Postmenopausal osteoporosis (PMOP) is a systemic metabolic bone disorder characterized by a reduction in bone mass per unit volume, deterioration of bone microstructure, decreased bone mineral density (BMD), and diminished bone strength. These pathological changes lead to increased bone fragility and a higher risk of fractures, primarily due to estrogen deficiency following menopause. PMOP is associated with accelerated bone turnover, in which bone resorption exceeds bone formation, resulting in rapid bone loss (Wang C. et al., 2024). This condition predominantly affects trabecular bone, rendering the vertebrae and long bones particularly susceptible to fractures. The pathogenesis of osteoporosis is multifactorial, involving factors such as aging, endocrine disturbances, and calcium homeostasis disruption, as well as influences from immune function, nutrition, and genetic factors (Wang Y. et al., 2024). In 2021, a total of 219,552 postmenopausal women died due to low BMD, which includes osteopenia and osteoporosis. Over a 31-year period, the number of such deaths has doubled, with an average annual increase of approximately 2.3% (Liang et al., 2025). Although bone loss initiates as early as 2–3 years before menopause, it peaks between 1 year before and 2 years after the final menstrual period (Lizneva et al., 2018). The mortality rate caused by PMOP is 15.17 times higher than that in premenopausal women, underscoring the postmenopausal period as a critical window of risk for metabolic bone diseases (Liang et al., 2025). As China transitions into an aging society, the incidence of osteoporosis has been steadily rising, with a prevalence of 37.7% among individuals aged 60 and older. This condition disproportionately affects women, emerging as a significant public health concern, ranking just after cardiovascular diseases, cerebrovascular diseases, hypertension, and diabetes (Quah et al., 2022; Xiao et al., 2022). By 2045, global deaths and disability attributable to PMOP among elderly women are projected to increase further, highlighting a growing worldwide demand for improved bone health management (Liang et al., 2025).

Minerals are essential for maintaining human health and play pivotal roles in numerous physiological processes. Among these, calcium is the most abundant mineral in the human body, second only to oxygen, carbon, hydrogen, and nitrogen. An adult human body typically contains between 1,200 and 2,400 g of calcium, accounting for 1.5%–2.0% of total body weight (Gordan and Vaughan, 1986). Calcium is involved in nearly every vital process, including the maintenance of bone health, cellular metabolism, enzyme regulation, muscle contraction, heart function, nerve transmission, and blood coagulation (Bass and Chan, 2006). Its primary function is to form and support the structure of bones and teeth, with bone tissue storing 99% of the body’s calcium in the form of hydroxyapatite (Deluque et al., 2025). In addition to its structural roles, adequate calcium intake is crucial for maintaining bone health, preventing osteoporosis and dental caries, and reducing bone loss (Marino et al., 2023). The kidneys, small intestine, and bones play pivotal roles in maintaining the body’s calcium homeostasis. Under normal conditions, these organs work in concert to regulate blood calcium levels, ensuring dynamic equilibrium (Papadopoulou et al., 2021). Dietary calcium serves as the primary source of calcium for the body, making the absorption of calcium in the small intestine crucial for maintaining calcium balance (Lieben et al., 2012). The small intestine, located in the abdomen, connects to the large intestine at its distal end, facilitating further digestion and absorption of food. The proximal end of the small intestine, the duodenum, is connected to the stomach through the pylorus. Additionally, the intestinal mucosal epithelium is the primary site of calcium (Ca²⁺) absorption, a critical mineral for bone health (Larussa et al., 2017). Intestinal dysfunction, which may arise from intense external stimuli, impairs the ability of the stomach and intestines to absorb calcium effectively. This results in impaired calcium synthesis in the blood and a diminished capacity to convert and absorb calcium from dietary sources, which can disrupt bone metabolism. Research indicates that individuals with Crohn’s disease (CD) and osteoporosis are more likely to experience chronic gastrointestinal disorders, highlighting the close relationship between the onset of osteoporosis and gastrointestinal dysfunction (Zhong et al., 2022). A 4-year, randomized, double-blind, placebo-controlled study involving 930 healthy older participants with an average age of 61 demonstrated that calcium supplementation is an effective strategy for preventing osteoporosis and fractures in individuals with senile osteoporosis (Bischoff-Ferrari et al., 2008). Consequently, preventing osteoporosis relies heavily on either enhancing the body’s calcium absorption capacity or ensuring adequate calcium intake. Research has shown that colostrum basic protein (CBP) modulates intestinal calcium transporters and calcium homeostasis, enhances bone calcium deposition, and supports bone growth via a tripartite regulatory mechanism encompassing “ileum-dominated paracellular calcium absorption, jejunal transcellular transport, and hormonal regulation of calcium homeostasis.” These findings designate CBP as a novel natural protein target for the prevention and therapy of osteoporosis (Zhang et al., 2024). Additionally, studies have investigated the synergistic application of probiotics and isoflavones in the treatment of PMOP. This method has demonstrated the ability to augment calcium absorption by increasing the expression of intestinal calcium transporters—TRPV6, CABP, and PMCA1b—thus mitigating bone loss and enhancing systemic calcium equilibrium and skeletal health (Harahap et al., 2024).

Currently, the pharmacological management of PMOP includes various agents, such as essential mineral supplements, bone resorption inhibitors, bone formation stimulators, and drugs with novel mechanisms of action. Nonetheless, each therapeutic option presents specific limitations (An et al., 2020). For instance, bisphosphonates, which inhibit osteoclast-mediated bone resorption, are currently first-line therapy for osteoporosis. However, both bisphosphonates and denosumab have been associated with adverse effects such as osteonecrosis of the jaw and atypical femoral fractures (Gehrke et al., 2023). Although cathepsin K inhibitors can reduce the risk of fractures, they are linked to an increased risk of cardiovascular events. Moreover, studies have indicated that oral calcitonin does not significantly reduce fracture incidence. While traditional hormone replacement therapy is effective in alleviating menopausal bone loss, its clinical use is limited by side effects, prompting increased interest in more natural treatment alternatives with improved safety profiles (Chen et al., 2021; Shen et al., 2022). Traditional Chinese Medicine (TCM) has shown considerable potential in the treatment of PMOP, with both compound formulations and single herbal extracts exhibiting scientifically validated efficacy (Wang L. et al., 2024). Despite numerous studies have reported on the use of Chinese patent medicines for PMOP, and several TCM formulas currently employed in clinical practice, most previous investigations have primarily focused on clinical outcomes rather than systematically elucidating the underlying molecular mechanisms or multi-organ crosstalk involved in bone metabolism (Liu et al., 2025). WSP is a classic Chinese patent medicine with a history dating back to the Tang Dynasty. It is currently approved for sale in the Chinese market and is manufactured by Hangzhou Hu Qing Yu Tang Pharmaceutical Co., Ltd. Composed of 12 botanical drugs, this preparation can alleviate renal tissue damage in aged mice, improve renal function, and effectively delay reproductive aging (Xiaohu et al., 2024).

Pharmacological research demonstrates that the principal chemical components of Rehmannia glutinosa (Gaertn.) Libosch. ex DC. exhibit notable therapeutic effects, such as osteoporosis prevention, anti-aging properties, anti-thrombotic activity, support for recovery from bone marrow suppression, and immune function modulation (Lim and Kim, 2013; Zhou et al., 2015). Dioscorea oppositifolia L., a prominent medicinal and culinary plant, is essential for tonifying the spleen, nourishing the stomach, and enhancing fluid production, notably concerning pulmonary health. The immunomodulatory impact of Dioscorea oppositifolia L. on the intestinal tract is chiefly evidenced by its capacity to restore the intestinal mucosa. This repair improves the mucosal protective function of the intestinal wall, consequently modulating immunological responses and allowing the intestine to efficiently execute its immune activities (Guo et al., 2024; Xue et al., 2019). Cuscuta australis var. australis exhibits anticancer, antioxidant, and endocrine-immune regulating characteristics (Moon et al., 2019). The extract of C. australis var. australis, as indicated by Liu et al. (2020), possesses therapeutic advantages in osteoporosis and can promote osteoblast proliferation. A possible mechanism for these effects includes suppressing osteoclast differentiation and alleviating estrogen deficiency, which contributes to bone loss; this pathway is also linked to the anti-osteoporotic properties of C. australis var. australis and its bioactive constituents (Wang et al., 2023). We anticipate that the synergistic effects of these botanical medications may effectively alleviate the symptoms of osteoporosis, based on their pharmacological qualities.

TCMs that tonify the kidney play a crucial role in promoting bone formation, inhibiting bone resorption, stimulating sex hormone secretion, suppressing oxidative stress, and regulating calcium-phosphorus metabolism. According to the TCM theory of “the kidney governing bone,” kidney-tonifying herbs are often employed in the treatment of PMOP. Additionally, TCM etiology identifies “deficiency of both the spleen and kidney” as a key pathological mechanism underlying osteoporosis. As a well-known kidney-tonifying formula, WSP has been a focus of our prior research. In prior studies, we found that WSP had a significant restorative impact on intestinal function in animal models of “Folium senna-induced diarrhea” and “dietary disorders” combined with “overwork.” Building on these findings, the present study utilized 13-month-old female mice, which were fed a low-calcium, high-phosphorus diet to better replicate the clinical features of menopausal osteoporosis. We systematically evaluated the physical manifestations of the disease, changes in bone mass and mineral content, and the expression of calcium absorption mechanisms in the kidneys, intestines, and bones. This comprehensive approach was used to investigate the effects and underlying mechanisms of WSP in the context of diet-induced PMOP mice. By examining these multiple aspects, we aimed to elucidate how WSP may modulate calcium metabolism and bone health, giving us valuable knowledge about its potential therapeutic role in the management of PMOP.

Osteoporosis is categorized into two primary classifications: primary and secondary. Primary osteoporosis is largely a degenerative condition linked to the aging process, including postmenopausal, senile, and idiopathic osteoporosis (which primarily affects adolescents). Secondary osteoporosis is generally instigated by pharmacological interventions or other underlying medical conditions (Zhou et al., 2025). Calcium is a vital mineral in the human body, playing a crucial role in maintaining bone density. A reduction in calcium levels can lead to decreased bone density, ultimately contributing to the development of osteoporosis (Shen et al., 2022; Topolska et al., 2020; Wang and Li, 2022). The calcium intake and subsequent absorption are primarily allocated to skeletal mineralization, whereas unabsorbed calcium is primarily eliminated through fecal and urinary excretion. Consequently, calcium homeostasis is principally governed by the skeletal system, small intestine, and kidneys. During menopause, the reduction in estrogen levels precipitates osteoporosis, which is distinguished by heightened bone resorption, resulting in the mobilization of calcium and phosphorus from the skeletal matrix into the systemic circulation. This disruption of calcium balance leads to disorders in calcium metabolism. The regulation of calcium metabolism in the body involves several key factors, including PTH, CT, vitamin D₃, and E₂. PTH is a single-chain polypeptide hormone synthesized and secreted by the parathyroid glands. It plays a crucial role in regulating extracellular calcium and phosphorus levels. Elevated levels of PTH enhance bone resorption and increase calcium loss from the bones (Rejnmark and Ejlsmark-Svensson, 2020), thereby disrupting calcium homeostasis. CT primarily functions to inhibit the absorption of calcium ions in the small intestine and reduce calcium and phosphorus reabsorption in the distal renal tubules. Additionally, 1,25-(OH)₂D₃, the active form of vitamin D, regulates calcium absorption in the intestine. In postmenopausal women and during aging, estrogen deficiency can lead to relative insufficiency or resistance to active vitamin D, specifically 1,25(OH)₂D₃, which subsequently downregulates the expression of intestinal TRPV6 and renal TRPV5. This process ultimately impairs calcium absorption efficiency and elevates calcium loss. When intestinal calcium absorption is inadequate to maintain serum calcium levels, PTH secretion is upregulated. PTH enhances renal calcium reabsorption but primarily stimulates osteoclast-mediated bone resorption, resulting in the mobilization of bone calcium to stabilize serum calcium and promote bone loss (Hao et al., 2024). Physiologically, elevated serum calcium stimulates thyroid C cells to release CT, which counteracts PTH. CT significantly inhibits bone resorption by reducing osteoclast activity and proliferation, while also promoting renal calcium excretion; these actions collectively facilitate calcium deposition in bone, reduce serum calcium, and preserve bone integrity. In the postmenopausal state, in addition to the aforementioned calcium absorption deficits, estrogen deficiency may reduce basal CT secretion and attenuate its effects, thereby depriving the body of a critical endogenous mechanism to counteract bone resorption. This was additionally supported by increased serum concentrations of calcium and phosphorus (Luo et al., 2023).

The small intestine is recognized as the primary site for calcium absorption, with regional variations in absorptive capacity. The duodenum exhibits the highest calcium absorption rate, followed by the jejunum and ileum. (Fleet, 2022). These segments display an inverse relationship between absorption rate and retention time, with the ileum having the longest retention period, succeeded by the jejunum and duodenum (Bronner, 2009). An analysis of absorption efficiency and transit time indicates that the ileum and jejunum are the major sites for calcium absorption, accounting for approximately 65% and 17% of total intestinal calcium uptake, respectively (Christakos, 2012). The intestinal mucosa, particularly in the vicinity of the ileum, facilitates the absorption of calcium ions (Ca²⁺) through calcium transport channels associated with epithelial tight junction complexes. The only identified Ca²⁺-selective channels within this family are TRPV6 and TRPV5, which are members of the transient receptor potential (TRP) families (Clapham, 2003; Wang et al., 2025). TRPV6, a six-transmembrane domain epithelial calcium channel predominantly expressed in intestinal villi, plays a critical role in facilitating active calcium absorption by mediating Ca²⁺ influx from the intestinal lumen into enterocytes (Lieben et al., 2010). Simultaneously, CABP, characterized by its strong calcium affinity, is localized within enterocytes and the brush border epithelial cells of the small intestine. Following intracellular Ca²⁺ influx, CABP facilitates ion sequestration and promotes transcellular transport from the apical membrane to the basolateral compartment (Li et al., 2014).

Moreover, TRPV5 is predominantly situated in the distal and connecting tubules of the kidney, where it is instrumental in renal calcium reabsorption (Zhang et al., 2023). The kidney’s distal tubules facilitate calcium reabsorption via TRPV5, responsible for 15% of total calcium recycling; TRPV5 knockdown leads to elevated urine calcium, reduced blood calcium, and impaired bone mineralization (Luo et al., 2023). Renal calcium reabsorption is a tightly controlled process involving two main pathways: paracellular passive transport across the renal epithelium and transcellular active transport. The transcellular pathway occurs in three steps: first, calcium enters the epithelial cells through the apical membrane via TRPV5 channels; then, calcium binds to CABP and is transported across the cytoplasm; finally, calcium exits the cell via plasma membrane calcium ATPases (PMCA1 and PMCA4) and sodium-calcium exchanger isoform 1 (NCX1) (van Goor et al., 2017). A study on PMOP mice revealed substantial renal injury, characterized by increased renal volume, glomerular enlargement, collagen fiber aggregation, inflammatory cell infiltration, and tubular dilation into cystic structures. Moreover, the expression of calcium channel proteins, including TRPV5 and CABP, was markedly diminished. Ultimately, VDR serves as a nuclear receptor for 1,25-(OH)₂D₃, which, upon activation, enhances the absorption of osteocalcin and subsequently diminishes its secretion into the bloodstream (Benn et al., 2008; Gasperini et al., 2023). 1,25-(OH)₂D₃ can create a hormone-receptor complex with VDR, which subsequently interacts with additional regulatory variables to modulate VDR expression (Yang et al., 2015).

Previous studies have identified that the botanical components of WSP regulate pathways involved in bone metabolism, supporting its traditional use in strengthening kidney and spleen function (Han et al., 2021; Tang et al., 2023). However, the specific pharmacological mechanisms underlying the efficacy of WSP, as an integrated botanical compound, in the treatment of PMOP remain unclear. Current research indicates that numerous TCM compound prescriptions exert significant ameliorative effects on PMOP, with distinct mechanisms of action. Nevertheless, substantial gaps still exist in the understanding of their roles in regulating calcium-phosphorus absorption and bone metabolism (Liu et al., 2023). Distinct from most herbal interventions that primarily target anti-inflammatory pathways or exhibit estrogen-mimetic effects, WSP has demonstrated preliminary evidence of modulating calcium reabsorption in the intestine, kidney, and bone. In this study, we sought to develop a PMOP model by screening female mice exhibiting estrous cycle irregularities and subsequently administering a calcium-deficient, phosphorus-enriched diet. Assessment of general signs in these mice revealed clinical presentations consistent with human PMOP. These results validate the model’s capacity to replicate the features of primary menopausal osteoporosis observed in the current human population. Although the PMOP model established by combining natural menopause with dietary intervention avoids the invasiveness of ovariectomy, several limitations remain. The extreme calcium-phosphorus imbalance in the diet may overactivate PTH; meanwhile, individual variability in the extent of ovarian decline among mice contributes to phenotypic inconsistency and reduced reproducibility. Nevertheless, this model can still serve as a valuable complement to the ovariectomy model. Additionally, using 6-8-week-old mice as the normal control group introduces a confounding age effect. This cross-age experimental design results in differences in baseline bone metabolism, endocrine status, and physiological function between groups, which is also an issue that should be considered in subsequent studies.

In conclusion, our data indicate that WSP mitigates osteoporotic alterations in a diet-induced murine model by augmenting calcium absorption via the gut-kidney-bone axis. These results offer initial mechanistic understanding of calcium regulation in PMOP, while validation in clinical trials is warranted.

In summary, this study reveals that WSP treatment mitigates osteoporotic manifestations in a diet-induced murine model of PMOP. The observed therapeutic effects are associated with augmented calcium absorption, substantiated by the upregulation of critical calcium transport proteins (TRPV5/6, CABP, and VDR) within the gut–kidney–bone axis. Concurrently, WSP improved bone mass, restored calcium–phosphorus homeostasis, ameliorated systemic metabolism, and reversed anemia in this model. These observations suggest that WSP facilitates structural repair in the ileum, kidney, and femur, thereby integrating multi-tissue regeneration with calcium regulatory pathways (Figure 8). Collectively, these preclinical findings offer mechanistic insights into the role of calcium absorption in PMOP pathophysiology and support further exploration of WSP as a potential therapeutic intervention. However, given that the current evidence is limited to a diet-induced mouse model, additional studies are warranted to validate its translational applicability in human PMOP.