B cells contribute to humoral immunity through antibody production and regulate bone metabolism. Studies have highlighted their osteoclastogenic potential, particularly under erythropoietin stimulation, where bone marrow B cells express receptor activator of nuclear factor kappa-Β ligand (RANKL), modulating bone metabolism via the RANKL/osteoprotegerin axis. High erythropoietin concentrations induce B cell differentiation into osteoclasts, resulting in bone loss (15). Additionally, estrogen downregulates hypoxia-inducible factor-1α expression by upregulating heat shock protein 70 production. In ovariectomized mice, elevated hypoxia-inducible factor-1α levels activate downstream pathways, upregulating RANKL gene expression in B cells and promoting osteoclastogenesis, thus accelerating PMOP progression (16).

T cells, originating from bone marrow lymphoid stem cells, mature in the thymus before circulating through systemic immune organs and tissues. Under normal conditions, T cells maintain bone homeostasis via multiple mechanisms. Estrogen presence facilitates T cell-derived CD40 ligand interactions with B cell surface CD40, enhancing osteoprotegerin mRNA expression and protecting bone (17). However, in PMOP, this protective mechanism is disrupted. Following ovariectomy, activated CD4+ and CD8+ T cells increase Dickkopf-1 production, inhibiting osteoblast Wnt signaling via paracrine effects (18). Simultaneously, these T cells secrete tumor necrosis factor-α (TNF-α) and RANKL, accelerating bone resorption (19). PMOP patients also exhibit elevated LIGHT expression in circulating monocytes and T cells, which enhances osteoclastogenesis by modulating TNF and RANKL expression (20).

Treg cells and Th17 cells, key subsets of T cells, exhibit differentiation plasticity but serve opposing functions. Treg cells suppress osteoclast differentiation by inhibiting IL-17 expression, whereas Th17 cells promote osteoclastogenesis via RANKL signaling (21). Maintaining the balance between these populations is critical for preserving normal bone mass (22). Emerging research demonstrates that gut microbiota significantly influences Treg and Th17 cell functions. For example, Bifidobacterium longum modulates Breg cell expression, establishing a Breg-Treg-Th17 axis that enhances Treg cell function and suppresses Th17 activity. This modulation reduces pro-inflammatory cytokines, such asInterleukin-6 (IL-6), Interleukin-17 (IL-17), and TNF-α, while increasing anti-inflammatory factors, including Interleukin-10 (IL-10) and IFN-γ, thereby protecting bone and alleviating PMOP symptoms (23).

Chronic inflammation-induced bone metabolic dysregulation is a fundamental pathological feature of PMOP, with the Th17/Treg cell balance and associated cytokine networks orchestrating bone remodeling. Treg cells inhibit bone resorption through two mechanisms: secretion of anti-resorptive factors such as IL-10 and transforming growth factor-β (TGF-β), and interaction of their surface cytotoxic T-lymphocyte-associated protein 4 with CD80/CD86 on osteoclast precursors, which activates indoleamine-2,3-dioxygenase. This activation triggers tryptophan catabolism, inducing precursor cell apoptosis (24). Conversely, Th17 cells enhance osteoclastogenesis by expressing RANKL and secreting IL-17, which stimulates macrophages to produce pro-inflammatory mediators, including TNF-α and IL-6, further upregulating RANKL expression in osteoclast-supporting cells (22).

Estrogen deficiency disrupts the Th17/Treg balance. In estrogen-depleted conditions, heightened Th17 cell activity increases IL-17 production, promoting bone marrow mesenchymal stem cell proliferation and osteogenic differentiation while inducing macrophage colony-stimulating factor and RANKL secretion, thereby accelerating osteoclastogenesis (22, 25). Clinical studies report significantly elevated IL-17 levels in postmenopausal osteoporotic vertebral compression fracture patients (26). Additionally, Interleukin-23 exacerbates bone loss through two mechanisms: enhancing Th17 cell activity and inducing T cell RANKL expression. Targeting these pathways offers therapeutic potential, as anti-IL-17 antibodies promote bone regeneration via forkhead box o1 and activating transcription factor 4 activation (27), while anti-Interleukin-23 antibodies prevent estrogen deficiency-induced bone loss (28). Recent studies show that B10 cell adoptive transfer reduces Th17 cell populations and inhibits alveolar bone osteoporosis in ovariectomized mice (29). At the molecular level, estrogen stimulates Treg cells to produce IL-10 and TGF-β1, suppressing osteoclast differentiation and bone resorption. TGF-β, a key bone repair regulator, balances osteoblast differentiation and osteoclast formation (30). Molecular studies reveal that TGF-β1 enhances osteoblast survival, differentiation, and migration by activating the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin/ribosomal protein S6 kinase beta-1 signaling pathway (31). These findings suggest that targeting Treg cell activity may provide effective therapeutic strategies for bone protection in inflammatory conditions.

In patients with PMOP, an increased abundance of clostridium species has been observed, with its relative abundance negatively correlated with bone mineral density (BMD) (57). Research suggests that clostridium activates the protein kinase B beta signaling pathway, promoting M1 macrophage production, thereby triggering inflammation and accelerating osteoporosis progression (58, 59). In a randomized trial involving postmenopausal Japanese women, participants treated with the probiotic Bacillus subtilis showed a significant increase in total hip BMD compared to the placebo group (60). After 12 and 24 weeks of Bacillus subtilis treatment, the abundance of clostridium species significantly decreased, potentially improving BMD by reducing pro-resorptive cytokines (60). Additionally, lactobacillus reuteri (american type culture collection PTA-6475) has been shown to reduce bone loss in elderly women with low BMD (61).

Lactobacillus acidophilus mitigates bone loss and enhances bone heterogeneity in osteoporotic mice by modulating the Treg-Th17 cell balance (62). Similarly, lactobacillus rhamnosus reduces bone loss and preserves bone health in ovariectomized mice (63). Probiotics such as Bifidobacterium and clostridium species promote Treg cell differentiation, thereby inhibiting excessive bone resorption. Intervention with Bifidobacterium longum significantly increases Breg cell proportions and the levels of IL-10 and interferon-gamma while decreasing the production of TNF-α, IL-6, and IL-17. Clostridium-stimulated Bregs also significantly enhance Treg cell proportions and IL-10 expression while reducing Th17 cells and IL-17 levels, indicating a potent regulatory role in Th17/Treg differentiation. Pathogenic bacteria such as clostridium and Ruminococcus are associated with Th17 cell activation, exacerbating osteoclastogenesis and bone resorption (64). Toxins produced by Bacteroides fragilis activate intestinal Th17 cell recruitment via the JAK-STAT3 pathway (65–67). Thus, alterations in gut microbiota composition disrupt the Th17/Treg balance, affecting bone metabolism and contributing to osteoporosis progression (68).

Gut microbiota-derived metabolites play a pivotal role in regulating immune cell development and function, including both adaptive and innate immune responses. IL-10, produced by effector T cells, acts as a key self-regulatory mechanism for maintaining immune homeostasis (68). Short-chain fatty acids (SCFAs) enhance IL-10 production in differentiated Th1 cells via a G-protein-coupled receptor 43-dependent pathway and induce IL-10 during Th1 and Th17 cell differentiation by inhibiting histone deacetylase activity (69). SCFAs and other metabolites directly influence osteoblast and osteoclast differentiation and activation, with butyrate playing a crucial role in osteoclast metabolism regulation (33). Butyrate-treated dendritic cells induce the expression of immunosuppressive enzymes, such as indoleamine 2,3-dioxygenase 1 and aldehyde dehydrogenase 1 family member A2, in a solute carrier family 5 member 8-dependent manner. This promotes Treg differentiation while inhibiting Th1 differentiation (70). Microbial tryptophan metabolites, such as indole and its derivatives, bind to aryl hydrocarbon receptors, influencing B cell development (71), differentiation (72), and cytokine regulation (73) via aryl hydrocarbon receptor signaling. LPS, components of Gram-negative bacteria, trigger cytokine cascades driving T cell-mediated inflammation (74). LPS also inhibits osteoblast maturation and activates osteoclasts, enhancing bone resorption and exacerbating PMOP progression. Additionally, bile acids and their metabolites regulate host immune responses by modulating the Th17/Treg cell balance (75). These metabolites provide mechanistic insights into the interplay between gut homeostasis and immune regulation.

Disruption of gut barrier function can lead to leakage of intestinal contents, creating a pro-inflammatory environment (76). In mouse models, elevated zonulin levels degrade essential tight junction proteins and increase the expression of claudin-2 and claudin-15, compromising tight junction integrity and severely impairing gut barrier function Increased intestinal permeability triggers T cell-mediated mucosal inflammation and facilitates the migration of autoreactive T cells, such as Th1 and Th17 cells, from the gut to other sites like joints, potentially contributing to PMOP onset (77). Estrogen plays a key role in maintaining intestinal epithelial barrier function, and its deficiency increases gut permeability (78). This enhanced permeability triggers inflammatory responses that promote osteoclast formation, ultimately leading to bone loss (79, 80). Sufficient estrogen levels activate Tregs, inhibit osteoclastogenesis, and prevent osteoblast destruction, contributing to the maintenance of bone mass (79, 81, 82). Tight junctions between intestinal epithelial cells are essential for preserving gut barrier integrity. Lactobacillus rhamnosus supports epithelial integrity through carbohydrate transport and metabolism (83). Specifically, lactobacillus rhamnosus GG regulates both the gut microbiota and gut barrier, modulating the Th17/Treg balance and alleviating osteoporosis induced by estrogen deficiency.

Probiotics have demonstrated potential to enhance the growth and metabolic activity of beneficial bacteria, offering significant benefits for bone health (16). They produce metabolites and genetic products that directly interact with epithelial and immune cells, improving gut function, reducing inflammation, lowering intestinal pH to promote calcium absorption, and preventing the colonization of harmful bacteria. Collectively, these effects support osteoblast activity and contribute to the maintenance of bone health (84, 85). Probiotic supplementation has been shown to increase bone density and promote fracture healing. Animal studies have further demonstrated that probiotics enhance bone mass by inhibiting CD4+ T cell proliferation in the bone marrow and reducing the expression of pro-inflammatory cytokines such as TNF-α (11, 86).

Additionally, the gut microbiota regulates immune balance and microbial stability through tryptophan and its metabolites, including indole and serotonin (87). Exogenous supplementation of tryptophan metabolites, such as indole acetic acid and indole-3-propionic acid, effectively restores intestinal barrier integrity in ovariectomy -induced PMOP mouse models and alleviates osteoporosis. This process critically depends on the activation of the aryl hydrocarbon receptor. Mechanistically, tryptophan metabolites, particularly indole acetic acid, activate intestinal AhR, which, in turn, stimulates the Wnt/β-catenin signaling pathway to restore intestinal barrier function. indole acetic acid and indole-3-propionic acid supplementation also enhances M2 macrophage secretion of IL-10, which diffuses from the intestinal lamina propria to the bone marrow, promoting osteogenesis while suppressing osteoclast formation. Notably, the therapeutic effects of tryptophan metabolites on intestinal homeostasis and osteoporosis symptoms are significantly reduced in ovariectomy mice lacking intestinal AhR. These findings highlight gut microbial tryptophan metabolites as promising therapeutic candidates for osteoporosis by modulating the AhR-mediated gut-bone axis.

By modulating the immune system, the gut microbiota influences bone metabolism, potentially affecting bone density and turnover. This highlights the gut microbiota as a novel therapeutic target for osteoporosis treatment and fracture prevention (5). For example, Lactobacillus casei modulates gut microbiota composition, reduces pro-inflammatory cytokines such as IL-17, interleukin-1β, IL-6, and TNF-α, and adjusts the Th1/Th17 ratio, thereby inhibiting the onset and progression of PMOP (88). Another byproduct of colonic microbial fermentation, hydrogen gas, is produced in significant amounts by certain strains of clostridium and may have potential effects on gut and bone health (89–91).

Fecal microbiota transplantation (FMT) has been shown to reshape gut microbiota and mitigate bone loss in ovariectomy-induced osteoporotic mice. The mechanisms underlying these effects include correcting gut microbiota dysbiosis, elevating SCFAs levels, improving gut permeability, and inhibiting the release of pro-osteoclastogenic factors, collectively suppressing excessive osteoclast formation (92). The circulatory system serves as a bridge connecting osteoclastogenic factors, transgenic cells, SCFAs, and the skeletal system. FMT effectively prevents ovariectomy-induced bone loss by limiting osteoclast overactivity (93). Compared to ovariectomized controls, FMT-treated mice exhibited increased expression of tight junction proteins such as occludin and reduced secretion of pro-osteoclastogenic factors, including TNF-α and interleukin-1β (94). Furthermore, FMT optimized gut microbiota composition and abundance, while increasing fecal SCFAs levels, particularly acetate and propionate (95). Consequently, FMT represents a promising alternative therapy and a potential strategy for preventing and treating PMOP in the future.

The integrity of the gut barrier is essential for the prevention and treatment of PMOP. An intact mucosal barrier prevents harmful substances, such as bacterial LPS, from entering systemic circulation (94). This, in turn, suppresses activation of the toll-like receptor 4 receptor signaling pathway in macrophages, thereby reducing the release of pro-inflammatory cytokines like TNF-α and inhibiting osteoclast differentiation and activation, which would otherwise accelerate bone resorption (96, 97). Additionally, a healthy gut microbiota produces beneficial metabolites, including SCFAs, that regulate bone metabolism. SCFAs inhibit osteoclast activity by downregulating key molecules such as TNF receptor-associated factor 6 and nuclear factor of activated T-cells 1, while simultaneously promoting bone formation by upregulating osteoblast differentiation-related genes (61, 98). Butyrate, a major SCFAs, promotes the differentiation of bone-protective Tregs, which, in turn, induce CD8+ T cells to release Wnt10b, activating the Wnt signaling pathway in osteoblasts (99, 100). Lucas et al. demonstrated that SCFAs supplementation or a high-fiber diet can prevent menopause and inflammation-induced bone loss, significantly increasing bone mass (70). Vegetarians and individuals adhering to a mediterranean diet tend to have higher SCFA levels, which are associated with improved bone health (101). Dietary supplementation with oligosaccharides enhances SCFA production, contributing to increased BMD (102). In antibiotic-treated mice, SCFA supplementation reduced bone loss without affecting bone turnover rates (103). Thus, maintaining gut barrier integrity and regulating gut microbial metabolites are critical for inhibiting bone resorption and promoting bone formation effectively.