Enhancing Bone Fracture Healing via Modulation of the Wnt/ERK Signaling Pathway and Mitochondrial Dynamics with Loxoprofen

Abstract

Background: Periosteal stem cells (PSCs) are pivotal in bone tissue repair and regeneration. This study explores the effect of Loxoprofen (Lox) on PSCs’ osteogenic differentiation, focusing on the Wingless/Integrated/Extracellular Signal-Regulated Kinase (Wnt/ERK) signaling pathway and mitochondrial dynamics. Methods: Initially, we induced osteogenic differentiation in PSCs and exposed them to varying concentrations of Lox (5 μg/mL, 10 μg/mL, 20 μg/mL). Employing a murine fracture healing model and Hematoxylin and Eosin (H&E) staining, we assessed Loxs in vivo impact on fracture healing in mice. PSCs proliferation, adhesion, and migration effects under Lox influence were scrutinized using Cell Counting Kit-8 (CCK-8), cell adhesion experiments, and Transwell assays. Furthermore, Loxs effects on Alkaline Phosphatase (ALP) activity, mineralized nodules, osteogenic markers, and Wnt/ERK pathway-associated proteins during PSCs osteogenic differentiation were analyzed via Enzyme-Linked Immunosorbent Assay (ELISA), ALP staining, Alizarin Red staining, western blot, and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Microscopy was utilized to explore Loxs impact on mitochondrial morphology and cytoskeleton in PSCs post-osteogenic differentiation. Additionally, we measured Loxs effects on adenosine triphosphate (ATP) levels, reactive oxygen species (ROS) levels, and mitochondrial membrane potential in PSCs after osteogenic differentiation with ELISA and flow cytometry. Finally, we demonstrated the inhibitory effect of Wnt/ERK pathway inhibitors (Dickkopf-1 (DKK1)/SCH772984) on Lox-induced Wnt/ERK signaling pathway activation using western blot. Results: Our findings confirm that Lox, at low, medium, and high doses, increases the proliferation, adhesion, and migration capacities of PSCs (p < 0.05). Lox demonstrates a pronounced elevation in the osteogenic potential of PSCs while significantly promoting mitochondrial activity, fusion, and progress of fracture healing (p < 0.05). Additionally, Lox treatment leads to significant enhancement in the activation of the Wnt/ERK signaling pathway (p < 0.05). The addition of Wnt/ERK pathway inhibitors significantly suppressed the effect of Lox on the Wnt/ERK signaling pathway (p < 0.05). Conclusions: Lox facilitates PSCs’ osteogenic differentiation by enhancing the Wnt and ERK signaling pathways and mitochondrial dynamics, contributing to improved fracture healing.