissues and knockdown of LINC00899 decreased the expression of osteogenesis-related genes. Moreover, LINC00899 was confirmed to inhibit miR-374a expression by direct interaction. Finally, we demonstrated that RUNX2 was a target of miR-374a, and the silencing of miR-374a partially abolished the inhibitory effect of LINC00899 knockdown on the expression of RUNX2, OPN and OCN.Conclusions: We demonstrated that LINC00899 facilitated the osteogenic differentiation of hBMSCs and prevented osteoporosis by sponging miR-374a and enhancing RUNX2 expression, which might provide a useful therapeutic strategy for osteoporosis patients.

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Mesoporous bioactive glass scaffold delivers salvianolic acid B to promote bone regeneration in a rat cranial defect model

Current Drug Delivery ◽

10.2174/1567201817666200916091253 ◽

2020 ◽

Vol 17 ◽

Author(s):

Lin Wu ◽

Zhanying Wei ◽

Siyu He ◽

Yunlong Bi ◽

Yang Cao ◽

...

Keyword(s):

Bioactive Glass ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Bone Defects ◽

Salvianolic Acid B ◽

Fluorescence Labeling ◽

High Dose ◽

Alizarin Red ◽

Salvianolic Acid ◽

Mesoporous Bioactive Glass

Background: Mesoporous bioactive glass (MBG) has been widely studied because of its excellent histocompatibility and degradability. However, due to the lack of good osteoinductive activity, the pure MBG scaffold is not effective in repairing large-scale bone defects. Objective: To observe the repair effect of MBG scaffolds delivering salvianolic acid B (SB) on critical bone defects in rats. Method: In this study, MBG scaffolds were used as delivery vehicle. SB, a small molecular active drug with good osteogenic differentiation ability, was loaded into the MBG scaffolds at low, medium and high doses. The effect of SB released from the MBG scaffolds on osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) was investigated using alkaline phosphatase staining, alizarin red staining and real-time quantitative polymerase chain reaction. Moreover, 8 weeks after implantation of the scaffolds, the bone regeneration was evaluated by micro-CT, sequential fluorescence labeling and histological staining analysis. Results: The in vitro results showed that different doses of SB had similar release rate from scaffolds and could be released from scaffolds continuously. The middle dose (MBG/MSB) and high dose (MBG/HSB) groups significantly promoted the osteogenic differentiation of rBMSCs when compared with low dose (MBG/LSB) group. Moreover, SB produced significant increases in newly formed bone of calvarial bone defects in rats. Conclusion: It’s concluded that the use of MBG scffolds delivering SB is an effective strategy for the treatment of bone defects.

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Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000491541 ◽

2018 ◽

Vol 47 (6) ◽

pp. 2307-2318 ◽

Cited By ~ 12

Author(s):

Geng-Yang Shen ◽

Hui Ren ◽

Jin-Jing Huang ◽

Zhi-Da Zhang ◽

Wen-Hua Zhao ◽

...

Keyword(s):

Protein Expression ◽

Osteogenic Differentiation ◽

Mrna Expression ◽

Signaling Pathway ◽

Quantitative Polymerase Chain Reaction ◽

Akt Signaling ◽

Cell Counting ◽

Luciferase Reporter ◽

Akt Signaling Pathway ◽

Alizarin Red

Background/Aims: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. Methods: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, β-catenin, Gsk3β, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-β-CATENIN, and p-GSK3β were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. Results: The optimum concentration for PTE was 30 μg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, β-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3β mRNA expression. PTE inhibited TNFR2, TRAF2, and p-β-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3β protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. Conclusions: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.

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