Effects Of Four Panaxatriol Saponins On The In Vitro Proliferation Of Murine Bone Marrow-derived MSCs And Related Technology

Background and punpouse:Little has been known about the pharmacological effects of ginsenosides on bone marrow-derived mesenchymal stem cells.It has been shown that deglycosylation of ginsenosides(GS) is closely related to pharmacological activity of them. This study was performed to examine the effect of panaxatriol saponins on proliferation of bone marrow-derived mesenchymal stem cells(MSCs) in vitro,and to clarify the relationship between pharmacological activity of panaxatriol saponins and their deglycosylation.A profound understanding of mechanisms underlying pharmacological actions of ginsenosides was expected.Methods:The following experimental methods were used in this study.The primary culture and subculture of murine bone marrow MSCs were performed.GS-Re,-Rg₁,-Rh₁ and-ppt were added to these cultures respectively.The clonogenic capacity of MSCs was evaluated by colony-forming assays.Cells were counted in the MSC subcultures. The grid film pasted to the bottom of the culture plate was designed and prepared.This grid was used to mark the fields of cell monolayer.In the selected fields cells were enumerated using the ocular grid in the eyepiece of inverted microscope.The average cell number of the ocular grid units from a well of culture plate was divided by the area of an ocular grid unit to calculate the cell count per unit area,which was multiplied by surface area of a culture well to result in the total number of cells for a well of culture plate.MTT test was used to assess cell viability.The mRNA levels for the genes cyclin E,CDK2,and p21 of cells in MSC subcultures were determined by fluorescent real-time quantitative RT-PCR.Resulds:The experimental results showed that the colony-forming efficiency of primary MSCs was dramatically decreased at concentrations of 10μmol/L and 20μmol/L of GS-Rh₁;the numbers of cells in MSC subcultures were obviously reduced at 20μmol/L of GS-Rh₁; MTT absorbance values of MSC subcultures were remarkably lower in range of 5 to 20μmol/L of GS-Rh₁,compared with the control group. The colony-forming efficiency of primary MSCs and the absorbance value in MTT assay of MSC subculture were dramatically decreased at a ppt concentration of 20μmol/L.However,no significant differences in the above-mentioned experimental measurements were observed between GS-Re and-Rg₁ groups and the control group.At 10μmol/L of GS-Re,-Rg₁,-Rh₁,-ppt,the level of cyclin E mRNA expression in MSC subcultures was decreased,while no significant changes in mRNA levels of CDK2 and p21 were found.Conclusion:These results indicate that GS-Rh₁ and -ppt at certain concentrations are able to reduce the colony-forming capacity of MSCs from murine bone marrow,and to inhibit proliferation of cells in the MSC subculture.But GS-Re and -Rg₁ have not any significant effects on the in vitro growth of these cells.It is suggested that the pharmacological activity of panaxatriol saponins may be dependent on deglycosylation of them to a certain degree.GS-Re,-Rg₁,-Rh₁ or -ppt has a down-regulating effect on mRNA expression of cyclin E in the MSC subculture,the significance of which remains to be assessed by further studies.