| Objective To explore the effect of EphB/EphrinB signaling pathway on the osteogenic differentiation of mouse bone marrow derived mesenchymal stem cells (BMSCs) and the postmenopausal osteoporosis animal model in mice. Methods 1) Twenty-four healthy 8-week-old BALB/c mice were randomly divided into castration group (OVX group) and sham operation group (Sham group). The expression level of Eph/Ephrin signaling pathway in BMSCs was detected. 2) The ovariectomized mice were divided into 4 groups. The Eph receptor agonists EfnB1-Fc and EfnB2-Fc, Eph receptor inhibitor EphB2-Fc, and human IgG-Fc as control, were intraperitoneally injected, respectively. The mice were sacrificed by cervical dislocation 10 weeks after the surgery. The femoral bones were compared under micro-CT analysis. The bone marrow of the femurs and the tibia was extracted from the ovariectomized and sham-operated mice. BMSCs were isolated and cultured by density gradient centrifugation and identified to P3. After osteogenic induction for 7 days, the expression levels of Runx2, ALP, Osterix, OPG, and RANKL were detected using real-time PCR. After 14 days and 21 days of osteogenic differentiation, the ability of osteogenic differentiation was observed with ALP staining and Alizarin red staining. Results Compared with those in the OVX group, the expressions of EfnA2, EphB4, EfnB2, EfnB1, and EphA4 in the Sham group increased significantly (P<0.01), and the expression difference was similar to those in humans. The expressions of EfnA2 and EfnB2 reduced significantly in the Sham group (P<0.01). After 10 weeks, the results of micro-CT showed that compared with the control group, the trabecular structure in the Sham group, and EfnB1-Fc, EfnB2-Fc, and EphB2-Fc in the castration group was relatively intact and the trabecular bone density was better (P<0.01). Compared with those in EfnB1-Fc and EfnB2-Fc castration groups, the bone mineral density and bone volume fraction in the Sham group and EphB2-Fc castration group increased significantly (P<0.01). The results of real-time PCR for detection of osteogenesis-related genes indicated that the expressions of ALP and Osterix in EfnB1-Fc, EfnB2-Fc, and EphB2-Fc castration groups were significantly higher than those in human IgG-Fc castration group. EfnB1-Fc and EphB2-Fc increased ALP activity and mineralization by BMSCs, and it is more obvious in EphB2-Fc group. Compared with that in the control group, the expression of RANKL in EfnB1-Fc, EfnB2-Fc, and EphB2-Fc castration groups was not significantly different (P>0.05), but the expression of OPG increased significantly (P<0.01). Among those, the increase of OPG/RANKL ratio in EfnB1-Fc and EphB2-Fc group was the most significant (P<0.01). Conclusion EphB2/EfnB1 signaling pathway can reverse the bone loss caused by castration, up-regulate ALP and Osterix expression, promote osteogenic differentiation of BMSCs, and affect the function of BMSCs in ovariectomized mice by regulating OPG/RNAKL ratio, indirectly affecting the differentiation of osteoclasts. |