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: Inflammatory microenvironment is significant to the differentiation and function of mesenchymal stem cells(MSCs). It evidentially influences the osteoblastogenesis of MSCs. IL-34, a newly discovered cytokine, playing a key role in metabolism. However, the research on its functional role in the osteogenesis of MSCs was rarely reported. Here, we described the regulatory effects of low-dose IL-34 on both osteoblastogenesis and osteoclastogenesis.Methods: We performed the osteogenic effects of hBMSCs by exogenous and overexpressed IL-34 in vitro, so was the osteoclastogenesis effects of mBMMs by extracellular IL-34. CCK-8 was used to assess the effect of IL-34 on the viability of hBMSCs and mBMMs. ALP staining, ARS and TRAP staining were used to evaluate ALP activity, mineral deposition and osteoclastogenesis, respectively. qRT-PCR and Western blotting analysis were performed to detect the expression of target genes and proteins. ELISA was used to evaluate the concentrations of IL-34. In vivo, a rat tibial osteotomy model and an OVX model were established. Radiographic analysis and histological evaluation were performed to confirm the therapeutic effects of IL-34 in fracture healing and osteoporosis. Statistical differences were evaluated by two-tailed Student’s t-test, one-way ANOVA with Bonferroni’s post hoc test and two-way ANOVA with Bonferroni multiple comparisons post hoc test in the comparison of 2 groups, more than 2 groups and different time points of treated groups, respectively. Results: Promoted osteoblastogenesis of hBMSCs was observed after treated by exogenous or overexpressed IL-34 in vitro, confirmed by increased mineral deposits and ALP activity. Furthermore, exogenous or overexpressed IL-34 enhanced the expression of p-AKT and p-ERK. The specific AKT and ERK signaling pathway inhibitors suppressed the enhancement of osteoblastogenesis induced by IL-34. In a rat tibial osteotomy model, imaging and histological analyses testified the local injection of exogenous IL-34 improved bone healing. However, the additional IL-34 has no influence on both osteoclastogenesis of mBMMs in vitro and osteoporosis of OVX model of rat in vivo. Conclusions: Collectively, our study demonstrate that low-dose IL-34 regulates osteogenesis of hBMSCs partly via the PIK/AKT and ERK signaling pathway and enhances fracture healing, with neither promoting nor preventing osteoclastogenesis in vitro and osteoporosis in vivo.
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: Inflammatory microenvironment is significant to the differentiation and function of mesenchymal stem cells(MSCs). It evidentially influences the osteoblastogenesis of MSCs. IL-34, a newly discovered cytokine, playing a key role in metabolism. However, the research on its functional role in the osteogenesis of MSCs was rarely reported. Here, we described the regulatory effects of low-dose IL-34 on both osteoblastogenesis and osteoclastogenesis.Methods: We performed the osteogenic effects of hBMSCs by exogenous and overexpressed IL-34 in vitro, so was the osteoclastogenesis effects of mBMMs by extracellular IL-34. CCK-8 was used to assess the effect of IL-34 on the viability of hBMSCs and mBMMs. ALP staining, ARS and TRAP staining were used to evaluate ALP activity, mineral deposition and osteoclastogenesis, respectively. qRT-PCR and Western blotting analysis were performed to detect the expression of target genes and proteins. ELISA was used to evaluate the concentrations of IL-34. In vivo, a rat tibial osteotomy model and an OVX model were established. Radiographic analysis and histological evaluation were performed to confirm the therapeutic effects of IL-34 in fracture healing and osteoporosis. Statistical differences were evaluated by two-tailed Student’s t-test, one-way ANOVA with Bonferroni’s post hoc test and two-way ANOVA with Bonferroni multiple comparisons post hoc test in the comparison of 2 groups, more than 2 groups and different time points of treated groups, respectively. Results: Promoted osteoblastogenesis of hBMSCs was observed after treated by exogenous or overexpressed IL-34 in vitro, confirmed by increased mineral deposits and ALP activity. Furthermore, exogenous or overexpressed IL-34 enhanced the expression of p-AKT and p-ERK. The specific AKT and ERK signaling pathway inhibitors suppressed the enhancement of osteoblastogenesis induced by IL-34. In a rat tibial osteotomy model, imaging and histological analyses testified the local injection of exogenous IL-34 improved bone healing. However, the additional IL-34 has no influence on both osteoclastogenesis of mBMMs in vitro and osteoporosis of OVX model of rat in vivo. Conclusions: Collectively, our study demonstrate that low-dose IL-34 regulates osteogenesis of hBMSCs partly via the PIK/AKT and ERK signaling pathway and enhances fracture healing, with neither promoting nor preventing osteoclastogenesis in vitro and osteoporosis in vivo.
Background and Purpose Glycyrrhizic acid (GA) is a major triterpene glycoside isolated from liquorice root that has been shown to inhibit osteoclastogenesis. However, there have been no reports regarding the effect of GA on osteogenic differentiation. Therefore, this study was performed to explore the effects and mechanism of action of GA on osteogenesis. Experimental Approach A CCK-8 array was used to assess cell viability. The osteogenic capability was investigated by real-time quantitative PCR, western blotting and immunofluorescence analyses. ALP staining and ARS were used to evaluate ALP activity and mineralisation, respectively. GA-GelMA hydrogels were designed to verify the therapeutic effects of GA in vivo by radiographic analysis and histological evaluation. Key Results Our results show that GA had no significant influence on the viability or proliferation of human bone mesenchymal stem cells (hBMSCs). GA promoted osteogenic differentiation and enhanced calcium deposition. Furthermore, levels of active β-catenin protein increased after treatment w ith GA. Wnt/catenin signalling inhibitor partially reversed the effects of GA on osteogenic differentiation. In a mouse femoral fracture model, GA-GelMA hydrogels accelerated bone healing. Conclusion and Implications Our results show that GA promotes the osteogenic differentiation of hBMSCs by modulating the Wnt/β-catenin signalling pathway. GA-GelMA hydrogels promoted bone fracture healing. GA has potential as a cost-effective treatment of bone defects. Title:Glycyrrhizic acid promotes osteogenic differentiation of human bone mesenchymal stem cells by activating the Wnt/β-catenin signalling pathway Running title: Glycyrrhizic acid promotes osteogenesis through Wnt/β-catenin pathway
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