Juglans regia L. extract promotes osteogenesis of human bone marrow mesenchymal stem cells through BMP2/Smad/Runx2 and Wnt/β-catenin pathways.

Background: The present study investigates the effects of Juglans regia L. (walnut, JRL) leaves extract on osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs).

Methods: hBMSCs were incubated with different concentrations of JRL extract (10, 20, 40, or 80 μM). Cell proliferation was evaluated by Cell Counting Kit-8 assay (CCK-8) assay. ALP activity and Alizarin Red staining were used to assess the osteogenesis of BMSCs. Western blot was performed to measure the levels of proteins.

Results: Our results showed all concentrations of JRL extract had no significant effect on cell proliferation. JRL extract concentration-dependently promoted osteoblastic differentiation and cell autophagy of hBMSCs, characterized by the increased expression of pro-osteogenic markers alkaline phosphatase (ALP), osteocalcin (BGLAP), osterin, and osteoprotegerin (OPG) and autophagy marker proteins (LC3II, Beclin-1, and p62). Furthermore, JRL extract stimulated the activation BMP2/Smad/Runx2 and Wnt/β-catenin signaling pathways in hBMSCs, which play key roles in osteogenesis differentiation. Meanwhile, BMP inhibitor (Noggin) and Wnt antagonist Dickkopf-1 (DKK1) both reversed the increases of BGLAP, osterin, and OPG expression induced by JRL extract.

Conclusions: Our findings indicate that JRL extract regulated osteogenic differentiation and cell autophagy of hBMSCs through the BMP2/Smad/Runx2 and Wnt/β-catenin pathways.