Study on the Mechanism of Aerobic Exercise Alleviating Osteoporosis in Ovariectomized Mice through Regulating the RANKL/RANK/OPG Pathway

Objective This study aimed to explore the regulatory effects of aerobic exercise on the RANKL/RANK/OPG signaling pathway and evaluate its potential therapeutic effects on osteoporosis in ovariectomized mice (OVX).Methods:1.Animal Experiment: 40 2-month-old SPF male SD rats, weighing (225±25) g, were purchased from the Animal Experiment Center of Xi’an Jiaotong University (Qualification Certificate No.: Shaanxi Medical Practice No. 17). The experiment was conducted at the Experimental Center of Yan’an University Medical College. Rats had free access to food and water, and the circadian rhythm was 12 h. The experimental process strictly followed the Shaanxi Provincial Regulations on the Administration of Laboratory Animals.An ovariectomized mouse model was established. Histomorphological Assessment: HE staining was used to assess the morphological impact of aerobic exercise on osteoclasts, and TRAP staining and bone resorption pit assay were used to evaluate its effect on osteoclast function. Molecular Biological Analysis: qRT-PCR and Western blotting were employed to investigate the regulation of the RANKL/RANK/OPG pathway by aerobic exercise and its impact on the expression of RANKL, RANK, IκB, NF-κB, and p-IκB proteins and their mRNA levels in bone tissue.2. Cell Experiment: After 8 weeks of aerobic exercise intervention, mice were sacrificed, and bone macrophage precursor cells were extracted from the forelimbs’ femurs for the experiments, including: Cell Activity Assessment: CCK-8 assay was used to observe the effect of aerobic exercise on RANKL-induced osteoclast activity.Biochemical Detection: The effects of aerobic exercise on serum levels of calcium, ALP, BALP, PTH, phosphate, and calcitonin were evaluated. Results 1. Histomorphological Assessment: HE and TRAP staining results showed that the number of osteoclasts per bone surface (N.Oc/BS) in the aerobic exercise group was significantly lower than that in the model group (p <0.001). 2. Serum Biochemical Analysis: Compared with the model group, the aerobic exercise group showed: CCK-8 Assay: The activity of DMSO was significantly reduced (p <0.01, p <0.001).Serum Biochemical Levels: The levels of serum calcium, ALP, BALP, and PTH were significantly decreased (p <0.01), while the levels of serum phosphate and calcitonin were significantly increased (p <0.01).qRT-PCR and Western Blotting: The expression of E2 and OPG was significantly increased, while the protein and mRNA expression of TRAP, RANKL, and Cathepsin K were significantly decreased (p <0.001). The expression of NFATc1, c-Fos, ATP6V0D2, CTSK, DC-STAMP, and MMP9 proteins and their mRNA levels were significantly increased in the aerobic exercise group (p <0.01 or p <0.001). Aerobic exercise significantly inhibited IκB phosphorylation and increased OPG protein and mRNA expression (p<0.01).Conclusion:Aerobic exercise significantly inhibits osteoclast activity by regulating the RANKL/RANK/OPG pathway, thereby improving osteoporosis in ovariectomized mice. This finding providestheoretical support for clinical guidelines on the treatment of osteoporosis in postmenopausal women.