Abstract
Estrogen is very important to the differentiation of B lymphocytes; B lymphopoiesis induced by OVX was supposedly involved in osteoporosis. But the effects of B lymphocytes on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) are not clear. In this study, we detected bone quality and bone loss in a trabecular bone by electronic universal material testing machine and microcomputed tomography (micro-CT) in OVX and splenectomized-ovariectomy (SPX-OVX) rats. Additionally, changes in lymphocytes (B lymphocyte, CD4+ and CD8+ T lymphocytes, and macrophages) in the bone marrow were analyzed by flow cytometry. The osteogenesis of BMSCs cocultured with normal and LPS-pretreated B lymphocytes was detected by BCIP/NBT and Alizarin red S staining. Measurement of the Notch2, Notch4, Hey1, Hey2, Hes1, and runt-related transcription factor 2 (Runx2) expression in BMSCs cocultured with B lymphocytes was done using real-time PCR. The effects of dexamethasone and DAPT (inhibitor of Notch signaling) on osteogenesis of BMSCs were detected by BCIP/NBT, Alizarin red S staining, and real-time PCR. Osteoporosis happened in OVX rats, more serious in SPX-OVX rats, B lymphocytes increased in OVX rats, and sharply higher in SPX-OVX rats. Osteoporosis did not happen in SPX rats which is still companied with a high increase of B lymphocytes. LPS-pretreated B lymphocytes suppressed the osteogenesis of BMSCs, but the normal B lymphocytes could not. The LPS-pretreated B lymphocytes upregulated the expression of Notch4, Hes1, and Hey2 and downregulated the expression of Runx2 in BMSCs. Dexamethasone and DAPT could downregulate the high expression of Notch4, Hes1, Hey2 and upregulate the low expression of Runx2 in BMSCs which cocultured with LPS treated B lymphocytes, the inhibited ALP and Alizarin red staining in BMSCs which cocultured with LPS treated B lymphocytes also partly restored.
Highlights
It has become clear that complex interactions underlie the relationship between the skeletal and immune systems
We investigated the effects of dexamethasone in the differentiation of bone mesenchymal stem cells (BMSCs) which were cocultured with B lymphocytes and the changes of the Notch signaling in BMSCs; we used the inhibitor of Notch signaling to investigate the differentiation and the expression of Notch signaling in BMSCs
Bone mineral density (BMD) of the femur and lumbar, as determined by dual-energy X-ray absorptiometry (DXA), was decreased by 6% (p < 0 05) and 19% (p < 0 001), respectively, in OVX rats compared with Sham but was decreased by 6.5% (p < 0 01) and by 19.8% (p < 0 001) in OVX-SPX rats compared with Sham (Figure 1(d))
Summary
It has become clear that complex interactions underlie the relationship between the skeletal and immune systems. Estrogen deficiency caused by ovariectomy (OVX) results in a marked bone loss due to exceeded bone resorption by increased osteoclasts (OC), which are partly stimulated by the immune system [1]. As one of the important lymphocytes in the immune system, the role of B lymphocytes in bone mesenchymal stem cells of bone loss induced by estrogen deficiency remains unknown. These experiments were designed to investigate the skeleton phenotypes in splenectomized OVX female rats and the effects of B lymphocytes on OVX-induced bone loss. We investigated the effects of dexamethasone in the differentiation of BMSCs which were cocultured with B lymphocytes and the changes of the Notch signaling in BMSCs; we used the inhibitor of Notch signaling to investigate the differentiation and the expression of Notch signaling in BMSCs
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