Melatonin improves osteogenic differentiation in a high-glucose environment by activating NRF2 to promote autophagy through the regulation of cross-talk between macrophages and bone marrow mesenchymal stem cells.

Abstract

Background: Melatonin (MT) can regulate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), but its effect on the osteogenic differentiation of BMSCs under high glucose (HG) conditions is unclear. Therefore, in this study, the effect of MT on the osteogenic differentiation of BMSCs under HG conditions was investigated.

Methods: A mouse model of diabetic osteoporosis (DOP) was induced by the intraperitoneal injection of streptozotocin (STZ), and macrophages or BMSCs were cultured with 25 mM glucose to construct an in vitro cell model. Different doses of MT were used to treat the mice or cells. Genes and proteins were assessed through RT‒qPCR and Western blotting. ALP staining, alizarin red staining, and HE staining were used to assess the osteogenic differentiation of BMSCs and the advancement of DOP in mice.

Results: Under normal conditions, MT could increase the expression of osteogenic differentiation-related proteins RUNX2, OCN, and OPN, and enhance differentiation and mineralization levels in BMSCs; however, MT failed to stimulate osteogenic differentiation in BMSCs under HG conditions. Furthermore, regardless of whether under HG conditions, in macrophages, MT suppressed the expression of the M1 phenotype markers CD86, iNOS, and CCR7 while increasing the expression of the M2 phenotype markers CD206, Arg1, and Ym1. Subsequent experiments revealed that under HG conditions, MT indirectly promoted the osteogenic differentiation of BMSCs through the enhancement of the M2 polarization of macrophages; however, MT was unable to directly influence the osteogenic differentiation of BMSCs. Additionally, in mouse experiments, administering high doses of MT effectively mitigated DOP by lowering blood glucose levels, ameliorating pathological damage in femoral tissues, and enhancing collagen accumulation and osteogenic markers expression. From a mechanistic standpoint, MT triggered autophagy by counteracting the suppressive effect of HG on NRF2, thus reducing HG-triggered ROS generation and inflammation in macrophage, promoting the M2 polarization of macrophages, and mitigating the suppressive effect of HG on the osteogenic differentiation of BMSCs.

Conclusion: Our study indicates that under HG conditions, MT improves osteogenic differentiation by regulating the crosstalk between M2 macrophages and BMSCs.