Abstract: BACKGROUND: Osteoporosis is not only a metabolic disease, but also an inflammatory or autoimmune disease. Immune cells Treg and Th17 have a mutually antagonistic effect on the differentiation and maturation of osteoclasts, and the maintenance of cell balance between the two is the basis for ensuring normal bone metabolism
OBJECTIVE: To explore the immunomodulatory mechanism of Xuling Jiangu Formula on osteoporosis and its relationship with the balance of Treg/Th17 cells.
METHODS: Sprague-Dawley rats were randomly divided into a sham operation group, a model group, and a drug group, with eight rats in each group. The latter two groups underwent bilateral ovariectomy to establish the animal model; the sham operation group underwent the same procedure except for ovary removal. Four weeks after modeling, bone mineral density was measured using dual-energy X-ray absorptiometry to evaluate whether the modeling was successful. Drug intervention started 4 weeks after surgery: the Xuling Jiangu Formula group was administered Xuling Jiangu Formula by gavage once daily at 10 mL/kg for 16 consecutive weeks; the model group and sham operation group were given an equal volume of physiological saline by gavage. After 16 weeks of drug administration, samples were collected. Bone mineral density and routine blood indicators were measured; bone tissue morphology was observed by hematoxylineosin staining; serum immune factor levels were detected by ELISA; gene and protein expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, retinoic acidrelated orphan receptor γT, and forkhead transcription factor 3 were measured by quantitative PCR and western blot assays; and the number of regulatory T (Treg) cells and Th 17 cells was detected by flow cytometry.
RESULTS AND CONCLUSION: (1) After 16 weeks of drug administration, compared with the model group, the bone mineral density of rats in the Xuling Jiangu Formula group significantly increased (P < 0.05). The serum levels of immune cytokines interleukin10 and transforming growth factor β increased but without significant difference, while the levels of interleukin 17 and interleukin 6 significantly decreased (P < 0.05). (2) After 16 weeks of drug administration, the gene and protein expression of osteoprotegerin and the Treg cell marker gene forkhead transcription factor 3 in the Xuling Jiangu Formula group were significantly higher than those in the model group (P < 0.05), whereas the gene and protein expression of the Th17 cell marker gene retinoic acidrelated orphan receptor γT significantly decreased (P < 0.05). The protein expression of receptor activator of nuclear factorκB ligand significantly decreased (P < 0.05), while its gene expression showed a decreasing trend without significant difference. (3) Flow cytometry results showed that the number of Treg cells in the Xuling Jiangu Formula group significantly increased (P < 0.05), while the number of Th17 cells decreased but without statistical significance. These findings suggest that the immune mechanism of Xuling Jiangu Formula in treating osteoporosis involves increasing the number of Treg cells to influence the Treg/Th17 balance, subsequently reducing the secretion of interleukin17 and interleukin6, promoting upregulation of forkhead transcription factor 3 and downregulation of retinoic acidrelated orphan receptor γT, thereby altering the osteoprotegerin/receptor activator of nuclear factor κB ligand ratio and further inhibiting osteoclast differentiation.

Key words: Xuling Jiangu Formula, osteoporosis, Treg/Th17 balance, bone immunity, immunomodulation