Curcumin Ameliorates Osteoporosis via Gut Microbiota-Dependent Modulation of TMAO in Ovariectomized Rats

Curcumin Ameliorates Osteoporosis via Gut Microbiota-Dependent Modulation of TMAO in Ovariectomized Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Curcumin Ameliorates Osteoporosis via Gut Microbiota-Dependent Modulation of TMAO in Ovariectomized Rats Xin Hu, Yuchen Tang, Qiufu Wang, Guanyin Jiang, Miao Lei, Wen Dong, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9218781/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Despite its poor oral bioavailability, curcumin demonstrates efficacy against postmenopausal osteoporosis, presenting a paradox between its pharmacokinetics and pharmacological effects. This study elucidates a gut microbiota-dependent mechanism underlying this phenomenon. In ovariectomized rats, curcumin improved bone mass and microstructure, effects that were abolished upon gut microbiota depletion but partially transferable via fecal microbiota transplantation. Mechanistically, curcumin reversed estrogen deficiency-induced gut dysbiosis, enhanced microbial diversity, and significantly reduced levels of the gut-derived metabolite trimethylamine-N-oxide (TMAO) and the pro-inflammatory cytokine IL-1. Our findings establish that curcumin exerts its osteoprotective effects indirectly by remodeling the gut microbiome and its metabolic output, thereby proposing a new paradigm for its therapeutic action—not as a systemically available drug, but as a targeted gut microbiota modulator. Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Gastroenterology Health sciences/Medical research Biological sciences/Microbiology Curcumin Postmenopausal osteoporosis Gut microbiota TMAO Ovariectomized rat Inflammation Bone mineral density Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Osteoporosis is a prevalent metabolic bone disorder characterized by reduced bone mineral density and impaired bone strength, leading to an elevated risk of fractures. Postmenopausal osteoporosis (PMOP) represents one of the most common forms of the disease, primarily resulting from an imbalance in bone metabolism due to decreased estrogen levels following menopause. This hormonal decline tilts the balance toward increased bone resorption and reduced bone formation (1–3). The pathogenesis of PMOP is multifactorial, involving numerous biological pathways and molecular mechanisms. Estrogen deficiency is widely recognized as a key contributor to postmenopausal osteoporosis. It promotes the overexpression of pro-inflammatory cytokines and enhances osteoclast activity through activation of the nuclear factor kappa B (NF-κB) signaling pathway, thereby accelerating bone loss (2, 4) .Moreover, emerging evidence indicates that gut microbiota dysbiosis plays a critical role in the development of PMOP. Alterations in the gut microbial community can exacerbate bone loss by modulating systemic inflammation and bone metabolic processes(5).Consequently, the pathogenic model of PMOP has evolved from a primarily estrogen-centric perspective to a more comprehensive framework that incorporates the influence of gut microbiota. A study involving 106 postmenopausal women—classified as having osteopenia (n = 33), osteoporosis (n = 42), or normal bone mineral density (n = 31)—revealed significant alterations in fecal microbiota composition. Specifically, patients with osteoporosis exhibited reduced microbial diversity and abundance, including decreased levels of Bacteroides, along with a marked increase in Proteobacteria, such as the pro-inflammatory genera Klebsiella and Escherichia coli (6). In the postmenopausal state, estrogen deficiency may induce gut dysbiosis, which in turn triggers abnormal immune activation. This includes an expansion of T helper 17 (Th17) cells, release of pro-inflammatory cytokines, and activation of the RANKL signaling pathway, collectively promoting osteoclast differentiation and ultimately leading to bone loss and increased fracture risk (7). Current pharmacological management of PMOP includes traditional agents such as bisphosphonates and selective estrogen receptor modulators (SERMs). While these therapies are widely used, their long-term application is often limited by associated adverse effects. Trimethylamine N-oxide (TMAO) is an organic compound primarily derived from dietary precursors through microbial metabolism in the gut. Trimethylamine (TMA), generated by gut microbiota from substrates such as choline, carnitine, betaine, and creatinine, is absorbed and oxidized in the liver by flavin-containing monooxygenases (FMOs) to form TMAO, which subsequently enters the systemic circulation. Additionally, certain foods—particularly marine fish and shellfish—contain high levels of preformed TMAO and can contribute directly to its plasma concentrations via intestinal absorption (8). The production of TMA is facilitated by various gut bacteria, including species from the genera Clostridium, Proteus, Shigella, and Enterobacter (9). Both dietary intake and the composition of the gut microbiota significantly influence plasma TMAO levels. Currently, TMAO is widely regarded not merely as a metabolic by-product of choline metabolism, but as a molecule with substantial pathophysiological implications. Elevated circulating TMAO has been closely linked to an increased risk of cardiovascular diseases and osteoporosis, and it is also known to mediate inflammatory responses within the body (10–12). Estrogen deficiency-induced osteoporosis is recognized as a chronic inflammatory condition, frequently accompanied by elevated levels of reactive oxygen species (ROS) and pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α(13). Given that TMAO contributes to systemic inflammation and may exacerbate oxidative stress, it is plausible that TMAO plays a role in amplifying bone loss under estrogen-deficient conditions. Therefore, we hypothesize that modulating gut microbiota dysbiosis resulting from estrogen deficiency and reducing plasma TMAO levels may indirectly alleviate osteoporosis symptoms by attenuating inflammation and oxidative damage.However, it should be noted that the causal relationship between TMAO and bone metabolism remains incompletely elucidated. Most existing evidence is derived from observational or correlative studies, and further mechanistic investigations are necessary to validate whether TMAO directly influences osteoclastogenesis or bone remodeling. Curcumin, the principal bioactive compound derived from the rhizomes of Curcuma longa (a member of the Zingiberaceae family), is a lipophilic polyphenol with the chemical structure of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(14). It exhibits a wide range of pharmacological properties, including anti-inflammatory, antioxidant, anti-osteoarthritic, anticancer, neuroprotective, and cardioprotective effects(14, 15). In vitro studies indicate that curcumin stimulates osteoblast proliferation, upregulates bone formation-related gene expression, and suppresses osteoclastogenesis, thereby contributing to the maintenance of bone health(16). Furthermore, numerous animal studies have demonstrated the potential of curcumin to ameliorate osteoporosis(17, 18). The diverse biological activities of curcumin are largely attributed to its anti-inflammatory and antioxidant mechanisms. However, the clinical translation of curcumin is hampered by its poor aqueous solubility, low oral bioavailability, and suboptimal pharmacokinetic profile(14). These limitations result in minimal intestinal absorption and low systemic exposure, which contrast sharply with its broad pharmacological efficacy in vitro and in preclinical models. To investigate whether curcumin alleviates osteoporosis through gut microecological modulation and anti-inflammatory mechanisms, we employed an ovariectomized (OVX) rat model of postmenopausal osteoporosis. We hypothesized that curcumin improves bone mass by restoring gut microbiota homeostasis, reducing systemic inflammation, and modulating metabolite production. Femoral bone mineral density (BMD) measurements, combined with integrated analyses of the gut microbiome and metabolome, revealed that curcumin treatment significantly increased BMD in OVX rats. Additionally, curcumin enhanced microbial richness and diversity, ameliorated ovariectomy-induced dysbiosis, and reduced circulating levels of the gut-derived metabolite trimethylamine N-oxide (TMAO) and key pro-inflammatory cytokines. Collectively, these findings suggest that curcumin attenuates osteoporosis in OVX rats by modulating the gut microbiota and its metabolic output, particularly TMAO, and by dampening systemic inflammation. This study underscores the importance of the gut-bone axis as a potential therapeutic target for osteoporosis treatment. 2. Materials and Methods 2.1. Animals and Treatments All experimental Sprague-Dawley (SD) rats were obtained from the Animal Experiment Center of Chongqing Medical University. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University and were in compliance with relevant ethical guidelines. First Animal Experiment:A total of 25 eight-week-old female SD rats (approximately 200 g body weight) were acclimatized for 7 days before undergoing bilateral ovariectomy (OVX). The rats were then randomly assigned to five experimental groups (n = 5 per group):Sham surgery group,OVX group (bilateral oophorectomy only),Curcumin group (200 mg/kg/day by oral gavage after bilateral oophorectomy),Antibiotic-treated (ABX) group (pseudo-germ-free model),ABX + Curcumin group (antibiotics plus curcumin).Bilateral oophorectomy was performed in all groups except the sham surgery group. Curcumin was administered daily via oral gavage at approximately 10:00 AM for 12 weeks. To establish a pseudo-germ-free state, rats in the ABX and ABX + Curcumin groups received a quadruple antibiotic cocktail by gavage for 7 days prior to OVX, consisting of vancomycin (100mg/kg), neomycin sulfate (200mg/kg), metronidazole (200mg/kg), and ampicillin (200mg/kg) (19, 20). Throughout the subsequent 12-week experimental period, these animals received the same antibiotics dissolved in drinking water at the following concentrations: vancomycin (500mg/L), metronidazole (1g/L), ampicillin (1g/L), and neomycin sulfate (1g/L). Second Animal Experiment:An additional 6 six-week-old female SD rats (approximately 200 g body weight) were acclimatized and underwent OVX surgery. After antibiotic-induced microbiota depletion (as described above), they were divided into two groups:FMT(OVX) group: received fecal microbiota transplantation (FMT) from donor OVX rats,FMT(Cur) group: received FMT from donor curcumin-treated rats. Fecal suspensions were prepared daily from fresh feces collected from donor rats (OVX or Cur group of the first experiment), flash-frozen on dry ice, and thawed immediately before use. Feces were diluted 1:10 (w/v) in sterile saline, vortexed for 2 minutes, and centrifuged at 500 × g for 3 min. The supernatant was collected and administered via gavage within 20 minutes of preparation to minimize microbial compositional changes (19). Following the 12-week treatment period, all rats were euthanized via intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight) after being anesthetized with isoflurane (5% induction, 2–3% maintenance in 100% oxygen) to ensure deep anesthesia prior to euthanasia. Euthanasia was confirmed by the absence of pedal and corneal reflexes, followed by bilateral thoracotomy. Subsequently, femurs, fecal samples, organs (including liver, kidneys, and spleen), and blood were collected for subsequent analyses. Blood was obtained via cardiac puncture using a 21-gauge needle and collected into EDTA-coated tubes for hematological analysis, as well as into serum separator tubes for biochemical assays. All instruments used for dissection were sterilized with 70% ethanol and autoclaved prior to use. Tissue samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until further processing. 2.2. Micro-Computed Tomography (Micro-CT) Analysis Following euthanasia, femurs were harvested and fixed for micro-CT scanning. Scans were performed using a [NMC-200] with consistent scanning parameters across all samples. The acquired images were reconstructed using three-dimensional reconstruction software [Cruiser,Recon]. A standardized region of interest (ROI) was selected for all samples in the distal femoral metaphysis, located [e.g., 0.5–1.0 mm proximal to the growth plate]. Trabecular bone parameters, including bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), were analyzed using dedicated data analysis software [Avatar]. Data were exported for statistical evaluation to assess differences in trabecular bone microstructure, mineralization, and inferred bone strength. 2.3. Histological Analysis Major organs (heart, liver, spleen, lungs, and kidneys) were collected, fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) to evaluate potential toxicological effects of curcumin and/or antibiotic treatments. For bone histomorphometry, femurs were decalcified in 10% EDTA (pH 7.4) for 4 weeks. Subsequently, decalcified bones were processed, embedded in paraffin, and sectioned. Sections were stained with H&E and Masson’s trichrome to evaluate osteoblast activity, bone formation, turnover, and mineralization status. 2.4. Serum Enzyme-Linked Immunosorbent Assay (ELISA) Blood samples were centrifuged at [e.g., 3000 × g for 15 min at 4°C] to obtain serum. The concentrations of pro-inflammatory cytokines, including IL-1, IL-6, and G-CSF, were quantified using specific commercial ELISA kits according to the manufacturers' protocols. Absorbance was measured using a microplate reader, and cytokine concentrations were calculated from standard curves. 2.5. 16S rRNA Gene Sequencing Total genomic DNA was extracted from frozen fecal samples according to the manufacturer's instructions. The hypervariable region of the bacterial 16S rRNA gene were amplified using specific primers . PCR products were purified, quantified, and pooled in equimolar ratios to construct sequencing libraries. Then the library was constructed. The constructed library was quantified by Qubit and qPCR.Bioinformatic analysis was performed using QIIME2 or a similar pipeline to assess microbial community diversity (alpha and beta diversity), composition, and relative taxonomic abundance. 2.6. Quantitative Metabolomic Analysis of Fecal TMAO The concentration of trimethylamine N-oxide (TMAO) in fecal samples was quantified using targeted liquid chromatography-mass spectrometry (LC-MS). Briefly, samples were homogenized in liquid nitrogen, and a precise weight (e.g., 20 mg) was diluted with mass spectrometry-grade water. After vortexing, 50 µL of the diluted (d9-TMAO). The mixture was vortexed, incubated on ice for 30 minutes, and centrifuged (12,000 rpm, 10 min, 4°C). The supernatant was collected for LC-MS analysis. Quantification was achieved by comparing the peak area ratio of TMAO to the internal standard against a standard calibration curve. 2.7. Correlation Analysis Between Metabolome and Microbiome To explore potential functional relationships between the gut microbiota and host metabolism, integrated correlation analyses were performed. Statistically significant differential abundant microbes (from 16S sequencing) and metabolite levels ( TMAO) were included. Spearman or Pearson correlation coefficients were calculated to identify significant associations between specific bacterial taxa and metabolite concentrations, aiming to infer microbiota-driven phenotypic changes. 2.9. Statistical Analysis Data are presented as mean ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test. For comparisons among multiple groups, one-way or two-way ANOVA followed by an appropriate post-hoc test ( Tukey's or Sidak's) was used. Correlation analyses were performed using Spearman's or Pearson's method. A p-value < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism version 9 . 3. Results 3.1. Curcumin Attenuates Bone Loss and Ameliorates Osteoporosis in Ovariectomized Rats The ovariectomized (OVX) rat model is a well-established and widely used animal model for studying postmenopausal osteoporosis. Successful model induction was confirmed 1–3 weeks after bilateral ovariectomy, characterized by significant bone loss in the proximal tibia, lumbar vertebrae, and femur, as evidenced by reduced bone mineral density (BMD), decreased trabecular number, and increased trabecular separation (21).Micro-CT analysis revealed that, compared with the sham-operated group, the ovariectomy (OVX) group exhibited significant reductions in bone volume fraction (BV/TV, P < 0.05), trabecular number (Tb.N, P < 0.05), trabecular thickness (Tb.Th, P < 0.05), and bone mineral density (BMD, P < 0.05). At the same time, compared with the OVX control group, the trabecular separation (Tb.Sp, P < 0.05) in the sham-operated group was also significantly reduced..Collectively, these results indicate that bilateral ovariectomy in rats successfully established an osteoporosis model.(Fig. 0 ). In the present study, micro-CT analysis of femoral samples from the first experiment revealed that curcumin treatment significantly alleviated OVX-induced bone loss. Compared to the OVX group (which received normal saline via gavage), the curcumin-treated group exhibited a notable improvement in multiple bone microarchitectural parameters. Specifically, curcumin administration significantly increased the bone volume fraction (BV/TV, P < 0.05), trabecular number (Tb.N, P < 0.05), trabecular thickness (Tb.Th, P < 0.05), and bone mineral density (BMD, P < 0.05). Additionally, a significant decrease in trabecular separation (Tb.Sp, P < 0.05) was observed in the curcumin group relative to the OVX controls (Fig. 1 ). Consistent with these findings, other indices—including bone volume, bone surface area to tissue volume ratio, bone surface area to bone volume ratio, and trabecular bone pattern factor—also indicated that oral administration of curcumin ameliorated osteoporotic changes in OVX rats. Furthermore, histopathological evaluation through H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) showed no evidence of adverse effects or structural abnormalities resulting from curcumin treatment, indicating its safety profile within the experimental context. 3.2. The Osteoprotective Effect of Curcumin in Ovariectomized Rats is Gut Microbiota-Dependent Estrogen deficiency following ovariectomy has been reported to induce significant alterations in the gut microbial community, which may contribute to bone loss through modulation of inflammation and bone metabolism(22). To investigate whether the anti-osteoporotic effect of curcumin depends on the gut microbiota, we established a pseudo-germ-free (PGF) rat model via broad-spectrum antibiotic treatment (vancomycin: 100 mg/kg; neomycin sulfate: 200 mg/kg; metronidazole: 200 mg/kg; ampicillin: 200 mg/kg) to deplete gut microbiota (20). Rats were divided into two groups: ABX group (antibiotic-treated OVX rats) and ABX + Cur group (antibiotic-treated OVX rats receiving curcumin). Micro-CT analysis of femoral microstructure revealed no significant differences in bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), or trabecular separation (Tb.Sp) between these two groups (Fig. 1 ). Notably, when the ABX + Cur group was compared with the curcumin-treated non-antibiotic group (Cur group) from the first experiment, significant reductions in BV/TV, Tb.Th, and BMD were observed in the microbiota-depleted animals (P < 0.05). These results indicate that the beneficial effects of curcumin on bone microarchitecture—including improvements in BV/TV, Tb.N, Tb.Th, and BMD—were abolished in the absence of a functional gut microbiota. Histological analysis of femoral sections stained with H&E and Masson's trichrome revealed pronounced microstructural improvements in the Cur group compared to the OVX, ABX, and ABX + Cur groups. Specifically, the Cur group exhibited increased trabecular bone density, greater trabecular thickness, and significantly reduced inter-trabecular spacing with a more compact architectural organization. Notably, these trabeculae maintained structural integrity without evidence of fragmentation, and demonstrated better preservation of bone matrix(Fig. 2 ).These findings indicate that curcumin treatment effectively restored bone microstructure in OVX rats. Conversely, microbiota-depleted rats (ABX and ABX + Cur groups) showed no significant structural improvements regardless of curcumin administration, confirming the essential role of gut microbiota in mediating curcumin's osteoprotective effects. Thus, we conclude that the amelioration of OVX-induced osteoporosis by curcumin is gut microbiota-dependent. 3.3 Curcumin Alters the Intestinal Microbial Composition in OVX Rats Estrogen deficiency-induced osteoporosis has been previously associated with significant alterations in gut microbial composition(6, 7). To investigate whether curcumin modulates the gut microbiota in OVX rats, we performed 16S rRNA gene amplicon sequencing on fecal samples collected from the four experimental groups in the first animal trial.After processing and analyzing the sequencing data, we identified unique amplicon sequence variants (ASVs) across groups. The curcumin-treated (Cur) group exhibited 926 unique ASVs, substantially higher than the 448 observed in the OVX group. In contrast, both antibiotic-treated groups (ABX and ABX + Cur) showed a marked reduction in total ASVs, confirming successful establishment of a pseudo-germ-free state through antibiotic administration (Fig. 3 A).We further evaluated microbial composition at the phylum, family, and genus levels using relative abundance bar plots and clustered heatmaps. Comparative analysis revealed pronounced differences in the abundance of dominant bacterial taxa among the groups (Fig. 3 B–D). These results indicate that curcumin significantly restructures the gut microbial community in OVX rats. At the phylum level, significant alterations in microbial composition were observed between the Cur and OVX groups. The relative abundance of Verrucomicrobiota was significantly lower in the Cur group compared to the OVX group (p < 0.05; Fig. 3 E). Although not statistically significant, the Firmicutes population showed an increasing trend in the Cur group (54.37% vs. 39.07% in OVX, p = 0.1092; Fig. 3 F). In contrast, the abundance of Bacteroidota remained comparable between the two groups (40.20% vs. 37.13%). Notably, several other phyla, including Cyanobacteria, Patescibacteria, Campylobacterota, Spirochaetota, and Elusimicrobiota, were significantly enriched in the Cur group. At the family level, curcumin treatment led to a significant increase in the relative abundance of Prevotellaceae (p < 0.05), Oscillospiraceae (p < 0.05), and Spirochaetaceae (p < 0.05). Conversely, a marked reduction was observed in Muribaculaceae (p < 0.05), Akkermansiaceae (p < 0.05), and Lactobacillaceae (p = 0.086; Fig. 4 C–H). No significant differences were detected in other families such as Lachnospiraceae.Further analysis at the genus level revealed that curcumin supplementation significantly increased the abundance of UCG-005 (p = 0.0884) and Prevotellaceae_UCG-001 (p < 0.05). In contrast, the genera Akkermansia, Lactobacillus, and Lachnospira exhibited a significant decrease (p < 0.05; Fig. 5 A–B). To identify specific microbial taxa associated with curcumin-induced improvement in osteoporosis, we performed linear discriminant analysis effect size (LEfSe) analysis. The Cur group was characterized by a higher abundance of p_Firmicutes, followed by c_Clostridia, c_Bacteroidia, p_Bacteroidota, and o_Oscillospirales. In the OVX group, the most discriminative features were f_Muribaculaceae, f_Akkermansiaceae, p_Verrucomicrobiota,g_Akkermansia, and c_Verrucomicrobiae, all with LDA scores > 4 (Fig. 5 C–D). These results demonstrate that curcumin significantly restructures the gut microbiota across multiple taxonomic levels and suggest that the osteoprotective effects of curcumin may be mediated through specific changes in microbial community composition.Through lefse analysis, we can speculate that it is precisely because of the changes in the composition of these flora that the osteoporosis of OVX rats is improved. 3.4 Curcumin Alters the Intestinal Microbial Diversity in OVX Rats Previous studies have indicated that ovariectomy induces significant dysbiosis of the gut microbiota, characterized by altered species richness and compositional shifts compared to sham-operated controls(23). To evaluate the effect of curcumin on the gut microbial community, we performed alpha diversity analysis on fecal samples from each group. Alpha diversity metrics provide insights into the complexity of microbial communities. The Chao1 and observed features indices estimate community richness. The Shannon and Simpson indices represent community diversity, incorporating both richness and evenness, while the Pielou-e index specifically measures species evenness. The Goods coverage index evaluates Community coverage, indicating the completeness of sampling.A rarefaction curve was generated for each alpha diversity metric (Fig. 6 A). The plateauing of these curves with increasing sequencing depth confirms that the obtained data sufficiently captured the microbial diversity within the samples. Notably, compared to the OVX group, the curcumin-treated (Cur) group exhibited significant increases in all measured alpha diversity indices, including Chao1 (P < 0.05), observed features (P < 0.05), Shannon (P < 0.05), Simpson (P < 0.05), and Pielou-e (P < 0.05) (Fig. 6 B–F). These results demonstrate that curcumin treatment effectively restores the richness, evenness, and overall diversity of the gut microbiota in OVX rats, suggesting a reversal of OVX-induced microbial dysbiosis. Beta diversity measures the compositional dissimilarity between microbial communities from different samples or groups. To assess the structural differences in gut microbiota, we performed beta diversity analysis based on both weighted and unweighted UniFrac distances, which incorporate phylogenetic information to quantify the degree of community separation. A higher distance value indicates greater dissimilarity in microbial composition between samples.The distance matrix heatmap demonstrated that the weighted UniFrac distance between the Cur group and the OVX group was 0.231, while the unweighted UniFrac distance was 0.578, indicating substantial differences in microbial community structure.Furthermore, principal coordinate analysis (PCoA) and principal component analysis (PCA) were employed to visualize sample clustering based on microbial composition. Ellipses representing the 95% confidence interval for each group (with n ≥ 4 biological replicates per group) revealed clear and significant separation between the Cur and OVX groups, with no overlapping regions or closely clustered points (Fig. 7 A–D). These results collectively demonstrate that curcumin treatment significantly alters the overall structure of the gut microbiota in OVX rats, resulting in a microbial community that is phylogenetically distinct from that of the untreated OVX controls. 3.5 Curcumin Treatment Reduces TMAO Abundance but Does Not Affect SCFA Levels in OVX Rats Trimethylamine-N-oxide (TMAO), a gut microbiota-derived metabolite generated from dietary choline, has been implicated in accelerated tissue metabolism and bone remodeling. Elevated TMAO levels are associated with osteoporosis, particularly in the context of estrogen deficiency, where it promotes bone loss by inhibiting mineral acquisition and osteogenic differentiation while enhancing osteoclast activity (11, 12).Short-chain fatty acids (SCFAs), also products of microbial fermentation, play a beneficial role in bone homeostasis by serving as an energy source for osteoblasts and positively influencing bone mineral density. Altered SCFA profiles have been documented in postmenopausal osteoporotic patients, underscoring their link with gut microbiota composition(24–26). To determine whether curcumin-induced microbial changes influence these metabolites, we performed targeted metabolomic analyses of TMAO-related metabolites and SCFAs. Curcumin treatment significantly reduced the abundance of TMAO and its precursors, including choline hydroxide (p < 0.05), creatinine (p < 0.05), and betaine (p < 0.05). A decreasing trend was also observed for L-carnitine, though it did not reach statistical significance (p = 0.3741; Fig. 8 A–D). These results suggest that curcumin remodels the gut microbiota, leading to suppressed production of TMAO and related metabolites. In contrast, no significant differences were detected in the levels of major SCFAs—such as acetate, propionate, and butyrate—between the Cur and OVX groups . To further elucidate the relationship between microbiota composition and TMAO metabolism, we conducted an integrated microbiome–metabolome analysis using Pearson correlation. At the genus level, five differentially abundant bacteria showed correlations with TMAO and its metabolites (Fig. 8 E–F). Among these, four genera—Alloprevotella,Defluviitaleaceae_UCG-011,Quinella, and an unidentified Muribaculaceae—were negatively correlated with TMAO levels. In contrast, Akkermansia exhibited a positive correlation.A metabolite–bacteria interaction network was constructed based on correlation coefficients to visualize key nodes and potential regulatory relationships within the microbiota–metabolite axis. These findings partially elucidate the microbial origins of TMAO and provide a basis for further mechanistic investigation into how curcumin modulates gut microbiota to influence bone metabolism. 3.6 Curcumin Treatment Attenuates Systemic Inflammation in OVX Rats by Reducing Pro-Inflammatory Cytokines Postmenopausal osteoporosis is a systemic disorder influenced not only by estrogen deficiency and gut microbiota dysbiosis but also by chronic low-grade inflammation. Elevated levels of pro-inflammatory cytokines—such as IL-1, IL-6, and G-CSF—following menopause contribute to impaired bone formation and enhanced osteoclastogenesis (27). Furthermore, TMAO has been shown to promote vascular inflammation and increase circulating pro-inflammatory cytokines (28). To evaluate the anti-inflammatory effects of curcumin, we measured serum levels of IL-1, IL-6, and G-CSF using ELISA. Curcumin treatment significantly reduced the concentration of IL-1 compared to the OVX group (p < 0.05). However, no significant differences were observed in IL-6 or G-CSF levels (Fig. 9 A–C). Notably, elevated inflammatory cytokine levels were detected in both antibiotic-treated groups (ABX and ABX + Cur), likely resulting from antibiotic-induced dysbiosis and gastrointestinal disturbance. IL-1 is a potent immunomodulatory cytokine often referred to as an “osteoclast-activating factor” and is strongly associated with the pathogenesis of postmenopausal osteoporosis (29). The observed reduction in IL-1 suggests that curcumin may mitigate bone loss partly through suppression of systemic inflammation. Although no significant changes in IL-6 or G-CSF were detected, the decrease in IL-1 represents a promising anti-inflammatory mechanism. This effect may be indirectly mediated through curcumin’s modulation of TMAO production and gut microbial composition, ultimately contributing to improved bone outcomes in OVX rats. 3.7 Fecal Microbiota Transplantation from Curcumin-Treated Rats Partially Rescues Bone Microarchitecture Based on our findings that the osteoprotective effects of curcumin are gut microbiota-dependent, we further investigated whether fecal microbiota transplantation (FMT) from curcumin-treated donors could attenuate bone loss in ovariectomized (OVX) recipients. Gut microbial dysbiosis is implicated in the pathogenesis of multiple diseases, and FMT from healthy donors has been shown to improve bone mass in OVX rats by remodeling the gut microbial community (30). To test this hypothesis, we conducted a second animal experiment in which microbiota-depleted OVX rats received daily FMT from one of two donor groups: FMT(OVX) (receiving microbiota from OVX rats) and FMT(Cur) (receiving microbiota from curcumin-treated OVX rats). Following the 12-week transplantation period, micro-CT analysis revealed that the FMT(Cur) group exhibited significantly greater trabecular thickness (Tb.Th) and bone mineral density (BMD) compared to the FMT(OVX) group (P < 0.05). However, no significant differences were observed in other trabecular parameters such as bone volume fraction (BV/TV) or trabecular number (Tb.N) (Fig. 10 ). The partial recovery of bone microarchitectural parameters suggests that microbiota derived from curcumin-treated rats possesses protective capacity against OVX-induced bone loss. The limited effect observed may be attributable to technical limitations of FMT, including potential microbial loss during transplant preparation or incomplete engraftment. Nevertheless, these results provide further evidence that the anti-osteoporotic effect of curcumin is mediated, at least in part, through structural and functional modifications of the gut microbiota. 4. Discussion Postmenopausal osteoporosis, primarily driven by the decline in estrogen levels following ovarian dysfunction, poses a significant global health burden, affecting over 200 million women worldwide and accounting for nearly one-third of fracture risks in women over 50 years of age (31, 32). The ovariectomized (OVX) rat model, which faithfully recapitulates key features of human postmenopausal osteoporosis—including estrogen deficiency and gut microbial dysbiosis—serves as a validated experimental system for investigating microbiota-targeted therapeutic interventions (21). The central paradox in curcumin research lies in the disconnect between its potent in vitro bioactivity and its negligible systemic bioavailability. Our study resolves this paradox by demonstrating that curcumin's in vivo osteoprotective effects are not dependent on its systemic circulation, but are instead mediated entirely through its local action on the gut microbiota. This represents a significant shift in understanding, positioning curcumin not as a failed systemic drug, but as a highly effective, locally-acting gut microbiota modulator. The most compelling evidence for this gut-dependent mechanism comes from our dual-approach validation. The complete abolition of curcumin's benefits in pseudo-germ-free rats unequivocally establishes the gut microbiota as a necessary component. More importantly, the partial transfer of these osteoprotective effects via FMT provides direct, causal evidence that the curcumin-modified microbiota is, in itself, sufficient to confer a therapeutic benefit. This FMT approach moves beyond correlation and firmly establishes a causal role for the microbiota in mediating curcumin's effects on bone. Patients with postmenopausal osteoporosis exhibit reduced gut microbial diversity, and animal studies corroborate that microbial composition influences bone strength (33). The gut–bone axis modulates bone homeostasis through multiple mechanisms, including short-chain fatty acid production, mineral absorption, immune regulation, and endocrine signaling(34). Our 16S rRNA sequencing revealed that curcumin significantly altered the abundance of key taxa, including Firmicutes, Clostridia, Bacteroidia, and Oscillospirales, and enhanced both alpha and beta diversity, indicating a reversal of OVX-induced dysbiosis.Our findings challenge the traditional drug development paradigm that prioritizes oral bioavailability above all else. For a growing class of compounds like curcumin, low systemic exposure may not be a limitation but rather an indication of a different mode of action—one that operates within the gut ecosystem. This redefines curcumin from a "poor drug" to a pioneering "microbiota-therapeutic." The clinical implication is profound: rather than futilely attempting to improve curcumin's absorption, a more effective strategy may be to leverage its gut-restricted action, potentially by developing advanced delivery systems that target its release to the colon. Elevated circulating TMAO has been associated with inflammatory bone loss and represents a potential biomarker for osteoporosis(35, 36). Curcumin treatment significantly reduced fecal TMAO and its precursors, which correlated with decreased systemic inflammation, as evidenced by lower IL-1 levels. These findings suggest that curcumin attenuates osteoporosis partly through suppressing TMAO-induced inflammation.Mechanistically, we identified a specific gut-bone signaling axis centered on TMAO. Curcumin robustly remodeled the gut ecosystem, enriching for beneficial taxa and suppressing putative TMA-producers, which in turn led to a marked reduction in TMAO levels. The correlation between these microbial shifts, lower TMAO, and decreased systemic IL-1 illuminates a clear pathway: curcumin alleviates the chronic inflammatory state of estrogen deficiency by suppressing a gut-derived pro-osteoclastogenic signal. It is noteworthy that this effect was specific to the TMAO-inflammation axis, as SCFA levels remained unchanged, distinguishing curcumin's mechanism from other microbiota-targeting therapies. A limitation of our study is the partial bone rescue by FMT, which may reflect technical challenges in microbial engraftment or the involvement of other non-transferable factors. Furthermore, while we established a strong association, future studies using TMAO supplementation or specific inhibition of TMA-lyases are needed to cement the causal role of TMAO in this pathway. In summary, we have elucidated a previously unrecognized therapeutic pathway for curcumin that bypasses the classical pharmacokinetic constraints. By acting as a gut microbiota modulator, curcumin suppresses the production of TMAO, thereby dampening systemic inflammation and protecting against bone loss. This work not only provides a novel mechanism for curcumin but also establishes a new strategic framework for developing microbiota-targeted therapies for osteoporosis. 5. Conclusions Although curcumin itself suffers from poor absorption, its potent modulation of the gut microbiota makes it a promising candidate for the treatment of osteoporosis. Innovative delivery systems—such as bone-targeted nanoparticles—may further enhance its efficacy by improving bioavailability and tissue-specific distribution(37). In conclusion, this study fundamentally redefines the mechanism of action of curcumin in osteoporosis. We demonstrate that its osteoprotective efficacy is entirely dependent on its capacity to remodel the gut microbiota, a property that renders its low systemic bioavailability irrelevant to its therapeutic function. The causal role of the microbiota was conclusively shown through depletion and transplantation experiments. The downstream mechanism involves the suppression of the gut-derived metabolite TMAO and a key pro-inflammatory cytokine, IL-1, creating an environment less conducive to bone resorption. These findings pivot the perspective on curcumin from a challenging systemic drug candidate to a potent gut-centric therapeutic. They underscore the "gut-bone" axis as a fertile ground for intervention and advocate for a paradigm shift in evaluating natural products, where local gut effects may be more therapeutically relevant than systemic exposure. Declarations Acknowledgements All authors thank the respective editors and professional reviewers for their efforts on improving this manuscript during the peer review process. Author contributions Xin Hu: conceptualization, methodology, formal analysis, data curation, writing—original draft, writing—review & editing; Yuchen Tang: conceptualization, methodology, formal analysis, data curation, writing—original draft, writing—review & editing; Qiufu Wang: conceptualization, methodology, formal analysis, data curation, writing—original draft, writing—review & editing; Guanyin Jiang: writing—review & editing; Miao Lei: writing—review & editing; Wen Dong: review & editing; Yongle Wu: conceptualization, data curation, methodology, writing—review & editing, supervision; Jie Hao: conceptualization, methodology, funding acquisition, writing—review & editing, supervision; Zhenming Hu: conceptualization, methodology, writing—review & editing, supervision. Funding This study was supported by the Special Project for the Discipline Summit Program of the First Clinical College, Chongqing Medical University (No. CYYY-XKDFJH-DSTD-202404). Data availability statement The data that support the findings of this study are openly available in Science Data Bank at https://doi.org/10.57760/sciencedb.33829. Ethics approval and consent to participate All animal procedures to be employed in the project was approved by Institutional Animal Care and Use of Chongqing Medical University(IACUS-CQMU) Approval number:IACUC-CQMU-2025-0656 Consent for publication All the authors consented for publication. Competing interests The authors declare no competing interests Author details Department of Orthopedics, University-Town Hospital of Chongqing Medical University, Daxuecheng Middle Road No. 55, Shapingba District, Chong qing 401331, China. Department of Orthopedics, The First Affiliated Hospital of Chongqing Medical University, Yuanjiagang Youyi Road No. 1, Yuzhong District, Chongqing 400010, China. References Lane JM, Russell L, Khan SN. Osteoporosis. Clin Orthop Relat Res. 2000(372):139–50. 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Bone. 2020;135:115317. Thomas MS, Fernandez ML. Trimethylamine N-Oxide (TMAO), Diet and Cardiovascular Disease. Curr Atheroscler Rep. 2021;23(4):12. Li L, An J, Bai J, Zhang Y, Li X, Lv H. Association between systemic immune-inflammation index and trimethylamine N-oxide levels in peripheral blood and osteoporosis in overweight and obese patients. Front Endocrinol (Lausanne). 2025;16:1539594. Li Y, Cai Z, Ma W, Bai L, Luo E, Lin Y. A DNA tetrahedron-based ferroptosis-suppressing nanoparticle: superior delivery of curcumin and alleviation of diabetic osteoporosis. Bone Res. 2024;12(1):14. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Apr, 2026 Reviews received at journal 15 Apr, 2026 Reviews received at journal 11 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 06 Apr, 2026 Editor assigned by journal 06 Apr, 2026 Editor invited by journal 01 Apr, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 31 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9218781","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":621713338,"identity":"60631855-9c8f-46dc-8e93-7a90ecdeb341","order_by":0,"name":"Xin 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University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Hao","suffix":""},{"id":621713346,"identity":"1371793c-5266-4d06-a2de-6d08ddb1aad4","order_by":8,"name":"Zhenming Hu","email":"","orcid":"","institution":"University-Town Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhenming","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2026-03-25 06:10:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9218781/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9218781/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106799429,"identity":"a4ac4c77-d5bc-4148-8a62-85b07b5512df","added_by":"auto","created_at":"2026-04-13 14:30:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157451,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative micro-CT images of femoral sagittal sections, three-dimensional trabecular architecture\u003cbr\u003e\n.(B-G) Quantitative Analysis of Femoral Microstructural Parameters in the OVX Group and Sham Group, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineral density (BMD).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/99386acbdec677bec959dd3d.png"},{"id":106959864,"identity":"c3c642fc-a45c-4819-8c76-afc7a20cf6b1","added_by":"auto","created_at":"2026-04-15 09:16:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209266,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative micro-CT images of femoral sagittal sections, three-dimensional trabecular architecture from the Cur group, OVX group, ABX group, and ABX+Cur group, respectively.(B-G) Quantitative analysis of femoral microstructural parameters in rats subjected to four different treatments (saline, curcumin, quadruple antibiotics, and quadruple antibiotics + curcumin, respectively), including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineral density (BMD).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/34f1730ee4a1fa69008e4f16.png"},{"id":106799432,"identity":"0f1b0ed7-999a-418c-873c-c2725861a8fc","added_by":"auto","created_at":"2026-04-13 14:30:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":632008,"visible":true,"origin":"","legend":"\u003cp\u003e(A-B) Histological sections of femoral bone from rats in four different treatment groups stained with hematoxylin and eosin (H\u0026amp;E) and Masson's trichrome.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/a6f107f395bd31d0acd88cf5.png"},{"id":106799431,"identity":"7528cf24-ceae-4eac-a616-3d1285300dcd","added_by":"auto","created_at":"2026-04-13 14:30:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":147912,"visible":true,"origin":"","legend":"\u003cp\u003e(A) In the \u0026nbsp;\u0026nbsp;Venn diagram, each circle represents a sample group. The numbers in \u0026nbsp;\u0026nbsp;overlapping areas indicate the count of shared features (e.g., ASVs/OTUs) \u0026nbsp;\u0026nbsp;between groups, while numbers in non-overlapping sections represent features \u0026nbsp;\u0026nbsp;unique to each group.(B-C) Panels B and C display phylum-level taxonomic \u0026nbsp;\u0026nbsp;profiles, illustrating between-group variations and inter-sample differences. \u0026nbsp;\u0026nbsp;The x-axis (Sample Name) denotes individual samples, while the y-axis \u0026nbsp;\u0026nbsp;(Relative Abundance) represents taxonomic proportions. \"Others\" \u0026nbsp;\u0026nbsp;indicates the aggregated relative abundance of all phyla beyond the top 10 \u0026nbsp;\u0026nbsp;most abundant ones.(D) Based on phylum-level annotations and abundance data, \u0026nbsp;\u0026nbsp;a heatmap was generated using the top most abundant phyla. Hierarchical \u0026nbsp;\u0026nbsp;clustering was performed based on abundance patterns across sample groups to \u0026nbsp;\u0026nbsp;visualize phylogenetic composition similarities.(E-F) Comparative analysis of \u0026nbsp;\u0026nbsp;relative abundance for the phyla Verrucomicrobiota (E) and Firmicutes (F) \u0026nbsp;\u0026nbsp;between the two experimental groups. Data are presented as mean ± SD, with \u0026nbsp;\u0026nbsp;statistical significance determined by Student's t-test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/022c654b9a51882ccfe50e47.png"},{"id":106799433,"identity":"bf97a13c-98e2-4c57-b631-167df5e4a952","added_by":"auto","created_at":"2026-04-13 14:30:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112630,"visible":true,"origin":"","legend":"\u003cp\u003e(A-B) Bar \u0026nbsp;\u0026nbsp;plots illustrating taxonomic composition at the family level, demonstrating \u0026nbsp;\u0026nbsp;inter-group variations and inter-sample differences. The x-axis (Sample Name) \u0026nbsp;\u0026nbsp;identifies individual samples, while the y-axis (Relative Abundance) \u0026nbsp;\u0026nbsp;indicates proportional representation of taxonomic groups. \"Others\" \u0026nbsp;\u0026nbsp;denotes the cumulative relative abundance of all families beyond the top 10 \u0026nbsp;\u0026nbsp;most abundant taxa.(C-F) Comparative analysis of relative abundance for \u0026nbsp;\u0026nbsp;specific bacterial families between the two experimental groups: \u0026nbsp;\u0026nbsp;Prevotellaceae (C), Oscillospiraceae (D), Spirochaetaceae (E), Muribaculaceae \u0026nbsp;\u0026nbsp;(F), Akkermansiaceae (G), and Lactobacillaceae (H). Data are presented as \u0026nbsp;\u0026nbsp;mean ± SD, with statistical significance determined by Student's t-test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/526a85ca32cd0ae45d862bd2.png"},{"id":106960218,"identity":"ab985ed7-07fc-46ef-9dfd-db8ec743a941","added_by":"auto","created_at":"2026-04-15 09:19:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":191180,"visible":true,"origin":"","legend":"\u003cp\u003e(A-B) \u0026nbsp;\u0026nbsp;Taxonomic composition analysis at the genus level, presenting inter-group \u0026nbsp;\u0026nbsp;variations and inter-sample disparities. The x-axis (Sample Name) designates \u0026nbsp;\u0026nbsp;individual samples, while the y-axis (Relative Abundance) indicates \u0026nbsp;\u0026nbsp;proportional representation of taxonomic groups. \"Others\" \u0026nbsp;\u0026nbsp;represents the cumulative relative abundance of all genera beyond the top 10 \u0026nbsp;\u0026nbsp;most abundant taxa.(C) The LDA score distribution histogram displays species \u0026nbsp;\u0026nbsp;with LDA scores exceeding the threshold value (default setting: 4), identifying \u0026nbsp;\u0026nbsp;statistically significant biomarkers demonstrating inter-group \u0026nbsp;\u0026nbsp;differences.(D) In the evolutionary cladogram, concentric circles radiating \u0026nbsp;\u0026nbsp;from the center represent taxonomic hierarchies from phylum to genus (or \u0026nbsp;\u0026nbsp;species). Each node at different taxonomic levels corresponds to a specific \u0026nbsp;\u0026nbsp;classification, with node diameter proportional to relative abundance. \u0026nbsp;\u0026nbsp;Statistically non-significant species are uniformly colored yellow, while \u0026nbsp;\u0026nbsp;differential biomarkers are colored according to their respective groups. Red \u0026nbsp;\u0026nbsp;nodes indicate microbial taxa playing significant roles in the red group, \u0026nbsp;\u0026nbsp;whereas green nodes represent those in the green group. Absence of a colored \u0026nbsp;\u0026nbsp;group in the diagram indicates that the abundance of significantly different \u0026nbsp;\u0026nbsp;species in that group was lower compared to other groups.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/520a72d4988e477e2681587b.png"},{"id":106799437,"identity":"29f1cecc-b505-4c30-998a-85dbc0cbe3ec","added_by":"auto","created_at":"2026-04-13 14:30:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":103400,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Rarefaction curves demonstrating sequencing depth adequacy. The x-axis represents sequencing depth (number of sequences), while the y-axis indicates corresponding alpha diversity indices. Curve plateauing suggests sufficient sequencing depth, as additional sequences would not significantly alter diversity estimates.(B-F) Comparative analysis of alpha diversity indices between Cur and OVX groups: (B) Chao1 index, (C) observed features, (D) Shannon index, (E) Simpson index, and (F) Pielou's evenness (pielou_e). Data are presented as mean ± SD, with statistical significance determined by Student's t-test.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/97f88cff62cde22275ca7402.png"},{"id":106799439,"identity":"3753b419-1933-4085-8a99-50db51ed644c","added_by":"auto","created_at":"2026-04-13 14:30:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":109233,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The \u0026nbsp;\u0026nbsp;values within the matrix represent pairwise \u0026nbsp;\u0026nbsp;dissimilarity coefficients between samples. Lower values indicate smaller \u0026nbsp;\u0026nbsp;differences in species diversity between corresponding samples. For each \u0026nbsp;\u0026nbsp;pairwise comparison, the upper and lower values denote Weighted Unifrac and \u0026nbsp;\u0026nbsp;Unweighted Unifrac distances, respectively.(B-D) Multivariate analysis of \u0026nbsp;\u0026nbsp;microbial community structure: (B) Principal Coordinate Analysis (PCoA) based \u0026nbsp;\u0026nbsp;on Weighted Unifrac distance, (C) PCoA based on Unweighted Unifrac distance, \u0026nbsp;\u0026nbsp;and (D) Principal Component Analysis (PCA) performed at the feature abundance \u0026nbsp;\u0026nbsp;level. The x-axis represents the first principal component with the \u0026nbsp;\u0026nbsp;percentage indicating its contribution to overall variance, while the y-axis \u0026nbsp;\u0026nbsp;represents the second principal component with corresponding variance \u0026nbsp;\u0026nbsp;contribution. Individual points represent samples, colored according to their \u0026nbsp;\u0026nbsp;respective experimental groups.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/31487375c88c5b5b28cd1cdb.png"},{"id":106799435,"identity":"89edc9d3-3f59-4742-b3d6-76a966a027b2","added_by":"auto","created_at":"2026-04-13 14:30:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":140020,"visible":true,"origin":"","legend":"\u003cp\u003e(A-D) Comparative analysis of TMAO-related metabolites between Cur and OVX groups: (A) Choline Hydroxide, (B) Creatinine, (C) Betaine, and (D) L-Carnitine. Data are presented as mean ± SD, with statistical significance determined by Student's t-test.(E) Heatmap visualization of Spearman correlations between differential bacterial genera (x-axis) and differential metabolites (y-axis). The color scale represents correlation coefficients, with red and blue indicating positive and negative correlations, respectively. Asterisks denote statistically significant correlations (P value \u0026lt; 0.05).(F) Network analysis of metabolite-microbe interactions. Rectangular nodes represent metabolites (yellow) and bacterial genera (blue). Connecting lines indicate significant correlations, with red and blue edges representing positive and negative relationships, respectively.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/65532f4b554bf0cadf9b7344.png"},{"id":106799440,"identity":"c64c8db1-b374-44d8-9b1f-b869a3cd5fc7","added_by":"auto","created_at":"2026-04-13 14:30:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":63515,"visible":true,"origin":"","legend":"\u003cp\u003e(A-C)Quantification of serum concentrations of pro-inflammatory cytokines IL-1, IL-6, and G-CSF across experimental groups. Data are presented as mean ± SD, with statistical significance determined by one-way ANOVA followed by Tukey's post-hoc test.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/df2b1d223e85bd399482dbf3.png"},{"id":106799436,"identity":"7e781b48-ec14-447a-9646-cb740aa1a8ca","added_by":"auto","created_at":"2026-04-13 14:30:48","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":134490,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Representative micro-CT images of the 3D reconstruction of femoral sagittal sections, 3D trabecular architecture, and the analyzed region of interest (ROI) from the FMT(OVX) and FMT(Cur) groups, respectively.(B-G) Quantitative analysis of femoral microstructural parameters in FMT-treated rats following microbiota depletion with quadruple antibiotic cocktail, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone mineral density (BMD).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/1f34e7fb29810b7ddc1a8efb.png"},{"id":106964206,"identity":"b6335de5-3ea3-4a5a-8670-f3da724c0702","added_by":"auto","created_at":"2026-04-15 09:49:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2575610,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9218781/v1/d888a592-22ff-4c01-8677-31098f2556c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Curcumin Ameliorates Osteoporosis via Gut Microbiota-Dependent Modulation of TMAO in Ovariectomized Rats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOsteoporosis is a prevalent metabolic bone disorder characterized by reduced bone mineral density and impaired bone strength, leading to an elevated risk of fractures. Postmenopausal osteoporosis (PMOP) represents one of the most common forms of the disease, primarily resulting from an imbalance in bone metabolism due to decreased estrogen levels following menopause. This hormonal decline tilts the balance toward increased bone resorption and reduced bone formation (1\u0026ndash;3). The pathogenesis of PMOP is multifactorial, involving numerous biological pathways and molecular mechanisms.\u003c/p\u003e \u003cp\u003eEstrogen deficiency is widely recognized as a key contributor to postmenopausal osteoporosis. It promotes the overexpression of pro-inflammatory cytokines and enhances osteoclast activity through activation of the nuclear factor kappa B (NF-κB) signaling pathway, thereby accelerating bone loss (2, 4) .Moreover, emerging evidence indicates that gut microbiota dysbiosis plays a critical role in the development of PMOP. Alterations in the gut microbial community can exacerbate bone loss by modulating systemic inflammation and bone metabolic processes(5).Consequently, the pathogenic model of PMOP has evolved from a primarily estrogen-centric perspective to a more comprehensive framework that incorporates the influence of gut microbiota.\u003c/p\u003e \u003cp\u003eA study involving 106 postmenopausal women\u0026mdash;classified as having osteopenia (n\u0026thinsp;=\u0026thinsp;33), osteoporosis (n\u0026thinsp;=\u0026thinsp;42), or normal bone mineral density (n\u0026thinsp;=\u0026thinsp;31)\u0026mdash;revealed significant alterations in fecal microbiota composition. Specifically, patients with osteoporosis exhibited reduced microbial diversity and abundance, including decreased levels of Bacteroides, along with a marked increase in Proteobacteria, such as the pro-inflammatory genera Klebsiella and Escherichia coli (6). In the postmenopausal state, estrogen deficiency may induce gut dysbiosis, which in turn triggers abnormal immune activation. This includes an expansion of T helper 17 (Th17) cells, release of pro-inflammatory cytokines, and activation of the RANKL signaling pathway, collectively promoting osteoclast differentiation and ultimately leading to bone loss and increased fracture risk (7).\u003c/p\u003e \u003cp\u003eCurrent pharmacological management of PMOP includes traditional agents such as bisphosphonates and selective estrogen receptor modulators (SERMs). While these therapies are widely used, their long-term application is often limited by associated adverse effects.\u003c/p\u003e \u003cp\u003eTrimethylamine N-oxide (TMAO) is an organic compound primarily derived from dietary precursors through microbial metabolism in the gut. Trimethylamine (TMA), generated by gut microbiota from substrates such as choline, carnitine, betaine, and creatinine, is absorbed and oxidized in the liver by flavin-containing monooxygenases (FMOs) to form TMAO, which subsequently enters the systemic circulation. Additionally, certain foods\u0026mdash;particularly marine fish and shellfish\u0026mdash;contain high levels of preformed TMAO and can contribute directly to its plasma concentrations via intestinal absorption (8).\u003c/p\u003e \u003cp\u003eThe production of TMA is facilitated by various gut bacteria, including species from the genera Clostridium, Proteus, Shigella, and Enterobacter (9). Both dietary intake and the composition of the gut microbiota significantly influence plasma TMAO levels. Currently, TMAO is widely regarded not merely as a metabolic by-product of choline metabolism, but as a molecule with substantial pathophysiological implications. Elevated circulating TMAO has been closely linked to an increased risk of cardiovascular diseases and osteoporosis, and it is also known to mediate inflammatory responses within the body (10\u0026ndash;12).\u003c/p\u003e \u003cp\u003eEstrogen deficiency-induced osteoporosis is recognized as a chronic inflammatory condition, frequently accompanied by elevated levels of reactive oxygen species (ROS) and pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α(13). Given that TMAO contributes to systemic inflammation and may exacerbate oxidative stress, it is plausible that TMAO plays a role in amplifying bone loss under estrogen-deficient conditions. Therefore, we hypothesize that modulating gut microbiota dysbiosis resulting from estrogen deficiency and reducing plasma TMAO levels may indirectly alleviate osteoporosis symptoms by attenuating inflammation and oxidative damage.However, it should be noted that the causal relationship between TMAO and bone metabolism remains incompletely elucidated. Most existing evidence is derived from observational or correlative studies, and further mechanistic investigations are necessary to validate whether TMAO directly influences osteoclastogenesis or bone remodeling.\u003c/p\u003e \u003cp\u003eCurcumin, the principal bioactive compound derived from the rhizomes of Curcuma longa (a member of the Zingiberaceae family), is a lipophilic polyphenol with the chemical structure of 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(14). It exhibits a wide range of pharmacological properties, including anti-inflammatory, antioxidant, anti-osteoarthritic, anticancer, neuroprotective, and cardioprotective effects(14, 15). In vitro studies indicate that curcumin stimulates osteoblast proliferation, upregulates bone formation-related gene expression, and suppresses osteoclastogenesis, thereby contributing to the maintenance of bone health(16). Furthermore, numerous animal studies have demonstrated the potential of curcumin to ameliorate osteoporosis(17, 18). The diverse biological activities of curcumin are largely attributed to its anti-inflammatory and antioxidant mechanisms.\u003c/p\u003e \u003cp\u003eHowever, the clinical translation of curcumin is hampered by its poor aqueous solubility, low oral bioavailability, and suboptimal pharmacokinetic profile(14). These limitations result in minimal intestinal absorption and low systemic exposure, which contrast sharply with its broad pharmacological efficacy in vitro and in preclinical models.\u003c/p\u003e \u003cp\u003eTo investigate whether curcumin alleviates osteoporosis through gut microecological modulation and anti-inflammatory mechanisms, we employed an ovariectomized (OVX) rat model of postmenopausal osteoporosis. We hypothesized that curcumin improves bone mass by restoring gut microbiota homeostasis, reducing systemic inflammation, and modulating metabolite production.\u003c/p\u003e \u003cp\u003eFemoral bone mineral density (BMD) measurements, combined with integrated analyses of the gut microbiome and metabolome, revealed that curcumin treatment significantly increased BMD in OVX rats. Additionally, curcumin enhanced microbial richness and diversity, ameliorated ovariectomy-induced dysbiosis, and reduced circulating levels of the gut-derived metabolite trimethylamine N-oxide (TMAO) and key pro-inflammatory cytokines. Collectively, these findings suggest that curcumin attenuates osteoporosis in OVX rats by modulating the gut microbiota and its metabolic output, particularly TMAO, and by dampening systemic inflammation. This study underscores the importance of the gut-bone axis as a potential therapeutic target for osteoporosis treatment.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e2.1. Animals and Treatments\u003c/p\u003e\n\u003cp\u003eAll experimental Sprague-Dawley (SD) rats were obtained from the Animal Experiment Center of Chongqing Medical University. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University and were in compliance with relevant ethical guidelines.\u003c/p\u003e\n\u003cp\u003eFirst Animal Experiment:A total of 25 eight-week-old female SD rats (approximately 200 g body weight) were acclimatized for 7 days before undergoing bilateral ovariectomy (OVX). The rats were then randomly assigned to five experimental groups (n = 5 per group):Sham surgery group,OVX group (bilateral oophorectomy only),Curcumin group (200 mg/kg/day by oral gavage after bilateral oophorectomy),Antibiotic-treated (ABX) group (pseudo-germ-free model),ABX + Curcumin group (antibiotics plus curcumin).Bilateral oophorectomy was performed in all groups except the sham surgery group.\u003c/p\u003e\n\u003cp\u003eCurcumin was administered daily via oral gavage at approximately 10:00 AM for 12 weeks. To establish a pseudo-germ-free state, rats in the ABX and ABX + Curcumin groups received a quadruple antibiotic cocktail by gavage for 7 days prior to OVX, consisting of vancomycin (100mg/kg), neomycin sulfate (200mg/kg), metronidazole (200mg/kg), and ampicillin (200mg/kg) (19, 20). Throughout the subsequent 12-week experimental period, these animals received the same antibiotics dissolved in drinking water at the following concentrations: vancomycin (500mg/L), metronidazole (1g/L), ampicillin (1g/L), and neomycin sulfate (1g/L).\u003c/p\u003e\n\u003cp\u003eSecond Animal Experiment:An additional 6 six-week-old female SD rats (approximately 200 g body weight) were acclimatized and underwent OVX surgery. After antibiotic-induced microbiota depletion (as described above), they were divided into two groups:FMT(OVX) group: received fecal microbiota transplantation (FMT) from donor OVX rats,FMT(Cur) group: received FMT from donor curcumin-treated rats.\u003c/p\u003e\n\u003cp\u003eFecal suspensions were prepared daily from fresh feces collected from donor rats (OVX or Cur group of the first experiment), flash-frozen on dry ice, and thawed immediately before use. Feces were diluted 1:10 (w/v) in sterile saline, vortexed for 2 minutes, and centrifuged at 500 \u0026times; g for 3 min. The supernatant was collected and administered via gavage within 20 minutes of preparation to minimize microbial compositional changes (19).\u003c/p\u003e\n\u003cp\u003eFollowing the 12-week treatment period, all rats were euthanized via intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight) after being anesthetized with isoflurane (5% induction, 2\u0026ndash;3% maintenance in 100% oxygen) to ensure deep anesthesia prior to euthanasia. Euthanasia was confirmed by the absence of pedal and corneal reflexes, followed by bilateral thoracotomy. Subsequently, femurs, fecal samples, organs (including liver, kidneys, and spleen), and blood were collected for subsequent analyses. Blood was obtained via cardiac puncture using a 21-gauge needle and collected into EDTA-coated tubes for hematological analysis, as well as into serum separator tubes for biochemical assays. All instruments used for dissection were sterilized with 70% ethanol and autoclaved prior to use. Tissue samples were immediately snap-frozen in liquid nitrogen and stored at \u0026minus;80\u0026deg;C until further processing.\u003c/p\u003e\n\u003cp\u003e2.2. Micro-Computed Tomography (Micro-CT) Analysis\u003c/p\u003e\n\u003cp\u003eFollowing euthanasia, femurs were harvested and fixed for micro-CT scanning. Scans were performed using a [NMC-200] with consistent scanning parameters across all samples. The acquired images were reconstructed using three-dimensional reconstruction software \u0026nbsp;[Cruiser,Recon]. A standardized region of interest (ROI) was selected for all samples in the distal femoral metaphysis, located [e.g., 0.5\u0026ndash;1.0 mm proximal to the growth plate]. Trabecular bone parameters, including bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp), were analyzed using dedicated data analysis software \u0026nbsp; [Avatar]. Data were exported for statistical evaluation to assess differences in trabecular bone microstructure, mineralization, and inferred bone strength.\u003c/p\u003e\n\u003cp\u003e2.3. Histological Analysis\u003c/p\u003e\n\u003cp\u003eMajor organs (heart, liver, spleen, lungs, and kidneys) were collected, fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H\u0026amp;E) to evaluate potential toxicological effects of curcumin and/or antibiotic treatments.\u003c/p\u003e\n\u003cp\u003eFor bone histomorphometry, femurs were decalcified in 10% EDTA (pH 7.4) for 4 weeks. Subsequently, decalcified bones were processed, embedded in paraffin, and sectioned. Sections were stained with H\u0026amp;E and Masson\u0026rsquo;s trichrome to evaluate osteoblast activity, bone formation, turnover, and mineralization status.\u003c/p\u003e\n\u003cp\u003e2.4. Serum Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/p\u003e\n\u003cp\u003eBlood samples were centrifuged at [e.g., 3000 \u0026times; g for 15 min at 4\u0026deg;C] to obtain serum. The concentrations of pro-inflammatory cytokines, including IL-1, IL-6, and G-CSF, were quantified using specific commercial ELISA kits according to the manufacturers\u0026apos; protocols. Absorbance was measured using a microplate reader, and cytokine concentrations were calculated from standard curves.\u003c/p\u003e\n\u003cp\u003e2.5. 16S rRNA Gene Sequencing\u003c/p\u003e\n\u003cp\u003eTotal genomic DNA was extracted from frozen fecal samples according to the manufacturer\u0026apos;s instructions. The hypervariable region of the bacterial 16S rRNA gene were amplified using specific primers . PCR products were purified, quantified, and pooled in equimolar ratios to construct sequencing libraries. Then the library was constructed. The constructed library was quantified by Qubit and qPCR.Bioinformatic analysis was performed using QIIME2 or a similar pipeline to assess microbial community diversity (alpha and beta diversity), composition, and relative taxonomic abundance.\u003c/p\u003e\n\u003cp\u003e2.6. Quantitative Metabolomic Analysis of Fecal TMAO\u003c/p\u003e\n\u003cp\u003eThe concentration of trimethylamine N-oxide (TMAO) in fecal samples was quantified using targeted liquid chromatography-mass spectrometry (LC-MS). Briefly, samples were homogenized in liquid nitrogen, and a precise weight (e.g., 20 mg) was diluted with mass spectrometry-grade water. After vortexing, 50 \u0026micro;L of the diluted (d9-TMAO). The mixture was vortexed, incubated on ice for 30 minutes, and centrifuged (12,000 rpm, 10 min, 4\u0026deg;C). The supernatant was collected for LC-MS analysis. Quantification was achieved by comparing the peak area ratio of TMAO to the internal standard against a standard calibration curve.\u003c/p\u003e\n\u003cp\u003e2.7. Correlation Analysis Between Metabolome and Microbiome\u003c/p\u003e\n\u003cp\u003eTo explore potential functional relationships between the gut microbiota and host metabolism, integrated correlation analyses were performed. Statistically significant differential abundant microbes (from 16S sequencing) and metabolite levels ( TMAO) were included. Spearman or Pearson correlation coefficients were calculated to identify significant associations between specific bacterial taxa and metabolite concentrations, aiming to infer microbiota-driven phenotypic changes.\u003c/p\u003e\n\u003cp\u003e2.9. Statistical Analysis\u003cbr\u003e Data are presented as mean \u0026plusmn; standard deviation (SD). Normality was assessed using the Shapiro-Wilk test. For comparisons among multiple groups, one-way or two-way ANOVA followed by an appropriate post-hoc test ( Tukey\u0026apos;s or Sidak\u0026apos;s) was used. Correlation analyses were performed using Spearman\u0026apos;s or Pearson\u0026apos;s method. A p-value \u0026lt; 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism version 9 .\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Curcumin Attenuates Bone Loss and Ameliorates Osteoporosis in Ovariectomized Rats\u003c/h2\u003e \u003cp\u003eThe ovariectomized (OVX) rat model is a well-established and widely used animal model for studying postmenopausal osteoporosis. Successful model induction was confirmed 1\u0026ndash;3 weeks after bilateral ovariectomy, characterized by significant bone loss in the proximal tibia, lumbar vertebrae, and femur, as evidenced by reduced bone mineral density (BMD), decreased trabecular number, and increased trabecular separation (21).Micro-CT analysis revealed that, compared with the sham-operated group, the ovariectomy (OVX) group exhibited significant reductions in bone volume fraction (BV/TV, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), trabecular number (Tb.N, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), trabecular thickness (Tb.Th, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and bone mineral density (BMD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At the same time, compared with the OVX control group, the trabecular separation (Tb.Sp, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the sham-operated group was also significantly reduced..Collectively, these results indicate that bilateral ovariectomy in rats successfully established an osteoporosis model.(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e0\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the present study, micro-CT analysis of femoral samples from the first experiment revealed that curcumin treatment significantly alleviated OVX-induced bone loss. Compared to the OVX group (which received normal saline via gavage), the curcumin-treated group exhibited a notable improvement in multiple bone microarchitectural parameters. Specifically, curcumin administration significantly increased the bone volume fraction (BV/TV, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), trabecular number (Tb.N, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), trabecular thickness (Tb.Th, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and bone mineral density (BMD, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, a significant decrease in trabecular separation (Tb.Sp, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed in the curcumin group relative to the OVX controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistent with these findings, other indices\u0026mdash;including bone volume, bone surface area to tissue volume ratio, bone surface area to bone volume ratio, and trabecular bone pattern factor\u0026mdash;also indicated that oral administration of curcumin ameliorated osteoporotic changes in OVX rats.\u003c/p\u003e \u003cp\u003eFurthermore, histopathological evaluation through H\u0026amp;E staining of major organs (heart, liver, spleen, lungs, and kidneys) showed no evidence of adverse effects or structural abnormalities resulting from curcumin treatment, indicating its safety profile within the experimental context.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. The Osteoprotective Effect of Curcumin in Ovariectomized Rats is Gut Microbiota-Dependent\u003c/h2\u003e \u003cp\u003eEstrogen deficiency following ovariectomy has been reported to induce significant alterations in the gut microbial community, which may contribute to bone loss through modulation of inflammation and bone metabolism(22). To investigate whether the anti-osteoporotic effect of curcumin depends on the gut microbiota, we established a pseudo-germ-free (PGF) rat model via broad-spectrum antibiotic treatment (vancomycin: 100 mg/kg; neomycin sulfate: 200 mg/kg; metronidazole: 200 mg/kg; ampicillin: 200 mg/kg) to deplete gut microbiota (20).\u003c/p\u003e \u003cp\u003eRats were divided into two groups: ABX group (antibiotic-treated OVX rats) and ABX\u0026thinsp;+\u0026thinsp;Cur group (antibiotic-treated OVX rats receiving curcumin). Micro-CT analysis of femoral microstructure revealed no significant differences in bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), or trabecular separation (Tb.Sp) between these two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, when the ABX\u0026thinsp;+\u0026thinsp;Cur group was compared with the curcumin-treated non-antibiotic group (Cur group) from the first experiment, significant reductions in BV/TV, Tb.Th, and BMD were observed in the microbiota-depleted animals (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicate that the beneficial effects of curcumin on bone microarchitecture\u0026mdash;including improvements in BV/TV, Tb.N, Tb.Th, and BMD\u0026mdash;were abolished in the absence of a functional gut microbiota.\u003c/p\u003e \u003cp\u003eHistological analysis of femoral sections stained with H\u0026amp;E and Masson's trichrome revealed pronounced microstructural improvements in the Cur group compared to the OVX, ABX, and ABX\u0026thinsp;+\u0026thinsp;Cur groups. Specifically, the Cur group exhibited increased trabecular bone density, greater trabecular thickness, and significantly reduced inter-trabecular spacing with a more compact architectural organization. Notably, these trabeculae maintained structural integrity without evidence of fragmentation, and demonstrated better preservation of bone matrix(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).These findings indicate that curcumin treatment effectively restored bone microstructure in OVX rats. Conversely, microbiota-depleted rats (ABX and ABX\u0026thinsp;+\u0026thinsp;Cur groups) showed no significant structural improvements regardless of curcumin administration, confirming the essential role of gut microbiota in mediating curcumin's osteoprotective effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThus, we conclude that the amelioration of OVX-induced osteoporosis by curcumin is gut microbiota-dependent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Curcumin Alters the Intestinal Microbial Composition in OVX Rats\u003c/h2\u003e \u003cp\u003eEstrogen deficiency-induced osteoporosis has been previously associated with significant alterations in gut microbial composition(6, 7). To investigate whether curcumin modulates the gut microbiota in OVX rats, we performed 16S rRNA gene amplicon sequencing on fecal samples collected from the four experimental groups in the first animal trial.After processing and analyzing the sequencing data, we identified unique amplicon sequence variants (ASVs) across groups. The curcumin-treated (Cur) group exhibited 926 unique ASVs, substantially higher than the 448 observed in the OVX group. In contrast, both antibiotic-treated groups (ABX and ABX\u0026thinsp;+\u0026thinsp;Cur) showed a marked reduction in total ASVs, confirming successful establishment of a pseudo-germ-free state through antibiotic administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).We further evaluated microbial composition at the phylum, family, and genus levels using relative abundance bar plots and clustered heatmaps. Comparative analysis revealed pronounced differences in the abundance of dominant bacterial taxa among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;D). These results indicate that curcumin significantly restructures the gut microbial community in OVX rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the phylum level, significant alterations in microbial composition were observed between the Cur and OVX groups. The relative abundance of Verrucomicrobiota was significantly lower in the Cur group compared to the OVX group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Although not statistically significant, the Firmicutes population showed an increasing trend in the Cur group (54.37% vs. 39.07% in OVX, p\u0026thinsp;=\u0026thinsp;0.1092; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In contrast, the abundance of Bacteroidota remained comparable between the two groups (40.20% vs. 37.13%). Notably, several other phyla, including Cyanobacteria, Patescibacteria, Campylobacterota, Spirochaetota, and Elusimicrobiota, were significantly enriched in the Cur group.\u003c/p\u003e \u003cp\u003eAt the family level, curcumin treatment led to a significant increase in the relative abundance of Prevotellaceae (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Oscillospiraceae (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Spirochaetaceae (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, a marked reduction was observed in Muribaculaceae (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Akkermansiaceae (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Lactobacillaceae (p\u0026thinsp;=\u0026thinsp;0.086; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;H). No significant differences were detected in other families such as Lachnospiraceae.Further analysis at the genus level revealed that curcumin supplementation significantly increased the abundance of UCG-005 (p\u0026thinsp;=\u0026thinsp;0.0884) and Prevotellaceae_UCG-001 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the genera Akkermansia, Lactobacillus, and Lachnospira exhibited a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify specific microbial taxa associated with curcumin-induced improvement in osteoporosis, we performed linear discriminant analysis effect size (LEfSe) analysis. The Cur group was characterized by a higher abundance of p_Firmicutes, followed by c_Clostridia, c_Bacteroidia, p_Bacteroidota, and o_Oscillospirales. In the OVX group, the most discriminative features were f_Muribaculaceae, f_Akkermansiaceae, p_Verrucomicrobiota,g_Akkermansia, and c_Verrucomicrobiae, all with LDA scores\u0026thinsp;\u0026gt;\u0026thinsp;4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;D).\u003c/p\u003e \u003cp\u003eThese results demonstrate that curcumin significantly restructures the gut microbiota across multiple taxonomic levels and suggest that the osteoprotective effects of curcumin may be mediated through specific changes in microbial community composition.Through lefse analysis, we can speculate that it is precisely because of the changes in the composition of these flora that the osteoporosis of OVX rats is improved.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Curcumin Alters the Intestinal Microbial Diversity in OVX Rats\u003c/h2\u003e \u003cp\u003ePrevious studies have indicated that ovariectomy induces significant dysbiosis of the gut microbiota, characterized by altered species richness and compositional shifts compared to sham-operated controls(23). To evaluate the effect of curcumin on the gut microbial community, we performed alpha diversity analysis on fecal samples from each group.\u003c/p\u003e \u003cp\u003eAlpha diversity metrics provide insights into the complexity of microbial communities. The Chao1 and observed features indices estimate community richness. The Shannon and Simpson indices represent community diversity, incorporating both richness and evenness, while the Pielou-e index specifically measures species evenness. The Goods coverage index evaluates Community coverage, indicating the completeness of sampling.A rarefaction curve was generated for each alpha diversity metric (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The plateauing of these curves with increasing sequencing depth confirms that the obtained data sufficiently captured the microbial diversity within the samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, compared to the OVX group, the curcumin-treated (Cur) group exhibited significant increases in all measured alpha diversity indices, including Chao1 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), observed features (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Shannon (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Simpson (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and Pielou-e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026ndash;F). These results demonstrate that curcumin treatment effectively restores the richness, evenness, and overall diversity of the gut microbiota in OVX rats, suggesting a reversal of OVX-induced microbial dysbiosis.\u003c/p\u003e \u003cp\u003eBeta diversity measures the compositional dissimilarity between microbial communities from different samples or groups. To assess the structural differences in gut microbiota, we performed beta diversity analysis based on both weighted and unweighted UniFrac distances, which incorporate phylogenetic information to quantify the degree of community separation. A higher distance value indicates greater dissimilarity in microbial composition between samples.The distance matrix heatmap demonstrated that the weighted UniFrac distance between the Cur group and the OVX group was 0.231, while the unweighted UniFrac distance was 0.578, indicating substantial differences in microbial community structure.Furthermore, principal coordinate analysis (PCoA) and principal component analysis (PCA) were employed to visualize sample clustering based on microbial composition. Ellipses representing the 95% confidence interval for each group (with n\u0026thinsp;\u0026ge;\u0026thinsp;4 biological replicates per group) revealed clear and significant separation between the Cur and OVX groups, with no overlapping regions or closely clustered points (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese results collectively demonstrate that curcumin treatment significantly alters the overall structure of the gut microbiota in OVX rats, resulting in a microbial community that is phylogenetically distinct from that of the untreated OVX controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Curcumin Treatment Reduces TMAO Abundance but Does Not Affect SCFA Levels in OVX Rats\u003c/h2\u003e \u003cp\u003eTrimethylamine-N-oxide (TMAO), a gut microbiota-derived metabolite generated from dietary choline, has been implicated in accelerated tissue metabolism and bone remodeling. Elevated TMAO levels are associated with osteoporosis, particularly in the context of estrogen deficiency, where it promotes bone loss by inhibiting mineral acquisition and osteogenic differentiation while enhancing osteoclast activity (11, 12).Short-chain fatty acids (SCFAs), also products of microbial fermentation, play a beneficial role in bone homeostasis by serving as an energy source for osteoblasts and positively influencing bone mineral density. Altered SCFA profiles have been documented in postmenopausal osteoporotic patients, underscoring their link with gut microbiota composition(24\u0026ndash;26).\u003c/p\u003e \u003cp\u003eTo determine whether curcumin-induced microbial changes influence these metabolites, we performed targeted metabolomic analyses of TMAO-related metabolites and SCFAs. Curcumin treatment significantly reduced the abundance of TMAO and its precursors, including choline hydroxide (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), creatinine (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and betaine (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A decreasing trend was also observed for L-carnitine, though it did not reach statistical significance (p\u0026thinsp;=\u0026thinsp;0.3741; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;D). These results suggest that curcumin remodels the gut microbiota, leading to suppressed production of TMAO and related metabolites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, no significant differences were detected in the levels of major SCFAs\u0026mdash;such as acetate, propionate, and butyrate\u0026mdash;between the Cur and OVX groups .\u003c/p\u003e \u003cp\u003eTo further elucidate the relationship between microbiota composition and TMAO metabolism, we conducted an integrated microbiome\u0026ndash;metabolome analysis using Pearson correlation. At the genus level, five differentially abundant bacteria showed correlations with TMAO and its metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eE\u0026ndash;F). Among these, four genera\u0026mdash;Alloprevotella,Defluviitaleaceae_UCG-011,Quinella, and an unidentified Muribaculaceae\u0026mdash;were negatively correlated with TMAO levels. In contrast, Akkermansia exhibited a positive correlation.A metabolite\u0026ndash;bacteria interaction network was constructed based on correlation coefficients to visualize key nodes and potential regulatory relationships within the microbiota\u0026ndash;metabolite axis. These findings partially elucidate the microbial origins of TMAO and provide a basis for further mechanistic investigation into how curcumin modulates gut microbiota to influence bone metabolism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Curcumin Treatment Attenuates Systemic Inflammation in OVX Rats by Reducing Pro-Inflammatory Cytokines\u003c/h2\u003e \u003cp\u003ePostmenopausal osteoporosis is a systemic disorder influenced not only by estrogen deficiency and gut microbiota dysbiosis but also by chronic low-grade inflammation. Elevated levels of pro-inflammatory cytokines\u0026mdash;such as IL-1, IL-6, and G-CSF\u0026mdash;following menopause contribute to impaired bone formation and enhanced osteoclastogenesis (27). Furthermore, TMAO has been shown to promote vascular inflammation and increase circulating pro-inflammatory cytokines (28).\u003c/p\u003e \u003cp\u003eTo evaluate the anti-inflammatory effects of curcumin, we measured serum levels of IL-1, IL-6, and G-CSF using ELISA. Curcumin treatment significantly reduced the concentration of IL-1 compared to the OVX group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant differences were observed in IL-6 or G-CSF levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;C). Notably, elevated inflammatory cytokine levels were detected in both antibiotic-treated groups (ABX and ABX\u0026thinsp;+\u0026thinsp;Cur), likely resulting from antibiotic-induced dysbiosis and gastrointestinal disturbance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIL-1 is a potent immunomodulatory cytokine often referred to as an \u0026ldquo;osteoclast-activating factor\u0026rdquo; and is strongly associated with the pathogenesis of postmenopausal osteoporosis (29). The observed reduction in IL-1 suggests that curcumin may mitigate bone loss partly through suppression of systemic inflammation. Although no significant changes in IL-6 or G-CSF were detected, the decrease in IL-1 represents a promising anti-inflammatory mechanism. This effect may be indirectly mediated through curcumin\u0026rsquo;s modulation of TMAO production and gut microbial composition, ultimately contributing to improved bone outcomes in OVX rats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Fecal Microbiota Transplantation from Curcumin-Treated Rats Partially Rescues Bone Microarchitecture\u003c/h2\u003e \u003cp\u003eBased on our findings that the osteoprotective effects of curcumin are gut microbiota-dependent, we further investigated whether fecal microbiota transplantation (FMT) from curcumin-treated donors could attenuate bone loss in ovariectomized (OVX) recipients. Gut microbial dysbiosis is implicated in the pathogenesis of multiple diseases, and FMT from healthy donors has been shown to improve bone mass in OVX rats by remodeling the gut microbial community (30).\u003c/p\u003e \u003cp\u003eTo test this hypothesis, we conducted a second animal experiment in which microbiota-depleted OVX rats received daily FMT from one of two donor groups: FMT(OVX) (receiving microbiota from OVX rats) and FMT(Cur) (receiving microbiota from curcumin-treated OVX rats). Following the 12-week transplantation period, micro-CT analysis revealed that the FMT(Cur) group exhibited significantly greater trabecular thickness (Tb.Th) and bone mineral density (BMD) compared to the FMT(OVX) group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, no significant differences were observed in other trabecular parameters such as bone volume fraction (BV/TV) or trabecular number (Tb.N) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe partial recovery of bone microarchitectural parameters suggests that microbiota derived from curcumin-treated rats possesses protective capacity against OVX-induced bone loss. The limited effect observed may be attributable to technical limitations of FMT, including potential microbial loss during transplant preparation or incomplete engraftment. Nevertheless, these results provide further evidence that the anti-osteoporotic effect of curcumin is mediated, at least in part, through structural and functional modifications of the gut microbiota.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePostmenopausal osteoporosis, primarily driven by the decline in estrogen levels following ovarian dysfunction, poses a significant global health burden, affecting over 200\u0026nbsp;million women worldwide and accounting for nearly one-third of fracture risks in women over 50 years of age (31, 32). The ovariectomized (OVX) rat model, which faithfully recapitulates key features of human postmenopausal osteoporosis\u0026mdash;including estrogen deficiency and gut microbial dysbiosis\u0026mdash;serves as a validated experimental system for investigating microbiota-targeted therapeutic interventions (21).\u003c/p\u003e \u003cp\u003eThe central paradox in curcumin research lies in the disconnect between its potent in vitro bioactivity and its negligible systemic bioavailability. Our study resolves this paradox by demonstrating that curcumin's in vivo osteoprotective effects are not dependent on its systemic circulation, but are instead mediated entirely through its local action on the gut microbiota. This represents a significant shift in understanding, positioning curcumin not as a failed systemic drug, but as a highly effective, locally-acting gut microbiota modulator.\u003c/p\u003e \u003cp\u003eThe most compelling evidence for this gut-dependent mechanism comes from our dual-approach validation. The complete abolition of curcumin's benefits in pseudo-germ-free rats unequivocally establishes the gut microbiota as a necessary component. More importantly, the partial transfer of these osteoprotective effects via FMT provides direct, causal evidence that the curcumin-modified microbiota is, in itself, sufficient to confer a therapeutic benefit. This FMT approach moves beyond correlation and firmly establishes a causal role for the microbiota in mediating curcumin's effects on bone.\u003c/p\u003e \u003cp\u003ePatients with postmenopausal osteoporosis exhibit reduced gut microbial diversity, and animal studies corroborate that microbial composition influences bone strength (33). The gut\u0026ndash;bone axis modulates bone homeostasis through multiple mechanisms, including short-chain fatty acid production, mineral absorption, immune regulation, and endocrine signaling(34). Our 16S rRNA sequencing revealed that curcumin significantly altered the abundance of key taxa, including Firmicutes, Clostridia, Bacteroidia, and Oscillospirales, and enhanced both alpha and beta diversity, indicating a reversal of OVX-induced dysbiosis.Our findings challenge the traditional drug development paradigm that prioritizes oral bioavailability above all else. For a growing class of compounds like curcumin, low systemic exposure may not be a limitation but rather an indication of a different mode of action\u0026mdash;one that operates within the gut ecosystem. This redefines curcumin from a \"poor drug\" to a pioneering \"microbiota-therapeutic.\" The clinical implication is profound: rather than futilely attempting to improve curcumin's absorption, a more effective strategy may be to leverage its gut-restricted action, potentially by developing advanced delivery systems that target its release to the colon.\u003c/p\u003e \u003cp\u003eElevated circulating TMAO has been associated with inflammatory bone loss and represents a potential biomarker for osteoporosis(35, 36). Curcumin treatment significantly reduced fecal TMAO and its precursors, which correlated with decreased systemic inflammation, as evidenced by lower IL-1 levels. These findings suggest that curcumin attenuates osteoporosis partly through suppressing TMAO-induced inflammation.Mechanistically, we identified a specific gut-bone signaling axis centered on TMAO. Curcumin robustly remodeled the gut ecosystem, enriching for beneficial taxa and suppressing putative TMA-producers, which in turn led to a marked reduction in TMAO levels. The correlation between these microbial shifts, lower TMAO, and decreased systemic IL-1 illuminates a clear pathway: curcumin alleviates the chronic inflammatory state of estrogen deficiency by suppressing a gut-derived pro-osteoclastogenic signal. It is noteworthy that this effect was specific to the TMAO-inflammation axis, as SCFA levels remained unchanged, distinguishing curcumin's mechanism from other microbiota-targeting therapies.\u003c/p\u003e \u003cp\u003eA limitation of our study is the partial bone rescue by FMT, which may reflect technical challenges in microbial engraftment or the involvement of other non-transferable factors. Furthermore, while we established a strong association, future studies using TMAO supplementation or specific inhibition of TMA-lyases are needed to cement the causal role of TMAO in this pathway.\u003c/p\u003e \u003cp\u003eIn summary, we have elucidated a previously unrecognized therapeutic pathway for curcumin that bypasses the classical pharmacokinetic constraints. By acting as a gut microbiota modulator, curcumin suppresses the production of TMAO, thereby dampening systemic inflammation and protecting against bone loss. This work not only provides a novel mechanism for curcumin but also establishes a new strategic framework for developing microbiota-targeted therapies for osteoporosis.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eAlthough curcumin itself suffers from poor absorption, its potent modulation of the gut microbiota makes it a promising candidate for the treatment of osteoporosis. Innovative delivery systems\u0026mdash;such as bone-targeted nanoparticles\u0026mdash;may further enhance its efficacy by improving bioavailability and tissue-specific distribution(37).\u003c/p\u003e \u003cp\u003eIn conclusion, this study fundamentally redefines the mechanism of action of curcumin in osteoporosis. We demonstrate that its osteoprotective efficacy is entirely dependent on its capacity to remodel the gut microbiota, a property that renders its low systemic bioavailability irrelevant to its therapeutic function. The causal role of the microbiota was conclusively shown through depletion and transplantation experiments. The downstream mechanism involves the suppression of the gut-derived metabolite TMAO and a key pro-inflammatory cytokine, IL-1, creating an environment less conducive to bone resorption. These findings pivot the perspective on curcumin from a challenging systemic drug candidate to a potent gut-centric therapeutic. They underscore the \"gut-bone\" axis as a fertile ground for intervention and advocate for a paradigm shift in evaluating natural products, where local gut effects may be more therapeutically relevant than systemic exposure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors thank the respective editors and professional reviewers for their efforts on improving this manuscript during the peer review process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXin Hu: conceptualization, methodology, formal analysis, data curation, writing\u0026mdash;original draft, writing\u0026mdash;review \u0026amp; editing;\u003cbr\u003e\u0026nbsp;Yuchen Tang: conceptualization, methodology, formal analysis, data curation, writing\u0026mdash;original draft, writing\u0026mdash;review \u0026amp; editing;\u003c/p\u003e\n\u003cp\u003eQiufu Wang: conceptualization, methodology, formal analysis, data curation, writing\u0026mdash;original draft, writing\u0026mdash;review \u0026amp; editing;\u003c/p\u003e\n\u003cp\u003eGuanyin Jiang: writing\u0026mdash;review \u0026amp; editing;\u003cbr\u003e\u0026nbsp;Miao Lei: writing\u0026mdash;review \u0026amp; editing;\u003c/p\u003e\n\u003cp\u003eWen Dong: review \u0026amp; editing;\u003cbr\u003e\u0026nbsp;Yongle Wu: conceptualization, data curation, methodology, writing\u0026mdash;review \u0026amp; editing, supervision;\u003cbr\u003e\u0026nbsp;Jie Hao: conceptualization, methodology, funding acquisition, writing\u0026mdash;review \u0026amp; editing, supervision;\u003cbr\u003e\u0026nbsp;Zhenming Hu: conceptualization, methodology, writing\u0026mdash;review \u0026amp; editing, supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Special Project for the Discipline Summit Program of the First Clinical College, Chongqing Medical University\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(No. CYYY-XKDFJH-DSTD-202404).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eThe data that support the findings of this study are openly available in Science Data Bank at https://doi.org/10.57760/sciencedb.33829.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures to be employed in the project was approved by Institutional Animal Care and Use of Chongqing Medical University(IACUS-CQMU)\u003c/p\u003e\n\u003cp\u003eApproval number:IACUC-CQMU-2025-0656\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors consented for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Orthopedics, University-Town Hospital of Chongqing Medical\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUniversity, Daxuecheng Middle Road No. 55, Shapingba District, Chong\u003c/p\u003e\n\u003cp\u003eqing 401331, China. Department of Orthopedics, The First Affiliated Hospital\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eof Chongqing Medical University, Yuanjiagang Youyi Road No. 1, Yuzhong\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDistrict, Chongqing 400010, China.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLane JM, Russell L, Khan SN. Osteoporosis. Clin Orthop Relat Res. 2000(372):139\u0026ndash;50.\u003c/li\u003e\n \u003cli\u003eZhong Z, Qian Z, Zhang X, Chen F, Ni S, Kang Z, et al. Tetrandrine Prevents Bone Loss in Ovariectomized Mice by Inhibiting RANKL-Induced Osteoclastogenesis. Front Pharmacol. 2019;10:1530.\u003c/li\u003e\n \u003cli\u003eWu D, Cline-Smith A, Shashkova E, Perla A, Katyal A, Aurora R. T-Cell Mediated Inflammation in Postmenopausal Osteoporosis. Front Immunol. 2021;12:687551.\u003c/li\u003e\n \u003cli\u003eTang Y, Lv XL, Bao YZ, Wang JR. 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Bone. 2020;135:115317.\u003c/li\u003e\n \u003cli\u003eThomas MS, Fernandez ML. Trimethylamine N-Oxide (TMAO), Diet and Cardiovascular Disease. Curr Atheroscler Rep. 2021;23(4):12.\u003c/li\u003e\n \u003cli\u003eLi L, An J, Bai J, Zhang Y, Li X, Lv H. Association between systemic immune-inflammation index and trimethylamine N-oxide levels in peripheral blood and osteoporosis in overweight and obese patients. Front Endocrinol (Lausanne). 2025;16:1539594.\u003c/li\u003e\n \u003cli\u003eLi Y, Cai Z, Ma W, Bai L, Luo E, Lin Y. A DNA tetrahedron-based ferroptosis-suppressing nanoparticle: superior delivery of curcumin and alleviation of diabetic osteoporosis. Bone Res. 2024;12(1):14.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Curcumin, Postmenopausal osteoporosis, Gut microbiota, TMAO, Ovariectomized rat, Inflammation, Bone mineral density","lastPublishedDoi":"10.21203/rs.3.rs-9218781/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9218781/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite its poor oral bioavailability, curcumin demonstrates efficacy against postmenopausal osteoporosis, presenting a paradox between its pharmacokinetics and pharmacological effects. This study elucidates a gut microbiota-dependent mechanism underlying this phenomenon. In ovariectomized rats, curcumin improved bone mass and microstructure, effects that were abolished upon gut microbiota depletion but partially transferable via fecal microbiota transplantation. Mechanistically, curcumin reversed estrogen deficiency-induced gut dysbiosis, enhanced microbial diversity, and significantly reduced levels of the gut-derived metabolite trimethylamine-N-oxide (TMAO) and the pro-inflammatory cytokine IL-1. Our findings establish that curcumin exerts its osteoprotective effects indirectly by remodeling the gut microbiome and its metabolic output, thereby proposing a new paradigm for its therapeutic action\u0026mdash;not as a systemically available drug, but as a targeted gut microbiota modulator.\u003c/p\u003e","manuscriptTitle":"Curcumin Ameliorates Osteoporosis via Gut Microbiota-Dependent Modulation of TMAO in Ovariectomized Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 14:30:37","doi":"10.21203/rs.3.rs-9218781/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T06:50:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T18:42:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-11T13:25:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172088133182351266763393133331423583743","date":"2026-04-09T09:20:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"80388000201179707076869199453243838147","date":"2026-04-09T08:42:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233284025183834895143654486504650203797","date":"2026-04-07T16:31:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T03:40:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T03:33:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-01T05:42:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T07:23:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-31T04:47:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"80be9078-56dd-4b68-9856-16e894307c2f","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66159246,"name":"Health sciences/Diseases"},{"id":66159247,"name":"Biological sciences/Drug discovery"},{"id":66159248,"name":"Health sciences/Gastroenterology"},{"id":66159249,"name":"Health sciences/Medical research"},{"id":66159250,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-12T03:38:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 14:30:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9218781","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9218781","identity":"rs-9218781","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}