Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling
Background: Eurycomanone (EN) is a diterpenoid compound isolated from the roots of Eurycoma longifolia (E. longifolia). Previous studies have confirmed that E. longifolia can enhance bone regeneration and bone strength. We previously isolated and identified ten quassinoids from E. longifolia, and th...
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| Format: | Article |
| Language: | English |
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Elsevier
2023-05-01
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| Series: | Journal of Orthopaedic Translation |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214031X23000335 |
| _version_ | 1797791883622088704 |
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| author | Yan-ting Zhong Hong-bo Liao Zhi-qiang Ye Hua-sheng Jiang Jia-xiao Li Lin-mao Ke Jun-ying Hua Bo Wei Xin Wu Liao Cui |
| author_facet | Yan-ting Zhong Hong-bo Liao Zhi-qiang Ye Hua-sheng Jiang Jia-xiao Li Lin-mao Ke Jun-ying Hua Bo Wei Xin Wu Liao Cui |
| author_sort | Yan-ting Zhong |
| collection | DOAJ |
| description | Background: Eurycomanone (EN) is a diterpenoid compound isolated from the roots of Eurycoma longifolia (E. longifolia). Previous studies have confirmed that E. longifolia can enhance bone regeneration and bone strength. We previously isolated and identified ten quassinoids from E. longifolia, and the result displayed that five aqueous extracts have the effects on promotion of bone formation, among whom EN showed the strongest activity. However, the molecular mechanism of EN on bone formation was unknown, and we further investigated in this study. Methods: After the verification of purity of extracted EN, following experiments were conducted. Firstly, the pharmacologic action of EN on normal bone mineralization and the therapeutic effect of EN on Dex-induced bone loss using zebrafish larvae. The mineralization area and integral optical density (IOD) were evaluated using alizarin red staining. Then the vital signaling pathways of EN relevant to OP was identified through network pharmacology analysis. Eventually in vitro, the effect of EN on cell viability, osteogenesis activities were investigated in human bone marrow mesenchymal stem cells (hMSCs) and C3H10 cells, and the molecular mechanisms by which applying AKT inhibitor A-443654 in hMSCs. Results: In zebrafish larvae, the administration in medium of EN (0.2, 1, and 5 μM) dramatically enhanced the skull mineralization area and integral optical density (IOD), and increased mRNA expressions of osteoblast formation genes (ALP, RUNX2a, SP7, OCN). Meanwhile, exposure of EN remarkably alleviated the inhibition of bone formation induced by dexamethasone (Dex), prominently improved the mineralization, up-regulated osteoblast-specific genes and down-regulated osteoclast-related genes (CTSK, RANKL, NFATc1, TRAF6) in Dex-treated bone loss zebrafish larvae. Network pharmacology outcomes showed the MAPK and PI3K-AKT signaling pathways are closely associated with 10 hub genes (especially AKT1), and AKT/GSK-3β/β-catenin was selected as the candidate analysis pathway. In hMSCs and C3H10 cells, results showed that EN at appropriate concentrations of 0.008–5 μM effectively increased the cell proliferation. In addition, EN (0.04, 0.2, and 1 μM) significantly stimulated osteogenic differentiation and mineralization as well as significantly increased the protein phosphorylation of AKT and GSK-3β, and expression of β-catenin, evidencing by the results of ALP and ARS staining, qPCR and western blotting. Whereas opposite results were presented in hMSCs when treated with AKT inhibitor A-443654, which effectively inhibited the pro-osteogenesis effect induced by EN, suggesting EN represent powerful potential in promoting osteogenesis of hMSCs, which may be closely related to the AKT/GSK-3β/β-catenin signaling pathway. Conclusions: Altogether, our findings indicate that EN possesses remarkable effect on bone formation via activating AKT/GSK-3β/β-catenin signaling pathway in most tested concentrations. The translational potential of this article: This study demonstrates EN is a new effective monomer in promoting bone formation, which may be a promising anabolic agent for osteoporosis (OP) treatment. |
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| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-13T02:25:11Z |
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| series | Journal of Orthopaedic Translation |
| spelling | doaj.art-7a73337b656b4f2e983368db29599f8c2023-06-30T04:22:06ZengElsevierJournal of Orthopaedic Translation2214-031X2023-05-0140132146Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signalingYan-ting Zhong0Hong-bo Liao1Zhi-qiang Ye2Hua-sheng Jiang3Jia-xiao Li4Lin-mao Ke5Jun-ying Hua6Bo Wei7Xin Wu8Liao Cui9Guangdong Provincial Key Laboratory of Research and Development of Natural Drugs, And School of Pharmacy, Guangdong Medical University, Zhanjiang, China; The Affiliated Hospital of Guangdong Medical University, Zhanjiang, ChinaGuangdong Provincial Key Laboratory of Research and Development of Natural Drugs, And School of Pharmacy, Guangdong Medical University, Zhanjiang, China; The Second Affiliated Hospital of Guangdong Medical University, Zhanjiang, ChinaGuangdong Provincial Key Laboratory of Research and Development of Natural Drugs, And School of Pharmacy, Guangdong Medical University, Zhanjiang, ChinaThe Affiliated Hospital of Guangdong Medical University, Zhanjiang, ChinaDepartment of Nephrology, The Third Affiliated Hospital of Southern Medical University, Guangzhou, ChinaGuangdong Provincial Key Laboratory of Research and Development of Natural Drugs, And School of Pharmacy, Guangdong Medical University, Zhanjiang, ChinaGuangdong Provincial Key Laboratory of Research and Development of Natural Drugs, And School of Pharmacy, Guangdong Medical University, Zhanjiang, ChinaThe Affiliated Hospital of Guangdong Medical University, Zhanjiang, ChinaThe Second Affiliated Hospital of Guangdong Medical University, Zhanjiang, China; Corresponding author.Guangdong Provincial Key Laboratory of Research and Development of Natural Drugs, And School of Pharmacy, Guangdong Medical University, Zhanjiang, China; Corresponding author.Background: Eurycomanone (EN) is a diterpenoid compound isolated from the roots of Eurycoma longifolia (E. longifolia). Previous studies have confirmed that E. longifolia can enhance bone regeneration and bone strength. We previously isolated and identified ten quassinoids from E. longifolia, and the result displayed that five aqueous extracts have the effects on promotion of bone formation, among whom EN showed the strongest activity. However, the molecular mechanism of EN on bone formation was unknown, and we further investigated in this study. Methods: After the verification of purity of extracted EN, following experiments were conducted. Firstly, the pharmacologic action of EN on normal bone mineralization and the therapeutic effect of EN on Dex-induced bone loss using zebrafish larvae. The mineralization area and integral optical density (IOD) were evaluated using alizarin red staining. Then the vital signaling pathways of EN relevant to OP was identified through network pharmacology analysis. Eventually in vitro, the effect of EN on cell viability, osteogenesis activities were investigated in human bone marrow mesenchymal stem cells (hMSCs) and C3H10 cells, and the molecular mechanisms by which applying AKT inhibitor A-443654 in hMSCs. Results: In zebrafish larvae, the administration in medium of EN (0.2, 1, and 5 μM) dramatically enhanced the skull mineralization area and integral optical density (IOD), and increased mRNA expressions of osteoblast formation genes (ALP, RUNX2a, SP7, OCN). Meanwhile, exposure of EN remarkably alleviated the inhibition of bone formation induced by dexamethasone (Dex), prominently improved the mineralization, up-regulated osteoblast-specific genes and down-regulated osteoclast-related genes (CTSK, RANKL, NFATc1, TRAF6) in Dex-treated bone loss zebrafish larvae. Network pharmacology outcomes showed the MAPK and PI3K-AKT signaling pathways are closely associated with 10 hub genes (especially AKT1), and AKT/GSK-3β/β-catenin was selected as the candidate analysis pathway. In hMSCs and C3H10 cells, results showed that EN at appropriate concentrations of 0.008–5 μM effectively increased the cell proliferation. In addition, EN (0.04, 0.2, and 1 μM) significantly stimulated osteogenic differentiation and mineralization as well as significantly increased the protein phosphorylation of AKT and GSK-3β, and expression of β-catenin, evidencing by the results of ALP and ARS staining, qPCR and western blotting. Whereas opposite results were presented in hMSCs when treated with AKT inhibitor A-443654, which effectively inhibited the pro-osteogenesis effect induced by EN, suggesting EN represent powerful potential in promoting osteogenesis of hMSCs, which may be closely related to the AKT/GSK-3β/β-catenin signaling pathway. Conclusions: Altogether, our findings indicate that EN possesses remarkable effect on bone formation via activating AKT/GSK-3β/β-catenin signaling pathway in most tested concentrations. The translational potential of this article: This study demonstrates EN is a new effective monomer in promoting bone formation, which may be a promising anabolic agent for osteoporosis (OP) treatment.http://www.sciencedirect.com/science/article/pii/S2214031X23000335EurycomanoneBone formationZebrafish larvaeHuman mesenchymal stem cellsC3H10 cellsAKT/GSK-3β/β-catenin signaling pathway |
| spellingShingle | Yan-ting Zhong Hong-bo Liao Zhi-qiang Ye Hua-sheng Jiang Jia-xiao Li Lin-mao Ke Jun-ying Hua Bo Wei Xin Wu Liao Cui Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling Journal of Orthopaedic Translation Eurycomanone Bone formation Zebrafish larvae Human mesenchymal stem cells C3H10 cells AKT/GSK-3β/β-catenin signaling pathway |
| title | Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling |
| title_full | Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling |
| title_fullStr | Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling |
| title_full_unstemmed | Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling |
| title_short | Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling |
| title_sort | eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating akt gsk 3β β catenin signaling |
| topic | Eurycomanone Bone formation Zebrafish larvae Human mesenchymal stem cells C3H10 cells AKT/GSK-3β/β-catenin signaling pathway |
| url | http://www.sciencedirect.com/science/article/pii/S2214031X23000335 |
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