Zuogui Pill Modulates Osteoblast-Osteoclast Coupling in Ovariectomized Rats: A Three by Three Factorial Study

Postmenopausal osteoporosis is driven by estrogen-deficiency-associated uncoupling of bone formation and resorption. This study quantified the independent and interactive effects of Zuogui Pill Yin-nourishing subformulations in an ovariectomized rat model and examined associations with the EphB4/ephrinB2/RhoA/alkaline phosphatase pathway. In accordance with ARRIVE 2.0, 130 specific pathogen-free female Sprague-Dawley rats were randomized to 13 groups (n=10/group), including nine groups in a three-by-three factorial design. Endpoints included P1NP, CTX-1, femoral bone mineral density measured by dual-energy X-ray absorptiometry (DXA), trabecular histology, and messenger ribonucleic acid and protein expression measured by reverse transcription quantitative polymerase chain reaction and Western blotting. The full formula (I2A2) was associated with higher P1NP, lower CTX-1, and higher BMD than the model group. Factorial analysis identified a Yin-Yang interaction between P1NP and BMD, supporting pharmacological synergy. Molecular changes were associated with increased EphB4/ephrinB2/ALP expression and reduced RhoA expression. These findings support an association between Zuogui Pill treatment, improved bone phenotypes, and modulation of pathway markers; causal pathway dependence requires inhibitor, knockdown, and co-culture validation.