Effect Of Panax Notoginseng Saponins On Biological Characteristics Of Endothelial Progenitor Cells And Activation Of Wnt Signaling Pathway

Objective: Distraction osteogenesis(DO)is a kind of bone regeneration process in which the vascular pedicle is continuously stretched after osteotomy and the new bone is formed in the distraction space.In addition to the bone regeneration process,the angiogenesis process is also indispensable for distraction osteogenesis.Panax notoginseng saponins(PNS)is an effective ingredient of traditional Chinese medicine notoginseng.Our research team found that PNS can promote the osteogenesis process of distraction osteogenesis while high expression of EPCs’ surface markers were detected in the stretch zone,we speculate that PNS also has an effect on angiogenesis during distraction osteogenesis.So,in this study,we used culture medium containing different concentrations of PNS to culture the EPCs derived from canine bone marrow in vitro.We want to explore whether the appropriate concentration of PNS can affect the function of EPCs,and whether the appropriate concentration of PNS can activate the Wnt signaling pathway on endothelial progenitor cells,which can lay the foundation for further study of the effect of PNS on the angiogenesis process during distraction osteogenesis in vivo.Methods: 1.Isolative culture and function identification of canine’s EPCs and effect of PNS on the biological characteristics of EPCs: Firstly,the EPCswere separated and cultured by density gradient centrifugation combined with differential adherence method,and EPCs surface markers were identified by immunofluorescence.The migration function of EPCs,the tube-forming experiment to detect the ability of EPCs to form tubes,the immunofluorescence to detect the EPCs’ VEGF,bFGF expression;CCK-8 proliferation assay to detect the effect of different concentrations of PNS on the proliferation of EPCs;Cell migration assay to detect the migration of EPCs under different concentrations of PNS;The effect of concentrations of PNS on the gene expression of VEGF and bFGF in EPCs was detected by PCR.The effect of different concentrations of PNS on the expression of VEGF in EPCs was detected by immunohistochemistry.The effect of different concentrations of PNS on the expression of bFGF in EPCs was detected by immunofluorescence.2.Effect of PNS on the activation of Wnt signaling pathway in EPCs: The effect of different concentrations of PNS on the gene expression of β-catenin,negative regulatory protein GSK-3β and downstream target gene cyclin D1 in Wnt signaling pathway were detected by PCR.Immunohistochemistry and Western Blot were used to detect the effects of different concentrations of PNS on the expression of Wnt signaling pathway key protein β-catenin,negative regulatory protein GSK-3β and downstream target gene cyclin D1.Results: 1.Isolative culture and function identification of canine’EPCs and the effect of PNS on EPCs: Under the inverted microscope,the EPCs grew in clusters,and they were round when they were attached to the wall.Later,they were long fusiform,polygonal,and spindle.Immunofluorescence results showed that EPCs expresses CD133 and CD34.In the cell migration experiment,after the EPCs were implanted into the upper chamber of transwell chamber,cell migration was observed in the lower chamber after 24 hours,and migrationnumber increased after 48 hours.In the matrigel tube formation experiment,EPCs were observed to form a "lumen" like structure in the matrigel after 6hours;immunofluorescence showed that EPCs expressed VEGF and bFGF;CCK-8 proliferation assay showed that PNS concentration at 6.25mg/L or lower can promote the proliferation of EPCs.Above 6.25mg/L,it may inhibit the proliferation of EPCs.The results of cell migration test showed that PNS at6.25mg/L or 12.5mg/L can promote the migration of endothelial progenitor cells.The 6.25mg/L PNS can promote the migration promotion of EPCs better(P<0.05).The PCR results show that PNS at 6.25mg/L or 12.5mg/L can up-regulate the expression of VEGF and bFGF gene levels in EPCs(P< 0.05),PNS at 6.25mg/L could up-regulate the expression of VEGF and bFGF genes in EPCs better(P<0.05).The results of immunohistochemistry and fluorescence showed that PNS at 6.25mg/L or 12.5mg/L could promote the expression of VEGF and bFGF in EPCs(P<0.05),the 6.25 mg/L PNS could promote better than 12.5mg/L PNS(P<0.05).2.The relationship between the effect of PNS on EPCs and Wnt signaling pathway: PCR,ICC,WB results showed that PNS at 6.25mg/L and 12.5mg/L could up-regulate the expression of genes and protein of the key proteinβ-catenin and downstream target gene cyclin D1 of Wnt signaling pathway(P<0.05),6.25mg/L PNS could up-regulate better than 12.5mg/L(P<0.05);6.25mg/L and 12.5mg/L PNS could down-regulate the expression of GSK-3βgene and protein in Wnt signaling pathway(P<0.05),and 6.25mg/L PNS could down-regulate better than 12.5mg/L(P< 0.05).Conclusion: 1.The appropriate concentration of PNS can promote the proliferation and migration of endothelial progenitor cells,and the expression of VEGF and bFGF;2.PNS can up-regulate the expression of key proteinβ-catenin and downstream target gene cyclin D1 of Wnt signaling pathway,and down-regulate the expression of negative regulatory protein GSK-3β of Wnt signaling pathway.