Objective To investigate the mechanism of action of vanillic acid (VA) in regulating osteoclast differentiation and its effect on osteoporosis in ovariectomized (OVX) mice. Methods In vitro experiment: receptor activator for nuclear factor-κB ligand (RANKL)-treated RAW264.7 cells were used to establish an osteoclast differentiation model. The cells were divided into negative control (NC), RANKL- differentiated (Diff), and RANKL plus VA at 50, 100, and 200 μmol/L groups. Cell viability was assessed using the cell counting kit-8(CCK8) assay. The osteoclast differentiation was detected by tartrate resistant acid phosphatase (TRAP) staining and immunoblot analysis of osteoclast differentiation-related proteins. Cellular reactive oxygen species (ROS) levels were measured using a 2',7'-Dichlorodihydrofluorescein diacetate（DCFH-DA）probe, and Western blotting was performed to detect nuclear factor erythroid2-related factor 2(Nrf2), Kelch like epichlorohydrin related protein 1(Keap1), glutamate-cysteine ligase catalytic subunit (GCLC), superoxide dismutase (SOD), and catalase (CAT) protein expressions. Additionally, ML385 was used to inhibit Nrf2 expression to evaluate its role in osteoclast differentiation and oxidative stress. In vivo experiment: an osteoporosis model was established in OVX mice. The mice were divided into sham, OVX, OVX+estradiol (E2), OVX+VA50 mg/kg, and OVX+VA100 mg/kg (n= 6 per group) groups. Femoral bone structure was analyzed using micro computed tomography（Micro-CT）. Serum RANKL and osteoprotegerin (OPG) levels were assessed by enzyme linked immunosorbent assay (ELISA), and serum oxidative stress markers (SOD, CAT, and malondialdehyde (MDA)) were also quantified. Nrf2 expression in femur sections was evaluated immunofluorescently. Results In vitro: VA at concentrations of 1-500 μmol/L was non-toxic to RAW264.7 cells. Osteoclast differentiation, protein expression, and ROS levels were significantly elevated in the Diff group compared to the NC group (P<0.05). VA (200 μmol/L) significantly inhibited osteoclast differentiation, decreased protein expression and ROS levels, promoted Nrf2 nuclear translocation, and increased antioxidant enzyme (GCLC, SOD, CAT) expression (P<0.05). These changes were reversed after the inhibition of Nrf2 by ML385 (P<0.05). In vivo: compared to the sham group, OVX mice exhibited significantly reduced femoral structural mass, increased serum RANKL and MDA levels, and decreased serum OPG, SOD, and CAT levels, along with reduced Nrf2 expression (P<0.05). VA (100 mg/kg) treatment significantly improved bone structure, reduced serum RANKL and MDA levels, increased serum OPG, SOD, and CAT levels, and elevated Nrf2 expression compared to the OVX group (P<0.05). Conclusion VA mitigates osteoporosis in OVX mice by activating the Nrf2 signaling pathway, and thereby inhibiting osteoclast differentiation and oxidative stress.
Key words
vanillic acid /
Nrf2 /
oxidative stress /
osteoclast /
osteoporosis
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