Preconditioning With Ligustrazine Promote The Migration Of Bone Marrow Mesenchymal Stem Cells In Vitro

Objective To observe the effect of bone marrow mesenchymal stem cells(BMSCs) pretreatment by Ligustrazine on the migration and adhesion ability of BMSCs in vitro.Then we investigate CXCR4mRNA and protein expression, which is the specific receptor for stromal cell-derived factor-1(SDF-1) after preconditioning with Ligustrazine.We can elucidate the involved molecular mechanism which may mediate the effects on the enhanced migration ability of BMSCs induced by Ligustrazine.Methods Isolated and cultured bone marrow mesenchymal stem cells by using the whole bone marrow adherent method, and conduct phenotypic identification using Flow cytometry analysis of surface positive antigen of CD29,CD90and the negative antigen of CD34, CD45. Then assess the MTT cell proliferation assay after different dose of Ligustrazine (0.1-200uMol/L) preconditioning of BMSCs. Analysis the effect on the proliferation of BMSCs mediated by Ligustrazine. After incubation of BMSCs with various dose of Ligustrazine, we conduct the Transwell migration assay and Cell-matrix adhesion assay. Followed by staining with0.1%crystal violet and microscopic count at a×20magnification.To detect CXCR4mRNA and protein expression using qRT-PCR and Western blot assay after the following dose of Ligustrazine(25、50、100μMol/L) pretreatment of BMSCs at24h.Results The whole bone marrow adherent culture method can obtain the well-growing and stable rat bone marrow-derived mesenchymal stem cells, BMSCs forms the fibroblast-like cluster and get a homogeneous morphology in vitro. By flow cytometric analysis,the cell phenotype of passage3BMSCs show that CD29, CD90was positive for96.9%,97.3%and CD34,CD45was positive for0.2%,3.0%, which is suitable for the following experiments. The MTT assay show that compared with control group, preconditioning groups incubated with of Ligustrazine (0.1-200uMol/L) at6h,12h,24h do not have any impacts on the proliferation of BMSCs(p>0.05). The transwell migration assay and Cell-matrix adhesion assay shows that preconditioning with Ligustrazine (0.1-200μMol/L)at6h,12h,24h time points could significantly increase the migration and adhesion capacity of BMSCs (P<0.05). In addition, the effect on the migration and adhesion ability of BMSCs mediated by Ligustrazine show the dose-dependent manner The dose of100μMol/L Ligustrazine preconditioning24hour conducts the highest migration cell number. And this effect was completely blocked by AMD3100, the antagonist of CXCR4.The qRT-PCR and Western blot assay show that compared with control group, the following dose of Ligustrazine(25,50,100μMol/L) at24hour significantly improve CXCR4mRNA and protein expression (P<0.05).Conclusion:①Preconditioning BMSCs with increasing dose of Ligustrazine (0.1-200μMol/L) at6,12,24h do not have any impact on the cell viability.②Preconditioning BMSCs with increasing dose of Ligustrazine (0.1-200μMol/L) can significantly increase the migration and adhesion ability of BMSCs.③Preconditioning BMSCs with following dose of Ligustrazine(25,50,100μMol/L) can significantly upregulate CXCR4mRNA and protein expression.④The upregulated CXCR4protein expression may associate with the enhanced migration capacity of BMSCs preconditioning with Ligustrazine.⑤Ligustrazine may be the main effective ingredient of Chuanxiong,which can guide other medicine upgoing.