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 8-week-old female C57BL/6J mice were randomly divided into four groups: sham-operated group, ovarian extirpation group, 5 mg/kg Hernandezine group and 10 mg/kg Hernandezine group. Bilateral ovariectomy was performed to establish an osteoporosis mouse model in the latter three groups. Hernandezine at the dose of 5 and 10 mg/kg was intraperitoneally injected in the two drug-treated groups every 2 days after the operation. Femurs were collected after 8 weeks of modeling for Micro-CT scanning, bone parameter analysis and hematoxylin-eosin staining to assess bone loss.
RESULTS AND CONCLUSION: (1) Hernandezine could inhibit the expression of pro-inflammatory related genes induced by lipopolysaccharide, and exerted anti-inflammatory effects by down-regulating the transcription level of genes related to Toll-like receptor 4/nuclear factor-κB signaling pathway in a concentration-dependent manner. 5 μmol/L Hernandezine exhibited a more pronounced inhibitory effect. (2) Hernandezine could inhibit the expression of genes related to osteoclast activation and bone resorption, and inhibit osteoclast activation in vitro, also in a concentration-dependent manner. (3) Hernandezine could reduce bone loss in estrogen-deficient induced osteoporosis mice, and the recovery effect was more significant in the 10 mg/kg group. In conclusion, we confirmed that Hernandezine inhibits the conversion process of macrophage pro-inflammatory phenotype and osteoclast activation, and exerts anti-inflammatory effects by down-regulating the Toll-like receptor 4/nuclear factor-κB signaling pathway. Hernandezine has also been confirmed to attenuate excessive bone loss in estrogen-deficient osteoporosis.  

Key words: Hernandezine, macrophages, inflammation, osteoclast activation, osteoporosis