Comparative Investigation Of Icariin Naringin And Asperosaponin Ⅵ Bone Formation In Vitro

Objective:To compare the different osteogenic effects of active ingredients of three reinforcing kidney Chinese herbs:Icariin(ICA), Naringin(NAR) and Asperosaponin VI (ASA VI)on rats bone mesenchymal stem cells and mice preosteoblast cells MC3T3-E1in vitro.for providing clues of more effective drugs to promote bone regeneration.Methods:The method of whole marrows stick to the wall was used to separate and culture the MSCs,and their biological characteristics were dentified.MC3T3-E1were recovered and cultured. Solutions of ICA,NAR and ASA VI were prepared by dissolving in DMSO at concentrations of10-3～10-8mol/L.CCK-8method was used to survey the effect that three drugs on proliferation of MSCs and MC3T3-E1. Make sure which concentration is the best one, then assurance the dose-effect relationship between three drugs and the proliferation of two kinds of cells.The ALP activity and protein concentration were determined using an ALP activity assay kit and a BCA-protein assay kit to confirm whether MSCs and MC3T3-E1were induced differentiation into osteoblast by ICA,NAR and ASA VI, and make sure of the best concentration.Mineralization in osteoblast cultures was determined by staining with Alizarin Red-S. Results:1、Effects on MSCs(1) ICA10"6M,NAR10’6M and ASA Ⅵ10-5M were the best concentrations to promote the proliferation of MSCs. NAR10"6M promoted the proliferation of MSCs better than ICA10"6M and ASA Ⅵ10-5M.(P<0.001)(2) ICA10-3M、ICA1O-4M、NAR10-3M、ASA Ⅵ10-3M、ASA Ⅵ10-4M produced inhibition of cell proliferation(3) ICA10-6M,NAR10-7M,ASA Ⅵ10-5M were the best concentrations to promote the alkaline phosphatase activity of MSCs after14days intervention. ICA10"6M performed better then ASAⅥ10-5M and NAR10-7M (P<0.05). Additionally, ICA10-6M enhanced the maturation of cultured MSCs, better than ASA Ⅵ10-5M (P<0.05)), NAR10"7M is the weakest one (P<0.01)2, Effects on MC3T3-E1(1) ICA10-8M, NAR10-6M and ASA Ⅵ10-5M were the best concentrations to promote the proliferation of MC3T3-E1. ASA Ⅵ10-5M performed significantly better than NAR10-6M on the third day.(P<0.05)(2) ICA10-3M、ICA10-4M、NAR10-3M、NAR10-4M、ASA Ⅵ10-3M、ASA Ⅵ10-4M produced inhibition of cell proliferation(3) ICA10-5M,NAR10-8M,ASA Ⅵ10-5M were the best concentrations to promote the alkaline phosphatase activity of MC3T3-E1after7days intervention. NAR10-8M performed weaker than ASA Ⅵ10-5M and ICA10-5M (P<0.001).Additionally, ICA10-5M and ASA Ⅵ10-5M enhanced the maturation of cultured MSCs, better than NAR10-8M (P<0.01and P<0.05)Conclusions:1、Three drugs for both cell proliferation and toxic effects are reflected the trend that high concentrations inhibit the proliferation and the low ones promoted the proliferation.2、In promoting cell proliferation, NAR promoted of proliferation of MSCs cells was significantly stronger than ICA and ASA VI. For MC3T3-E1cells, the overall effect of the three drugs to promote proliferation of cells were similar to the primary, but no significant difference among the three.3. In promoting cell alkaline phosphatase activity and mineralization, ICA promoted of the role of MSCs strongest, ASA VI followed, NAR weakest.MC3T3-E1cells had reached similar conclusions.4. To sum up,Icariin could promote both cell proliferation,alkaline phosphatase activity and mineralization, and it’s effect was strongest among the three drugs. So Icariin may be the most valuable drug for bone rebuilding among the three.