Establishment of OVX-induced osteoporosis mouse model

All animal experiments were carried out with the approval of the Guangxi Medical University Ethics Committee, including constructing an OVX-induced osteoporosis mouse model. Thirty 11-weeks-old female C57BL6/J mice were randomly divided into five groups: sham operation + vehicle group (Sham group), OVX + vehicle group (OVX group), OVX + 100 ng/kg E₂ group (E₂ group), OVX + 15 mg/kg MRL group (low-concentration group), and OVX + 30 mg/kg MRL group (high-concentration group). The mice were anesthetized using tribromoethanol (200 μL/10 g body weight), and mice from four groups (except for the Sham group) were ovariectomized to induce osteoporosis. Berberine was administered for 1 week, and mice were treated with group-specific reagents for 6 weeks. The mice from the Sham and OVX groups were intraperitoneally injected with normal saline containing 4% DMSO. Mice from the E₂ group were intraperitoneally injected with 100 ng/kg E₂. Low- and high-concentration groups of mice were administered 15 mg/kg and 30 mg/kg MRL respectively. Finally, all mice were sacrificed, and the tibias were removed for histological and microscopic-computed tomography (micro-CT) analysis.