Humic Acid Therapy Mitigates Estrogen Deficiency-Induced Alveolar Bone Loss and Modulates the RANKL/OPG Balance

Background: Estrogen deficiency negatively affects alveolar bone by disrupting key regulators of bone remodeling. Humic acids (HAs) are natural compounds with recognized antioxidant and anti-inflammatory properties that may attenuate bone resorption. This study investigated the effects of HAs on alveolar bone in an experimental model of estrogen depletion. Methods: Female C57BL/6 mice were randomly assigned to four groups: Sham, Sham + HA, ovariectomized (OVX), and OVX + HA. Estrogen deficiency was induced by bilateral ovariectomy. HAs derived from vermicomposted biomass were administered daily by oral gavage (80 mg/kg) for 28 days. At the end of the experimental period, mandibles were collected for structural, mineral, and histological analyses. Bone elemental composition was assessed using scanning electron microscopy coupled with energy-dispersive spectroscopy (SEM/EDS). Alveolar bone loss was evaluated by histomorphometry, while RANKL and osteoprotegerin (OPG) expression were assessed by immunohistochemistry. Osteoclasts were quantified by tartrate-resistant acid phosphatase (TRAP) staining. Data were analyzed using two-way ANOVA followed by Bonferroni's post hoc test. Results: Ovariectomy resulted in reduced calcium and phosphorus content, increased alveolar bone loss, elevated RANKL immunolabeling, increased osteoclast numbers, and a higher RANKL/OPG ratio (p < 0.05). HA treatment increased calcium and phosphorus content and attenuated alveolar bone loss in OVX animals (p < 0.05). Additionally, HA treatment partially increased OPG expression and reduced the RANKL/OPG ratio (p < 0.05), without significantly affecting RANKL immunolabeling or osteoclast numbers. Conclusions: HA therapy attenuated alveolar bone resorption in a model of estrogen depletion, possibly associated with modulation of the RANKL/OPG balance.