Juglans regia L. extract promotes osteogenesis of human bone marrow mesenchymal stem cells through BMP2/Smad/Runx2 and Wnt/\u03b2-catenin pathways

Abstract

BackgroundThe present study investigates the effects of Juglans regia L. (walnut, JRL) leaves extract on osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs).MethodshBMSCs 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.ResultsOur 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.ConclusionsOur findings indicate that JRL extract regulated osteogenic differentiation and cell autophagy of hBMSCs through the BMP2/Smad/Runx2 and Wnt/β-catenin pathways.