Investigating the anti-osteoporotic mechanisms of Ficus Deltoidea: Modulation of The WNT/Β-Catenin signaling pathway in an oxidative stress-induced osteoblast cell culture

Abstract

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mass and microarchitectural deterioration, which increases bone fragility and the risk of fractures, particularly among the elderly and individuals exposed to oxidative stress. Oxidative stress has been shown to impair osteoblast function and disrupt bone remodeling. Despite the high prevalence of osteoporosis, there is a lack of therapeutic agents that effectively target both oxidative damage and bone formation pathways. Ficus deltoidea (FD) has been shown to possess bone-protective effects in postmenopausal animal models. However, experimental validation of its molecular mechanisms in bone protection remains limited. This study aimed to evaluate the effects of FD extracton bone-forming activity and Wnt/β-catenin signaling in an in vitro oxidative stress model using human fetal osteoblast (hFOB 1.19) cells. The hFOB 1.19 cells were divided into five groups (n=3 per group): (1) untreated control, (2) H₂O₂-stressed group, and three treatment groups exposed to H₂O₂ followed by Ficus deltoidea (FD) extract at concentrations of (3) 6.25 μg/mL, (4) 12.5 μg/mL, and (5) 25 μg/mL. Oxidative stress was induced using hydrogen peroxide (H₂O₂), and its IC₅₀ value was determined using the MTS assay prior to treatment. After oxidative stress induction, the cells were treated with FD extract for 3 and 7 days. At the end of each treatment period, Alizarin Red S staining and quantification with 10% cetylpyridinium chloride (CPC) were performed to assess calcium deposition. Total RNA and protein were then extracted for RT-qPCR and ELISA analyses, respectively. Protein expression analysis focused on oxidative stress markers (SOD, GPx, CAT, and MDA) and alkaline phosphatase (ALP). Gene expression analysis targeted Wnt signaling components (Wnt3a, LRP5/6, Frizzled-2, GSK3β, AXIN-2, and β-catenin), antioxidant markers (Nrf2 and ERK), and osteogenic markers (RUNX2, Osterix, and COL1A1). The results demonstrated that FD, particularly at 6.25 μg/mL, significantly mitigated H₂O₂-induced cytotoxicity, promoted calcium deposition, and upregulated key proteins and genes associated with oxidative markers, the Wnt signalling pathway, antioxidant defense, and osteogenic differentiation. Specifically, FD significantly increased the expression of CAT and decreased MDA levels, while having no significant effect on GPx. In the Wnt signaling pathway, FD showed no significant effect on Wnt3a in Day 3 and 7 but significantly upregulated LRP5/6, Frizzled-2, and β-catenin expression. It also downregulated GSK3β, with no significant change observed in AXIN-2. FD treatment significantly increased the expression of antioxidant regulators Nrf2 and ERK. Regarding osteogenic markers, FD markedly increased the expression of RUNX2, Osterix, and ALP, while COL1A1 expression remained unchanged. These findings suggest a dual mechanistic role of FD as both an antioxidant and osteo-inductive agent, mediated in part through modulation of the Wnt/β-catenin signaling pathway in oxidatively stressed osteoblasts. In conclusion, FD extract reversed oxidative damage and enhanced osteoblast function through modulation of the Wnt/β-catenin pathway in H₂O₂- stressed osteoblasts. This study provides molecular evidence supporting the therapeutic potential of Ficus deltoidea as a phytomedicine for osteoporosis management.