The Effect Of Ilexonin A On The Migration Of Bone Mesenchymal Stem Cells In Vitro

Objective : To observe the migration of IA on bone mesenchymal stem cells Ilexonin in vitro. Then we investigate CXCR4 and the expression of β-catenin and GSK3β of the Wnt pathway. we can elucidate the involved molecular mechanism which may mediate the effects on the enhanced migration ability of BMSCs induced by IA.Methods:1. By whole bone marrow adherent method isolation、cultivation 、purification and identification of rat BMSCs in vitro. 2. Then assess the MTT cell proliferation assay after different does of IA(3.125~800 μg/ml) preconditioning of BMSCs for 24 h, 48 h and 72 h. Analysis the effect on the proliferation of BMSCs mediated by IA,for best drug concentration and best time of screening. 3. The third generation of BMSCs was exposed to the optimal concentration of IA for 48 h.Subsequently, the transwell system was used to carry out the experiment of BMSCs migration. And Western blot was used to the expression of CXCR4. 4. Intervene by activator Wnt3 a and inhibitor Dkk1, and Western blot was used to observe the relation among the β-catenin, GSK3β and CXCR4. The expertments were divided into 6 groups:(1)control group(BMSCs+DMSO);(2)Wnt3a group(BMSCs+Wnt3a);(3) IA group(BMSCs+IA);(4)IA+Wnt3a combining group(BMSCs+ IA+Wnt3a);(5) IA+Dkk1combining group(BMSCs+ IA+Dkk1);(6) Dkk1 group(BMSCs+ Dkk1).Results : 1. BMSCs are active proliferation. The three generation of BMSCs surface receptors, CD29 、 CD90 are expressed and CD34, CD45 are not expressed.Osteogenesis induction by von Kossa’s and adipogenesis induction by oil red O staining are positive,stating the cells is relatively purified BMSCs. 2. Compared with control group,(1) the cells incubated with IA(100~800 μg/ml) proliferation significantly reduced at 24h(P<0.05);(2)the cells incubated with IA(100~800μg/ml)proliferation significantly reduced at 48h(P<0.05) 、IA(6.25、3.125 μg/ml)proliferation significantly increased;(3)the cells incubated with IA(12.5~800 μg/ml) proliferation significantly reduced at 72h(P<0.05). The above results show that the cells incubated with IA(6.25、3.125 μg/mL) at 48 h is optimal choice. 3. The transwell migration assay shows that the cells incubated with IA(6.25 、 3.125 μg/ml) at 48 h could significantly increase the migration capacity of BMSCs(P<0.05), and the migration rate has no concern with concentration of IA. The effect of IA(6.25μg/ml) was blocked by AMD3100, the antagonist of CXCR4. 4. Western blot shows that the cells incubated with IA(6.25 、 3.125 μg/ml) at 48 h could increase the expression of CXCR4 in BMSCs(P<0.05). 5. CXCR4 was expressed both in the cell membrane and the cell by fluorescence microscope. 6. Expression of CXCR4 and β-catenin protein: Compared with control group,Wnt3 a group and IA group are significantly increased, Dkk1 group is significantly reduced(P<0.05); Compared with Wnt3 a group 、IA group respectively,expression of CXCR4 IA+Wnt3a combining group is significantly increased(P<0.05);Compared with IA group, expression of β-catenin IA+Wnt3a combining is significantly increased(P<0.05);Compared with Dkk1 group, IA+Dkk1combining group is significantly increased(P<0.05);Expression of GSK3β protein:Compared with control group, Wnt3 a group and IA group are significantly reduced(P<0.05); Compared with Wnt3 a group 、 IA group respectively,IA+Wnt3a combining group is significantly reduced(P<0.05);Compared with Dkk1 group,IA+Dkk1 combining group is significantly reduced(P<0.05); Compared with IA group,IA+Dkk1 combining group is significantly reduced(P<0.05).Conclusion: 1. IA(6.25、3.125 μg/ml) can promote migration by improving the expression of CXCR4 in BMSCs. 2. IA(6.25μg/ml) may up-regulation the expression of CXCR4, connect with low-level of GSK3β 、high-level of β-catenin and activation of Wnt/β-catenin signaling pathway,play its role in promoting migration.