ED-71 Improves Bone Mass in Ovariectomized Rats by Inhibiting Osteoclastogenesis Through EphrinB2-EphB4-RANKL/OPG Axis

Abstract

Purpose

Estrogen deficiency is the main reason of postmenopausal osteoporosis. Eldecalcitol (ED-71) is a new active vitamin D analogue clinically used in the treatment of postmenopausal osteoporosis. We aimed to investigate whether EphrinB2-EphB4 and RANKL/RANK/OPG signaling cooperate in mediating the process of osteoporosis by ED-71.

Methods

In vivo, the ovariectomized (OVX) rats were administered orally with 30 ng/kg ED-71 once a day for 8 weeks. HE staining, Masson staining and Immunofluorescence staining were used to evaluate bone mass, bone formation, osteoclastogenesis associated factors and the expression of EphrinB2, EphB4, RANKL and OPG. In vitro, H₂O₂ stimulation was used to simulate the cell environment in osteoporosis. Immunofluorescence, quantitative real time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blot were applied to detect the expression of EphrinB2, EphB4, RANKL and OPG. In osteoblasts, EphB4 was knocked down by EphB4 small-interfering RNA (siRNA) transfection. LY294002 (PI3K inhibitor) or ARQ092 (AKT inhibitor) was used to block PI3K/AKT pathway. An indirect co-culture system of osteoblasts and osteoclasts was established. The mRNA and protein expression of osteoclastogenes is associated factors were tested by qRT-PCR and Western Blot.

Results

ED-71 increased bone mass and decreased the number of osteoclasts in OVX rats. Moreover, ED-71 promoted the expression of EphrinB2, EphB4, and decreased the RANKL/OPG ratio in osteoblasts. Osteoclastogenesis was restrained when osteoclasts were indirectly co-cultured with ED-71-treated osteoblasts. After silencing of EphB4 expression in osteoblasts, ED-71 inhibited the expression of P-PI3K and P-AKT and increased the ratio of RANKL/OPG. This reversed the inhibitory effect of ED-71 on osteoclastogenes. Therefore, in ED-71-inhibited osteoclastogenes, EphB4 is a key factor affecting the secretion of RANKL and OPG by osteoblasts. EphB4 suppressed the RANKL/OPG ratio through activating PI3K/AKT signaling in osteoblasts.

Conclusion

ED-71 inhibits osteoclastogenesis through EphrinB2-EphB4-RANKL/OPG axis, improving bone mass in ovariectomized rats. PI3K/AKT pathway is involved this process.

Keywords:

• osteoporosis
• eldecalcitol
• osteoclasts
• EphrinB2-EphB4
• osteoblasts
• RANKL/RANK/OPG

Ethics Approval

All animal experiments in this study were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC), School and Hospital of Stomatology, Shandong University (Approval No. 20230502).

Author Contributions

All authors made contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation. All authors took part in drafting, revising or critically reviewing the article, and gave final approval of the version to be published. All authors have agreed on the journal to which the article has been submitted, and agreed to be accountable for the contents of the article.

Disclosure

The authors declare that they have no conflicts of interest in this work.

Additional information

Funding

This study was partially supported by the National Natural Science Foundation of China (No. 81972072) and the TaiShan Scholars of Shandong Province (No. tstp20221160) to Li M, the National Natural Science Foundation of China (No. 81800982) and the Construction Engineering Special Fund of “Taishan Young Scholars” of Shandong Province (No. tsqn202103177) to Liu H.