Ginsenoside Rh2 alleviates osteoporosis by attenuating oxidative stress-induced osteoblast dysfunction via the FoxO1/β-catenin pathway

Abstract

The degree of oxidative stress decreases osteoblast function with age, which leads to a decline in bone compressive capacity. Ginsenoside Rh2 is a known clinical or adjuvant therapy for various tissues. In this study, we investigate the pharmacological effects of Rh2 against oxidative stress-induced osteoblasts. Osteoblasts are pretreated with Rh2 for 48 h and then exposed to hydrogen peroxide (H2O2), which results in significantly decreased ROS levels, increased antioxidant enzyme activity, and enhanced mitochondrial function. Functionally, Rh2 increases alkaline phosphatase (ALP) expression, together with enhanced mineralization and expression of osteogenesis-associated genes. Rh2 also promotes the nuclear translocation of FoxO1 and β-catenin, whereas it does not reverse reduced mineralization caused by decreased FoxO1 or β-catenin activity, indicating that its effect is mediated through the functional interaction between FoxO1 and β-catenin. In a mouse model of lipopolysaccharide (LPS)-induced bone loss, Rh2 administration improves trabecular microstructure, increases osteoblast numbers, and upregulates serum metabolites associated with bone formation. Immunofluorescence analysis further reveals that Rh2 promotes the nuclear co-localization of FoxO1 and β-catenin in femurs, indicating their coordinated action within this signaling axis. These findings indicate that Rh2 mitigates oxidative stress-induced osteoblast dysfunction via the FoxO1/β-catenin pathway, highlighting the pivotal role of redox balance in bone remodeling and suggesting a promising therapeutic strategy for osteoporosis.

1. Introduction

Osteoporosis is a degenerative disease characterized by progressive deterioration of bone microstructure, leading to reduced bone mass and an increased fracture risk [1]. It tends to be associated with age and thus represents a major problem as populations age and life expectancies increase. Bone remodeling involves two interconnected processes: osteoclast-mediated resorption or destruction of the mineralized bone matrix and osteoblast-mediated formation of new bone [2]. Imbalances in the differentiation of osteoblasts and osteoclasts, characterized by reduced osteoblast activity and increased osteoclast activity, result in osteoporosis. Accumulating evidence suggests an association between elevated oxidative stress and reduced osteoblast differentiation, demonstrating that higher levels of oxidative stress result in the dysfunction and apoptosis of bone cells, ultimately causing bone loss [3,4] Therefore, minimizing ROS production, scavenging free radicals, and enhancing antioxidant activity are important for preventing or delaying bone loss. Excessive accumulation of ROS leads to oxidative stress, which damages cellular macromolecules and ultimately results in cell death [5]. Forkhead box O (FoxO) proteins are highly conserved transcription factors that regulate biological processes, including proliferation, differentiation, oxidative stress resistance, apoptosis, and inflammation, in response to various endogenous and exogenous stimuli [6,7]. FoxOs are primarily phosphorylated by PI3K/Akt signaling, resulting in their translocation from the nucleus to the cytoplasm, inhibiting FoxO-dependent transcription of target genes [8]. Oxidative stress induces FoxO1 expression, which upregulates antioxidant enzymes to counteract ROS [9,10]. Therefore, FoxO1 acts as a crucial cellular sensor pivotal for mitigating oxidative damage. FoxOs have been shown to modify the pathogenesis of skeletal diseases by their ability to influence both bone mass and strength [11]. Specifically, FoxO1 is a master regulator of osteoblast function and redox balance according to cell-specific knockout and molecular analysis [12,13]. The interaction of FoxO1 with Runx2 or its promoter stimulates osteoblast differentiation and mineralization nodule formation [14]. β-Catenin functions as an essential cofactor for FoxO1, and their interaction is central to transducing ROS-induced downstream signaling [15]. This leads to altered transcription of target genes and the antagonization of Wnt/TCF pathways in osteoblast precursors under stress conditions [16]. Consequently, pharmacological modulation of FoxO1/β-catenin signaling may represent a promising therapeutic strategy for osteoporosis by preserving osteoblast function in an adverse redox environment.

Ginseng (Panax ginseng C.A. Meyer) is a traditional Chinese medicinal herb that contains ginsenosides as its principal bioactive constituents, which are widely utilized in clinical and adjuvant treatments for various diseases. Ginsenosides have beneficial effects through multiple molecular mechanisms, including anti-apoptotic, anti-oxidative, anti-aging, anti-cancer, and functional restoration activities [17–19]. Notably, recent studies have documented that ginsenosides such as Rh2 inhibit osteoclastogenesis while promoting osteoblast differentiation and mineralization [20]. Nevertheless, the specific mechanisms by which Rh2 regulates osteoblast function, particularly under conditions of oxidative stress, remain unclear, and current evidence is still limited. Herein, the protective effects of Rh2 on osteoblast function under conditions of oxidative stress were investigated. Osteoblasts were exposed to H2O2 in vitro to induce oxidative stress. The effects of Rh2 on osteoblast differentiation, oxidative damage resistance, and mineralization were evaluated, with a particular focus on its regulation of the FoxO1/β-catenin signaling. To further validate the protective effects in vivo, we employed a lipopolysaccharide (LPS)-induced bone loss model. LPS triggers systemic inflammation that activates NADPH oxidase in immune and bone cells, leading to a surge in ROS and subsequent oxidative stress. This state of oxidative stress impairs osteoblast function and promotes osteoclast formation, thereby recapitulating key pathological features of osteoporosis associated with oxidative stress. In this model, we evaluated the therapeutic and preventive effects of Rh2 on osteoporosis. The findings provide novel insights into the mechanism by which Rh2 protects osteoblasts from oxidative stress-induced damage.