Restoration of osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by 
cyclophosphamide with psoralen

doi:10.12307/2024.730

Abstract

Abstract:

BACKGROUND: Psoralen has a strong anti-osteoporotic activity and may have a restorative effect on chemotherapy-induced osteoporosis.

OBJECTIVE: To explore the restorative effect of psoralen on the osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by cyclophosphamide and its mechanism.

METHODS: C57BL/6 mouse bone marrow mesenchymal stem cells were isolated and cultured. Effect of psoralen on viability of bone marrow mesenchymal stem cells was detected by MTT assay. Osteogenic induction combined with alkaline phosphatase staining was used to determine the optimal dose of psoralen to restore the osteogenic differentiation of bone marrow mesenchymal stem cells inhibited by cyclophosphamide. The mRNA expression levels of Runx2, alkaline phosphatase, Osteocalcin, osteoprotegerin, and Wnt/β-catenin signaling pathway-related genes Wnt1, Wnt4, Wnt10b, β-catenin, and c-MYC were measured by RT-qPCR at different time points under the intervention with psoralen. The protein expression of osteogenic specific transcription factor Runx2 and Wnt/β-catenin signaling pathway related genes Active β-catenin, DKK1, c-MYC, and Cyclin D1 was determined by western blot assay at different time points under the intervention with psoralen.

RESULTS AND CONCLUSION: (1) There was no significant effect of different concentrations of psoralen on the viability of bone marrow mesenchymal stem cells. The best recovery of the inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells caused by cyclophosphamide was under the intervention of psoralen at a concentration of 200 μmol/L. (2) Psoralen reversed the reduction in osteogenic differentiation marker genes Runx2, alkaline phosphatase, Osteocalcin and osteoprotegerin mRNA expression and Runx2 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium. (3) Psoralen reversed the decrease in Wnt/β-catenin pathway-related genes Wnt4, β-catenin, c-MYC mRNA and Active β-catenin, c-MYC, and Cyclin D1 protein expression and the increase in DKK1 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium. (4) The results showed that cyclophosphamide inhibited osteogenic differentiation of bone marrow mesenchymal stem cells in mice, and psoralen had a restorative effect on it. The best intervention effect was achieved at a concentration of 200 μmol/L psoralen, and this protective effect might be related to the activation of Wnt4/β-catenin signaling pathway by psoralen.

Key words: cyclophosphamide, bone marrow mesenchymal stem cell, osteogenic differentiation, psoralen, Wnt4, β-catenin

CLC Number:

Wang Chenglong , Yang Zhilie , Chang Junli , Zhao Yongjian , Zhao Dongfeng , Dai Weiwei , Wu Hongjin , Zhang Jie , Wang Libo , Xie Ying , Tang Dezhi , Wang Yongjun , Yang Yanping. Restoration of osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by cyclophosphamide with psoralen[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(1): 16-23.

Figures/Tables 7

2.6 补骨脂素可以逆转环磷酰胺条件培养基导致的骨髓间充质干细胞 Wnt/β-catenin 通路相关基因 mRNA 和蛋白表达变化为了进一步检测补骨脂素逆转骨髓间充质干细胞成骨分化能力的机制，用 RT-qPCR 法检测了各组 Wnt/β-catenin 通路相关基因 Wnt1、Wnt4、Wnt10b、 β-catenin 和 c-MYC mRNA 表达变化，与对照组相比，环磷酰胺条件培养基组 β-catenin 和 Wnt10b mRNA 表达都显著降低 (P < 0.001)；与环磷酰胺条件培养基组相比，环磷酰胺条件培养基 + 补骨脂素 200 μmol/L 组干预 24，48，72 h 的 β-catenin mRNA 表达均显著升高 (P < 0.001)；环磷酰胺条件培养基 + 补骨脂素 200 μmol/L 组干预 24 h 使 Wnt1 和干预 24，72 h 使 Wnt4 mRNA 表达均显著升高 (P < 0.05，P < 0.05，P < 0.001)；环磷酰胺条件培养基 + 补骨脂素 200 μmol/L 组干预 24，48，72 h 的 Wnt10b mRNA 表达均显著降低 (P < 0.001，P < 0.001， P < 0.05)；环磷酰胺条件培养基 + 补骨脂素 200 μmol/L 组干预24，48 h的c-MYC mRNA表达均显著升高(P < 0.001， P < 0.01)，见图 7。 Western blot 检测各组 Wnt/β-catenin 通路相关蛋白表达变化，与对照组相比，环磷酰胺条件培养基组 Acttve β-catenin、c-MYC和CyclinD1蛋白表达显著降低(P < 0.001)， DKK1 蛋白表达显著升高 (P < 0.01)。与环磷酰胺条件培养基组相比，环磷酰胺条件培养基 + 补骨脂素 200 μmol/L 组干预 48，72 h 的 Acttve β-catenin 和 CyclinD1 蛋白表达均显著升高 (P < 0.001)；干预 24，72 h 的 c-MYC 蛋白表达均显著升高 (P < 0.05，P < 0.001)；干预 72 h 的 DKK1 蛋白表达显著降低 (P < 0.001)，见图 8。表明补骨脂素对环磷酰胺条件培养基导致的骨髓间充质干细胞 Wnt/β-catenin 通路相关基因 mRNA 和蛋白表达变化有逆转作用，推测补骨脂素可能通过调节 Wnt/β-catenin 通路相关蛋白 ( 促进 Wnt1、Wnt4、β-catenin、c-MYC mRNA 和 Acttve β-catenin、 c-MYC、CyclinD1 蛋白表达，抑制 DKK1 蛋白表达 ) 而促进成骨分化标志基因表达，最终达成恢复骨髓间充质干细胞成骨分化能力的效果。 "

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