Background Aucubin (AU), an iridoid glucoside isolated from many traditional herbal medicines, has anti-osteoporosis and anti-apoptosis bioactivities. However, the effect of AU on the treatment of bone-fracture remains unknown. In the present study, the aims were to investigate the roles and mechanisms of AU not only on osteoblastogenesis of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and anti-oxidative stress injury in vitro, but also on bone-fracture regeneration by a rat tibial fracture model in vivo. Methods CCK-8 assay was used to assess the effect of AU on the viability and proliferation of hBM-MSCs. The expression of specific genes and proteins on osteogenesis, apoptosis and signaling pathways was measured by qRT-PCR, western blotting and immunofluorescence analysis. ALP staining and quantitative analysis were performed to evaluate ALP activity. ARS and quantitative analysis were performed to evaluate calcium deposition. DCFH-DA staining was used to assess the level of reactive oxygen species (ROS). A rat tibial fracture model was established to validate the therapeutic effect of AU in vivo. Micro-CT with quantitative analysis and histological evaluation were used to assess the therapeutic effect of AU locally injection at the fracture site. Results Our results revealed that AU did not affect the viability and proliferation of hBM-MSCs. Compared with control group, western blotting, PCR, ALP activity and calcium deposition proved that AU-treated groups promoted osteogenesis of hBM-MSCs. The ratio of phospho-Smad1/5/9 to total Smad also significantly increased after treatment of AU. AU-induced expression of BMP2 signaling target genes BMP2 and p-Smad1/5/9 as well as of osteogenic markers COL1A1 and RUNX2 was downregulated after treating with noggin and LDN193189. Furthermore, AU promoted the translocation of Nrf2 from cytoplasm to nucleus and the expression level of HO1 and NQO1 after oxidative damage. In a rat tibial fracture model, local injection of AU promoted bone regeneration. Conclusions Our study demonstrates the dual effects of AU in not only promoting bone-fracture healing by regulating osteogenesis of hBM-MSCs partly via canonical BMP2/Smads signaling pathway but also suppressing oxidative stress damage partly via Nrf2/HO1 signaling pathway.
Background: Previous studies have demonstrated that PCSK9 was constitutively expressed in osteoclast precursors and played an important role in bone metabolism in periodontitis disease. But the function of PCSK9 in the osteogenesis of hBMSCs and specific mechanism still remains unknown. In this study, we aim to explore the function and mechanism of PCSK9 on regulating osteogenic differentiation and osteoclastic differentiation.Methods: The osteogenic effects of endogenous and exogenous PCSK9 on hBMSCs were performed, the effect on osteoclastic differentiation of mBMM too. A CCK-8 was used to assess the effect of PCSK9 on the proliferation of hBMSCs. qRT-PCR and Western blotting analysis were conducted to determine the expression of target genes and proteins. ALP, ARS staining were used to evaluate osteoblasts, the trap staining was used to evaluate osteoclasts. In vivo experiment, we constructed a femoral bone defect model, and injected Adeno associated virus at defect site. Radiographic analysis and histological evaluation were conducted to evaluate the union effect of PCSK9 at bone defect site. Student’s t test was used between two groups, one-way ANOVA or Bonferroni’s post-hoc test was used more than two groups, depending on the distribution of the tested population.Result: In vitro experiment, low dose exogenous and overexpress endogenous PCSK9 could enhance hBMSCs osteogenic differentiation via ERK signaling pathway, and knockdown PCSK9 would reduce this influence. The promoting effect could be weaken by ERK inhibitor PD98059 with an appropriate concentration. All of which was confirmed by q-PCR, Western blotting and immunofluorescence outcome. Besides, the ALP staining, ARS staining results prove that it could raise ALP activity and mineral deposits formation. But low dose PCSK9 has no obvious effect on osteoclastic differentiation. In vivo experiment, the radiographic, histological result prove overexpress PCSK9 could accelerate bone defect union. Conclusion: In summarize, PCSK9 could promote osteogenic differentiation via ERK pathway and has no effect on osteoclast differentiation. The vivo and vitro experiments outcomes indicate that it may be a novel target for treating bone defect union.
Background: The available therapeutic options for large bone defects remain extremely limited, requiring new strategies to accelerate bone healing. Genetically modified bone mesenchymal stem cells (BMSCs) with enhanced osteogenic capacity are recognised as one of the most promising treatments for bone defects.Method: We performed differential expression analysis of miRNAs between human BMSCs (hBMSCs) and human dental pulp stem cells (hDPSCs) to identify osteogenic differentiation-related microRNAs (miRNAs). Furthermore, we identified shared osteogenic differentiation-related miRNAs and constructed an miRNA transcription network. The Forkhead box protein A1 (FOXA1) knockdown strategy with a lentiviral vector was used to explore the role of FOXA1 in the osteogenic differentiation of MSCs. Cell Counting Kit-8 was used to determine the effect of the knockdown of FOXA1 on hBMSC proliferation; real-time quantitative reverse transcription PCR (qRT-PCR) and western blotting were used to investigate target genes and proteins; and alkaline phosphatase (ALP) staining and Alizarin Red staining (ARS) were used to assess ALP activity and mineral deposition, respectively. Finally, a mouse model of femoral defects was established in vivo, and histological evaluation and radiographic analysis were performed to verify the therapeutic effects of FOXA1 knockdown on bone healing.Result: We identified 22 shared and differentially expressed miRNAs between hDPSC and hBMSC, 19 of which were downregulated in osteogenically induced samples. The miRNA-transcription factor interaction network showed that FOXA1 is the most significant and novel osteogenic differentiation biomarker among more than 300 transcription factors that is directly targeted by 12 miRNAs. FOXA1 knockdown significantly promoted hBMSC osteo-specific genes and increased mineral deposits in vitro. In addition, p-ERK1/2 levels were upregulated by FOXA1 silencing. Moreover, the increased osteogenic differentiation of FOXA1 knockdown hBMSCs was partially rescued by the addition of ERK1/2 signalling inhibitors. In a mouse model of femoral defects, a sheet of FOXA1-silencing BMSCs improved bone healing, as detected by micro-computed tomography and histological evaluation.Conclusion: These findings collectively demonstrate that FOXA1 silencing promotes the osteogenic differentiation of BMSCs via the ERK1/2 signalling pathway, and silencing FOXA1 in vivo effectively promotes bone healing, suggesting that FOXA1 may be a novel target for bone healing.
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