Objective To investigate the regulatory effect of moxibustion, a traditional Chinese medicine therapy, on bone metabolism in ovariectomized (estrogen-deficient) mice and to explore its underlying mechanisms.

Methods Female C57BL/6J mice of 12 weeks old were randomly assigned to five groups, including a sham operation control group (SHAM), an ovariectomy group (OVX), a group given ovariectomy and broad-spectrum antibiotics (OVX-A), a group given ovariectomy and moxibustion (OVX-M), and a group given ovariectomy, broad-spectrum antibiotics, and moxibustion (OVX-A-M), with 5 mice in each group. Then, 4 weeks post-surgery, the mice in each group received broad-spectrum antibiotics and/or moxibustion intervention for an additional 4 weeks. After that, the mice were sacrificed, and samples were collected. Micro-CT was used to assess bone volume parameters in the distal femurs, including bone volume/tissue fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness (Ct.Th). Targeted metabolomics was used to measure serum tryptophan metabolites, and qPCR was performed to quantify serotonin (SER) receptors Htr2a and Htr2b mRNA levels in bone marrow. In addition, primary bone marrow mesenchymal stem cells (BMSCs) were treated with serotonin of varying concentration gradients (0, 0.01, 0.1, 1, and 10 μmol/L). Alkaline phosphatase (ALP) staining was performed to assess osteogenic differentiation, while qPCR was performed to assess the expression of Colla1, an osteogenesis-related gene, and serotonin receptors Htr2a and Htr2b.

Results Compared with the SHAM mice, the OVX mice exhibited significant deterioration in bone microarchitecture, showing decreased BV/TV (10.57 ± 2.82% vs. 4.20 ± 0.96%, P < 0.01), reduced Tb.N (3.16 ± 0.11 vs. 2.25 ± 0.15, P < 0.01), increased Tb.Sp (0.31 ± 0.01 vs. 0.45 ± 0.03, P < 0.01), and decreased levels of serum tryptophan metabolite SER. Compared with the OVX mice, the OVX-M mice showed a notable improvement in bone microarchitecture, with BV/TV increasing to (7.51 ± 1.42)% (P < 0.05), and elevated levels of serum SER and bone marrow Htr2a gene expression (P < 0.05). However, the effect of moxibustion in reversing bone loss in OVX mice disappeared when the gut microbiota was disrupted by broad-spectrum antibiotics. OVX-A-M mice had significantly lower serum serotonin levels compared to OVX-M mice (P < 0.001). According to the findings from the in vitro experiments, SER enhanced the osteogenic differentiation of BMSCs, with the optimal effect achieved at a concentration of 0.1 μmol/L. Furthermore, SER at 0.1 μmol/L significantly increased the expression levels of osteogenesis-related genes Colla1 and Htr2a (P < 0.05).

Conclusion Moxibustion therapy can inhibit postmenopausal bone loss, potentially by regulating gut microbiota-derived SER, activating the 5-HT2A receptor, and promoting the osteogenic differentiation of BMSCs.