BACKGROUND:Osteoporosis caused by chemotherapy has become one of the serious side effects that impact the skeletal system. Icarin shows a strong anti-osteoporosis activity, which can have protective effect on osteoporosis induced by chemotherapy. OBJECTIVE: To study the protective effect and mechanism of icarin against cyclophosphamide-induced obstacle of mouse bone marrow mesenchymal stem cels differentiating into osteoblasts. METHODS:MTT assay and alkaline phosphatase (ALP) staining were used to determine the optimal protective concentration of icarin against cyclophosphamide-induced obstacle of mouse bone marrow mesenchymal stem cels differentiating into osteoblasts. mRNA expressions of osteoblast-specific transcription factors, OC, ALP, Runx2, and Wnt/β-catenin signaling pathway target genes, β-catenin, C-Myc, cyclin D1, were determined using RT-PCR method at different time after intervention with the optimal concentration of icarin. Expressions of Runx2, β-catenin, c-Myc, cyclin D1 regulated by the optimal concentration of icarin were detected using western blot assay at the protein level. RESULTS AND CONCLUSION:Cel viability and ALP activity decreased significantly in the cyclophosphamide group compared with the control group, but there was no significant difference in cel viability between icarin group and cyclophosphamide group. Icarin at 100 μmol/L showed the best protective effect against cyclophosphamide-induced obstacle of osteogenic differentiation of bone marrow mesenhymal stem cels. Compared with the control group, cyclophosphamide chemotherapy reduced the expressions of ALP, OC, Runx2 at mRNA level and Runx2 at protein level, weakened the expressions ofβ-catenin, cyclin D1 at mRNA level and active β-catenin, Cyclin D1, c-myc at protein level, and increased the expression of DKK1. Compared with the cyclophosphamide group, 100 μmol/L icarin increased the expression of osteoblast-specific transcription factors and Wnt/β-catenin signaling pathway genes at mRNA and protein levels, and reduced the expression of DKK1 protein. These results show that cyclophosphamide can lead to osteogenic differentiation disorder of mouse bone marrow mesenchymal stem cels, and in contrast, icarin shows a protective effect and its optimal intervention concentration is 100 μmol/L. Additionaly, the protective roleof icarin is probably related to activation of Wnt/β-catenin signal pathway.

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.