Mechanism by which Hernandezine alleviates osteoporosis through macrophage polarization and osteoclast activation

Abstract

BACKGROUND: Hernandezine has shown promising therapeutic effects due to its anti-inflammatory bioactivity in diseases such as suppression of tumors, antiplatelet agglutination and diabetes. However, there are no basic studies on the effects and molecular mechanism of Hernandezine on macrophage phenotype and osteoclast activation. OBJECTIVE: To investigate the role of Hernandezine on the regulation of macrophage polarization, osteoclast activation and osteoporosis. METHODS: (1) Cellular experiments: RAW264.7 was used as macrophage model and divided into four groups: Control group, lipopolysaccharide group, lipopolysaccharide + 2.5 μmol/L Hernandezine group, lipopolysaccharide + 5 μmol/L Hernandezine group. Macrophage polarization was induced in the latter three groups using a complete medium supplemented with lipopolysaccharide. The two drug-treated groups received 2.5 and 5 μmol/L Hernandezine, respectively. RAW264.7 cells were induced toward osteoclast differentiation using a complete medium supplemented with nuclear factor κB receptor activator ligand. Macrophage polarization was assessed via qRT-PCR and immunofluorescence for inflammatory cytokine expression. The effects of Hernandezine on osteoclast differentiation were evaluated using qRT-PCR, tartrate-resistant acid phosphatase staining, and F-actin staining. (2) In vivo experiments: Twenty-four female C57BL/6J mice were randomly divided into four groups: sham operation, ovariectomy, ovariectomy + 5 mg/kg Hernandezine, and ovariectomy + 10 mg/kg Hernandezine. The latter three groups underwent bilateral ovariectomy to establish an osteoporosis model. The two drug-treated groups received intraperitoneal injections of Hernandezine at 5 or 10 mg/kg every two days post-surgery. After 8 weeks, femurs were collected for Micro-CT scanning, bone parameter analysis, and hematoxylin-eosin staining to evaluate bone loss. RESULTS AND CONCLUSION: Hernandezine inhibited lipopolysaccharide-induced pro-inflammatory gene expression in macrophages by downregulating the transcription of Toll-like receptor 4/nuclear factor κB signaling pathway-related genes, exhibiting a concentration-dependent effect, with 5 μmol/L showing more significant inhibition. Hernandezine also inhibited the expression of genes related to osteoclast activation and bone resorption, and suppressed osteoclast activation in vitro in a concentration-dependent manner. In vivo, Hernandezine reduced bone loss in estrogen-deficient osteoporotic mice, with the 10 mg/kg group showing better recovery. CONCLUSION: This study confirms that Hernandezine inhibits macrophage pro-inflammatory phenotype transformation and osteoclast activation by downregulating the Toll-like receptor 4/nuclear factor κB signaling pathway, and alleviates excessive bone loss in estrogen-deficient osteoporosis.

1. Introduction

Osteoporosis is a systemic skeletal disease characterized by reduced bone mass, deterioration of bone microarchitecture, and increased fracture risk, and has become a major public health problem in the aging global society [1-2]. Fragility fractures caused by osteoporosis are the leading cause of disability and death in elderly patients. The need for prevention and treatment of osteoporosis is increasingly urgent, but its pathogenesis is complex, involving interactions of genetic, hormonal, lifestyle and other factors [3]. Bone homeostasis depends on the dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Excessive bone resorption caused by overactivation of osteoclasts is the core pathological process of bone loss [4-5].

Osteoclasts are bone tissue-specific multinucleated cells derived from macrophage precursors, primarily from bone marrow hematopoietic stem cells. Macrophage-to-osteoclast differentiation depends on two key cytokines: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) [6]. In local pathological processes induced by estrogen deficiency, aging, and inflammation, various inflammatory cytokines such as tumor necrosis factor α, interleukin-1β, and interleukin-6 can enhance osteoclast differentiation and bone resorption [7]. Currently, the most commonly used clinical drugs for osteoporosis are anti-resorptive agents, typically bisphosphonates and denosumab. However, long-term use of these drugs can lead to adverse events such as osteonecrosis of the jaw, atypical fractures, and acute bone loss after discontinuation [8]. Therefore, new anti-resorptive therapeutic strategies are urgently needed.

In recent years, small-molecule drugs and natural compounds extracted from Chinese herbal medicines have shown potent therapeutic capabilities in inhibiting osteoclast differentiation [9]. Hernandezine (Her) is a bisbenzylisoquinoline alkaloid extracted from the traditional Chinese herb Thalictrum glandulosissimum. Studies have reported that Hernandezine has anti-tumor, anti-platelet aggregation, and calcium channel blocking effects [9], and can inhibit lipopolysaccharide-induced tumor necrosis factor α expression in lymphocytes [10]. SONG et al. [11] reported that Hernandezine is an AMP-activated protein kinase (AMPK) agonist that can induce apoptosis and autophagy and kill tumor cells. The AMPK signaling pathway plays a key role in regulating cellular energy homeostasis [12]. Previous studies have shown that AMPK activation can inhibit osteoclast differentiation and bone resorption. However, the effects of Hernandezine on macrophage polarization and osteoclast activation, and its potential molecular mechanisms, remain unclear. This study aims to investigate the effects of Hernandezine on macrophage polarization, osteoclast activation, and osteoporosis, providing a theoretical basis for its clinical application.