The Antagonistic Effect Of Icariin And Psoralen On Cyclophosphamide-induced Obstacle Of Mouse Bone Marrow Mesenchymal Stem Cells Differentiating Into Osteoblasts

OBJECTIVE:To study the antagonistic effect and mechanism of icariin and psoralen on cyclophosphamide-induced obstacle of mouse bone marrow mesenchymal stem cells(BMSCs)differentiating into osteoblasts.METHODS:MIT assay and alkaline phosphatase(ALP)staining were used to determine the optimal protective concentration of icariin and psoralen against cyclophosphamide-induced obstacle of mouse bone marrow mesenchymal stem cells differentiating into osteoblasts.mRNA expressions of osteoblast-specific transcription factors ALP,OC,Runx2,and Wnt/β-catenin signaling pathway target genes,β-catenin,cyclinDl,c-myc,were determined using RT-PCR method at different time after intervention with the optimal concentration of icariin.Expressions of Runx2,β-catenin,cyclinDl,c-myc regulated by the optimal concentration of icariin were detected using western blot assay at the protein level;and mRNA expressions of osteoblast-specific transcription factors,Runx2,OC,were determined using RT-PCR method at different time after intervention with the optinal concentration of psoralen.Expressions of Runx2,and Wnt/β-catenin signaling pathway target genes,β-catenin,c-myc,cyclinDl regulated by the optimal concentration of psoralen were detected using western blot assay at the protein levelRESULTS:(1)Cell viability and ALP activity decreased significantly in the cyclophosphamide group compared with the control group,but there was no significant difference in cell viability between icariin group and cyclophosphamide group.Icariin at 100μmol/L showed the best protective effect against cyclophosphamide-induced obstacle of osteogenic differentiation of bone marrow mesnhymal stem cells.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 of β-catenin,cyclinD1 at mRNA level and active β-catenin,CyclinD1,c-myc at protein level,and increased the expression of DKK1.Compared with the cyclophosphamide group,100 μmol/L icariin increased the expression of osteoblast-specific transcription factors and Wnt/-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 cells,and in contrast,icariin shows a protective effect and its optimal intervention concentraion is 100 μmol/L Additionally,the protective role of icariin is probably related to activation of Wnt/β-catenin signal pathway.(2)Cell viability and ALP activity decreased significantly in the cycbphosphamide group compared with the control group,but there was no significant difference in cell viability between psoralen group and cycbphosphamide group.psoralen at 200 μmol/L showed the best protective effect against cyclophosphamide-induced obstacle of osteogenic differentiation of bone marrow mesenhymal stem cells.Compared with the control group,cyclophosphamide chemotherapy reduced the expressions of Runx2,OC,at mRNA level and Runx2 at protein level,weakened the expressions of activeβ-catenin,CyclinD1,c-myc at protein level.Compared with the cyclophosphamide group,200 μmol/Lpsoralen increased the expression of osteoblast-specific transcription factors at mRNA and Wnt/-catenin signaling pathway genes at protein levels,and increased the expression of Runx2protein.CONCLUSION:These results show that cycbphosphamide can lead to osteogenic differentiation disorder of mouse bone marrow mesenchymal stem cells,and in contrast,icariin and psoralen shows antagonistic effect and separately their optimal intervention concentration are100 μmol/Land 200 μmol/L Additionally,the antagonistic role of icariin and psoralen are probably related to activation of Wnt/β-catenin signal pathway.