Murine ovariectomy (OVX)-induced osteoporosis model

Following the Institutional Animal Ethics Committee’s approved guidelines, animal experiments were conducted. A cohort of 36 female C57BL/6J mice was individually housed in ventilated cages under specific pathogen-free conditions. The mice were allocated into three groups through random assignment: the sham-operated group (n = 12), the OVX group (n = 12), and the OVX + LIQ group (n = 12). After a 1-week acclimatization, mice were anesthetized and underwent bilateral OVX surgery for the OVX and OVX + LIQ groups, while the sham group underwent a sham procedure (Hong et al., 2021). For the OVX + LIQ group, mice were intraperitoneally injected with 20 mg/kg of LIQ (dissolved in DMSO) every other day for 6 weeks. Prior to the formal experiment, a pre-experiment was conducted to determine the appropriate and effective dose of LIQ for our study. During this pre-experiment, we systematically varied the doses (ranging from 5 to 30 mg/kg) of LIQ to identify the optimal dosage (20 mg/kg) that exhibited the desired inhibitory effects on bone resorption. a The sham and OVX groups received an equal volume of vehicle, prepared as 1% DMSO in PBS, during the same period. At the study’s conclusion, all mice were euthanized. The mice were subjected to the respective treatments promptly following OVX to assess the preventive or ameliorative effects of LIQ in the development of osteoporosis induced by ovariectomy.

Half of the mice from each group were utilized to assess bone homeostasis post LIQ administration. Blood samples were collected from the abdominal aortas, then centrifuged to obtain serum, and analyzed for TRAcP and C-terminal telopeptide (CTX-1) levels using R&D Systems’ enzyme-linked immunosorbent assay (ELISA) kits. The left femur were utilized for micro-computed tomography (Micro-CT) scanning to assess microstructural changes and three-point bending tests. Histomorphology analysis was performed on the right femur. The remaining half of the mice underwent in vivo biosafety testing for LIQ. Organ samples were extracted for evaluation of liver, spleen, lung, heart, and renal toxicity based on organ size and surface appearance. Blood samples were collected from the aortas for complete blood count analysis using a BC6800 automated analyzer.