Leonurine hydrochloride promotes osteogenic differentiation and increases osteoblastic bone formation in ovariectomized mice by Wnt/β-catenin pathway

Highlights: • Leonurine hydrochloride promotes osteogenic differentiation of osteoblasts in vitro. • Wnt/β-catenin pathway acts as an important role in the osteogenic differentiation affected by Leonurine hydrochloride. • Leonurine hydrochloride alleviate the effect of osteoporosis by acclerating the bone formation. • Bone mineral density and micro-structures are improved by the treatment of Leonurine hydrochloride. Leonurine hydrochloride (LH) is a synthetic chemical compound derived from leonurine that can be extracted from Leonurus sibiricus and possesses antioxidant, anti-apoptosis, and neuroprotective activities. In previous studies, LH has been demonstrated to attenuate osteoclast activity and prevent bone loss. However, it is unknown whether LH accelerates bone formation and promotes osteogenic differentiation. We systematically examined the effects of LH on ovariectomized-induced osteoporotic mice and the MC3T3-E1 osteoblastic cell line. The results revealed that LH enhanced differentiation of MC3T3-E1 cells, with a dose-dependent increase in alkaline phosphatase (ALP) activity. Moreover, LH upregulated osteogenesis-related gene expression, including osterix, alpha 1 type 1 collagen, runt-related transcription factor 2 (Runx2) and ALP, as shown by quantitative reverse transcription-polymerase chain reaction analysis. At the same time, elevated expression of low-density lipoprotein receptor-related protein 5 and β-catenin mRNA was detected in the Wnt/β-catenin pathway. A western blot analysis revealed that LH dose-dependently increased the expression of Runx2 and β-catenin, and promoted phosphorylation of glycogen synthase kinase-3β in vitro. The in vivo results showed that administering LH (15 mg/kg/d) for 8 weeks alleviated destruction of the trabecular microstructure caused by osteoporosis. LH increased the bone mineral density and trabecular number, decreased trabecular separation according to a micro-computed tomography scan. In addition, LH enhanced the expression of β-catenin and Runx2 in vivo. In conclusion, LH promoted osteogenic differentiation and bone formation in vivo and in vitro, which alleviated osteoporosis through activation of the Wnt/β-catenin pathway.