Research On The Role Of ERK5 Signaling Pathway And Fluid Shear Stress In The Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective: The aim of this study is to investigate the effect of fluid shear stress on the osteogenic differentiation of bone marrow mesenchymal stem cells and the role of ERK5 signal pathway in the process of the differentiation.Methods: Bone marrow mesenchymal stem cells(BMSCs) were extracted from Kunming mice by the whole bone marrow adherent cell culture method, and the surface markers of BMSCs were identified by flow cytometry. The see-saw rocking table fluid shear stress(FSS) loading model was designed, and the force was calculated by ANSYS-CFX software. The inhibition effect of XMD8-92(a specific inhibitor of ERK5) on ERK5 phosphorylation was verified and the optimal FSS loading time was investigated. The experiment was divided into 8 groups according to different intervention measures: control group, XMD8-92 group, osteogenic inducer group, inducer+XMD8-92 group, FSS group, FSS+XMD8-92 group, FSS+inducer group, FSS+inducer+XMD8-92 group. The 8 groups were consistently cultured for14 days, then its alkaline phosphatase(ALP) activity were determined and alizarin red staining were conducted. The intracellular protein was extracted, and the change of expression on ERK5, p-ERK5, ERK1/2, p-ERK1/2, Runx-2, Col-1 in different groups were detected by western blot.Results: The extracted cells were spindle, uniform in shape, and growing fast,the surface marks were CD29+, CD44+, CD90+, CD34-, CD45-, the cells can differentiate into osteoblasts, so they were bone marrow mesenchymal stem cells.5μmol/L XMD8-92 for 1 hour can inhibit the phosphorylation of ERK5 well. The FSS of 12 dyn/cm2 loading 1 hour could effectively activate ERK5. The phosphorylation of ERK1/2 can be activated by the osteogenic inducer and FSS(P<0.001). The osteogenic differentiation indicators of ALP activity, calcium nodule content, and thelevels of Runx-2 and Col-1 were higher than those in the control group(P<0.01).Those osteogenic differentiation indicators in three groups of inducer+XMD8-92,FSS+XMD8-92, FSS+inducer+XMD8-92 were higher than the other three groups without XMD8-92(P<0.05). Those osteogenic differentiation indicators of FSS+inducer group were higher than that of single inducer or FSS group(P<0.05).Conclusions: Fluid shear stress can promote the osteogenic differentiation of bone marrow mesenchymal stem cells through the phosphorylation of ERK1/2.Meanwhile, FSS and the osteogenic inducer have synergistic effect between each other. ERK5 specific inhibitor XMD8-92 can promote the osteogenic differentiation of BMSCs, therefore, the phosphorylation of ERK5 can inhibit the osteogenic differentiation of BMSCs.