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Irisin mitigates osteoporotic-associated bone loss and gut dysbiosis in ovariectomized mice by modulating microbiota, metabolites, and intestinal barrier integrity

BMC Musculoskeletal Disorders volume 26, Article number: 374 (2025) Cite this article

Abstract

Background

Osteoporotic bone defects significantly affect patient health and quality of life. The gut-bone axis plays a crucial role in osteoporosis, and disruptions in gut microbiota are linked to systemic inflammation and compromised bone metabolism. Irisin, a myokine, has shown potential in protecting against osteoporosis, but its mechanisms of action on the gut-bone axis remain unclear. This study aimed to investigate the role of irisin in mitigating osteoporotic bone defects by examining its effects on gut microbiota, related metabolites, and intestinal barrier integrity.

Methods

An osteoporosis model was created using ovariectomized (OVX) mice. The mice were divided into Sham, OVX, and r-irisin groups. Mice in the r-irisin group received intraperitoneal injections of 100 μg/kg irisin twice weekly for five weeks. Bone parameters were analyzed by micro-CT and histological staining. Gut microbiota composition was examined via 16S rDNA sequencing. Intestinal cytokines and barrier proteins were measured using immunohistochemistry and ELISA. Fecal metabolomic profiling was conducted using liquid chromatography-tandem mass spectrometry (LC–MS/MS), and correlations between gut microbiota, metabolites, and bone metabolism markers were evaluated.

Results

Irisin treatment improved bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular bone thickness (Tb.Th), and trabecular number (Tb.N), and reduced trabecular separation (Tb.Sp) in OVX mice. It enhanced new bone formation and collagen deposition. Irisin restored intestinal barrier integrity by increasing tight junction protein expression and reducing inflammatory cytokines in intestinal tissues. It also modulated gut microbiota diversity, reducing Firmicutes and increasing Verrucomicrobiota abundance. Key fecal metabolites, including atractylon (r = − 0.60, P < 0.01) and enterodiol (r = + 0.83, P < 0.01), showed strong correlations with BMD.

Conclusion

Irisin mitigates osteoporotic bone defects by enhancing bone formation, restoring intestinal barrier integrity, modulating gut microbiota composition, and influencing fecal metabolites. These preclinical findings highlight irisin’s potential to mitigate osteoporosis via the gut-bone axis.

Peer Review reports

Background

Osteoporotic bone defects, which can affect various skeletal regions such as the spine, legs, arms, ribs, or skull, significantly impair patient health and quality of life [1, 2]. These defects often lead to chronic pain, disability, and increased mortality, particularly in older populations [3]. With an aging demographic, the prevalence of osteoporotic fractures in China has risen sharply, increasing from 13.2% (2000–2010) to 22.7% (2012–2022), with higher prevalence in women than in men [4, 5], imposing a substantial burden on healthcare systems [6, 7]. Despite advancements in treatment options—ranging from conservative pain management to surgical interventions like bone grafts—the challenges of delayed healing and non-union fractures remain prominent [8, 9]. Such complications are particularly severe in elderly patients with comorbidities, underscoring the need for innovative therapeutic approaches [10].

The gut-bone axis plays a crucial role in osteoporosis, where gut microbiota dysbiosis exacerbates systemic inflammation and bone loss [11,12,13]. Mechanistically, the microbiome influences skeletal health via nutrient absorption, immune regulation, and intestinal barrier integrity [14,15,16]. Microbiota-hormone interactions (estrogen, PTH, serotonin) modulate osteoblast-osteoclast coupling [17, 18], while microbiota-independent hormones (insulin, glucagon-like peptide 1, etc.) directly influence osteoclasts [19, 20]. Although preclinical models strongly support this axis, human studies show variability, with some cohorts reporting minimal microbiota-bone associations [21, 22]. Additionally, emerging evidence highlights alternative pathways, such as direct bone-immune signaling, underscoring the complexity of osteoporosis pathogenesis [23]. Dysbiosis (an imbalance in gut microbiota composition) disrupts this gut-bone axis by reducing microbial-derived metabolites critical for bone homeostasis, increasing intestinal permeability, and elevating systemic pro-inflammatory cytokines, thereby exacerbating bone loss and inflammatory responses in OVX mice [24, 25]. Thus, targeting the gut-bone axis—while integrating broader skeletal regulatory systems—may offer therapeutic opportunities for osteoporotic bone defects.

Irisin, a myokine cleaved from fibronectin type III domain-containing protein 5 (FNDC5), has garnered attention for its multifaceted physiological roles [26]. In addition to its well-known effects on adipose tissue browning, glucose metabolism, and inflammation, recent studies suggest that irisin may serve as a protective agent against osteoporosis [27,28,29,30,31]. It has been shown to promote osteoblast differentiation and inhibit osteoclastogenesis via pathways such as Wnt/β-catenin signaling and RANKL/OPG axis modulation [29, 30, 32]. Studies also indicate that irisin enhances bone formation by upregulating RUNX2 and COL1A1 expression in osteoblasts while suppressing NF-κB-mediated inflammatory responses that drive bone resorption [33,34,35]. Furthermore, irisin’s role in improving glucose metabolism and reducing systemic inflammation may indirectly benefit skeletal health by mitigating insulin resistance and oxidative stress, both of which exacerbate bone loss [36].

Irisin’s roles in modulating the composition of gut microbiota and enhancing intestinal barrier function have also been reported [37,38,39,40]. While recent reviews summarize irisin’s role in osteoblast/osteoclast regulation and cell death pathways, its mechanisms via the gut-bone axis remain unexplored [30]. This study hypothesized that irisin mitigates osteoporotic bone defects by restoring intestinal barrier integrity, modulating gut microbiota composition, and altering fecal metabolites, thereby reducing systemic inflammation and improving bone metabolism. We aimed to test this hypothesis in an ovariectomized mouse model by evaluating irisin’s effects on bone parameters, gut microbial diversity, metabolite profiles, and inflammatory markers. By elucidating these mechanisms, this research seeks to advance the understanding of irisin’s role in osteoporosis management.

Methods

Animal experiment

All animal experimental procedures were approved by Laboratory Animal Ethics Committee of Lai’an Technology (Guangzhou) Co., LTD (Approval number: G2024094).

Mouse model and experimental grouping

Two-month-old female C57BL/6 J mice from Guangdong Zhiyuan Biomedical Technology Co., Ltd. were housed under specific pathogen-free conditions with a 12-h light–dark cycle. A sample size of six mice per group (Sham, OVX, r-irisin) was selected based on previous studies in OVX murine models, which demonstrate sufficient power to detect differences in bone phenotypes [41,42,43]. Mice were quarantined for one week before the experiment, provided a standard rodent diet and water ad libitum, and housed in ventilated polycarbonate cages with bedding and enrichment materials. Temperature and humidity were maintained at 22 ± 2 °C and 50 ± 10%, respectively.

Surgical procedures and experimental interventions

Before surgery, mice underwent 12-h water deprivation. Mice were anesthetized with pentobarbital sodium (150 mg/kg) intraperitoneally. The surgical site was shaved and disinfected thrice with iodophor. A 1-cm incision was made on each side to access and remove the ovaries in the OVX and r-irisin groups, while the Sham group underwent the same procedure without ovariectomy. No postoperative analgesia was administered due to its potential to interfere with bone metabolism and gut microbiota [44, 45], consistent with prior studies [46, 47].

One week post-surgery, the r-irisin group received intraperitoneal injections of 100 μg/kg irisin (10μg/mL in saline, HY-P70664, MCE) twice a week for 5 weeks. The Sham and OVX groups received equal volumes of normal saline as negative controls. Mice were monitored daily, with humane euthanasia criteria set at severe distress or > 20% weight loss, though no early euthanasia was necessary.

Outcomes measured and sampling techniques

After five weeks, all mice were euthanized using an overdose of CO2 asphyxiation followed by cervical dislocation. Uteri, feces, small intestine, and femur were collected. Tissues were either stored at -80 °C for molecular analysis or fixed in 4% paraformaldehyde for histology. Measured outcomes included body weight changes, behavioral assessments, and tissue-specific analyses to evaluate the effects of ovariectomy and r-irisin treatment.

16S rDNA sequencing analysis

Fecal sample DNA was extracted using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany). The V3-V4 region was amplified with primers 341F (5-CCTACGGGNGGCWGCAG-3) and 805R (5-GACTACHVGGGTATCTAATCC-3) to construct 16S rDNA gene libraries, which were then sequenced on the Illumina PE250 platform. Sequencing generated an average of 84,000 reads per sample (range: 81,000–87,000), with 68,000–80,000 reads retained per sample after quality filtering.

Bioinformatics analysis was done with QIIME2. Adapter, primer, and low-quality sequences were removed using a Phred score threshold of < 20, and sequences shorter than 200 bp were also filtered out. Chimeras were filtered using Vsearch. Amplicon sequence variants (ASVs) were identified by the DADA2 algorithm. Alpha and beta diversity were analyzed based on ASV data. R and QIIME2 software were used for Chao1 index and principal coordinates analysis (PCoA) generation. Taxonomic annotation relied on the SILVA database (Release 138). Differential abundance analysis used Least Discriminant Analysis Effect Size (LEfSe) and Kruskal–Wallis tests, and the KEGG database explored microbiome functions.

Fecal metabolome analysis

Fecal metabolites from 100 mg samples were extracted with 50% methanol. The extract was processed with a pre-cooled 50% methanol–acetonitrile mix, incubated, centrifuged, and the residue was further treated. The final supernatant was analyzed by high-resolution mass spectrometry using a Waters ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) with a gradient mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), run at 0.3 mL/min. Mass spectrometry was performed on a Thermo Fisher Q Exactive Plus system with electrospray ionization (ESI) in positive and negative modes. Ionspray voltage was set at 3800 V (positive) and -3100 V (negative), with a scan range of m/z 70–1500 and capillary temperature maintained at 320°C.

Microstructure analysis of femur bone

Femur samples were scanned on a Micro CT instrument (NEMO NMC-200) at 10-μm resolution. The 3D reconstruction software Recon was used, and a 1.5-mm r distal femur region was selected as the ROI. Cruiser and Avatar software determined bone parameters including bone mineral density (BMD), bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp).

Histopathology analysis

Femoral and small intestinal tissues were rinsed, fixed in 4% paraformaldehyde, dehydrated, made transparent with xylene, and embedded in paraffin. Sections (4 μm) were prepared, dewaxed, and stained with hematoxylin and eosin (H&E).

For assessing collagen content and muscle fiber changes in bone tissue, Masson’s trichrome staining was done on femoral sections. The slides were fixed in Bouin’s solution, nuclei were stained black with Weigert’s iron hematoxylin, muscle fibers and cytoplasm were stained red with Biebrich scarlet—acid fuchsin. After differentiation with phosphomolybdic/phosphotungstic acid, collagen fibers were stained blue or green using aniline blue or light green.

To examine mineralized and non—mineralized bone components, Goldner’s trichrome staining was performed on undecalcified femoral sections. The slides were fixed in 10% NBF, dehydrated in graded ethanol, embedded in MMA, and then stained. Mineralized bone appeared green and non-mineralized osteoid appeared orange-red.

Immunohistochemistry analysis

Small intestinal sections were dewaxed, rehydrated through graded ethanol, and blocked with 3% BSA. Primary antibodies (anti-Occludin, ab216327; anti-ZO-1, ab221547; anti-Claudin-1, ab307692) (all from abcam, UK) were applied and incubated overnight at 4 °C. After washing, secondary antibodies were added, and DAB chromogen was used for visualization. Sections were counterstained and mounted for microscopy.

Enzyme-linked immunosorbent assay (ELISA)

Serum samples were obtained by centrifuging blood at 3000 rpm for 10 min. Serum levels of bone-forming factors (ALP, BGP, OPG) and bone resorption factors (CTX-1, TRACP-5b) were measured using ELISA kits according to the manufacturers’ instructions: Mouse BALP ELISA Kit (ml037754, mlbio, China), Mouse OC/BGP ELISA Kit (E-EL-M0864, Elabscience, China), Mouse OPG ELISA Kit (F1133, Westang Bio-tech, China), Mouse CTX-I ELISA Kit (E-EL-M3023, Elabscience, China), and Mouse TRACP-5b ELISA Kit (E-EL-M3100, Elabscience, China).

For cytokine analysis in small intestinal tissues, homogenates were prepared by ultrasonic trituration in normal saline and centrifuged at 3000 rpm for 10 min. Levels of IL-1β (EMC001b, Neobioscience, China), IL-6 (98027ES, Yeasen, China), and TNF-α (D721217, Sangon, China) were measured using ELISA kits according to the manufacturers’ instructions.

Statistical analysis

Data analysis was performed using GraphPad Prism 9.0 software. Statistical tests included Student’s t-test and one-way ANOVA followed by Tukey’s test for multiple comparisons, with results presented as mean ± standard deviation (SD). For correlation analysis of the 16S microbiome, metabolome, inflammatory factors, and bone phenotypes, the spearman rank correlation method was used due to non-normally distributed data and/or non-linear relationships between variables. This is conducted through the R package Hmisc, and results were visualized using R version 4.4.2. Statistical significance was defined as P-values < 0.05.

Results

Irisin mitigates ovariectomy-induced bone loss and enhances bone formation

We established an osteoporosis mouse model via ovariectomy (OVX) and treated the mice with irisin. Post-treatment, irisin reduced body weight in OVX mice to levels comparable to the Sham group (P < 0.05 vs. OVX; Fig. 1A). The uterine weight ratio numerically increased in the irisin-treated group compared to OVX (0.24 ± 0.02 vs. 0.16 ± 0.06 g/g body weight), though the difference was not statistically significant (P = 0.09) and remained below Sham levels (0.34 ± 0.06 g/g; Fig. 1A).

Fig. 1
figure 1

Irisin mitigates ovariectomy-induced bone loss and enhances bone formation. A Changes in body weight and uterine weight ratio across groups. Body weight decreased in irisin-treated mice, aligning with Sham levels, while the uterine weight ratio showed a non-significant increase compared to the OVX group but remained lower than Sham. B Micro CT images of femurs showing bone cavity expansion in OVX mice, which was partially reversed by irisin treatment. C Bone parameter analysis: Bone Mineral Density (BMD), Bone Volume/Tissue Volume ratio (BV/TV), trabecular bone thickness (Tb.Th), and trabecular number (Tb.N) were improved with irisin treatment. Trabecular separation (Tb.Sp) decreased in the irisin-treated group. D H&E staining of mouse femur indicated enhanced new bone trabeculae formation in irisin-treated mice. E MASSON staining highlights a denser collagen matrix and newly formed bone tissue in the irisin-treated group. F Goldner staining demonstrates increased osteoblast activity in irisin-treated bone sections compared to OVX mice. G-H Quantification of bone metabolic markers. CTX-1 and TRACP-5b levels (G) were elevated in OVX mice but reduced with irisin treatment. ALP, BGP, and OPG levels (H) were reduced in OVX mice but showed recovery with irisin treatment. Data are presented as mean ± standard deviation (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001 versus OVX group. Sham: Sham-operated mice; OVX: Ovariectomized mice; r-Irisin: Irisin-treated OVX mice

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The OVX group exhibited significant the bone cavity expansion (49% increase vs. Sham, P < 0.05), which was partially reversed with irisin treatment (17% reduction vs. OVX, P < 0.05; Fig. 1B). Bone parameters such as Bone Mineral Density (BMD), Bone Volume/Tissue Volume ratio (BV/TV), trabecular bone thickness (Tb.Th), and trabecular number (Tb.N) were reduced in OVX mice but improved with irisin. In addition, trabecular separation (Tb.Sp) increased in OVX mice but decreased with irisin treatment (Fig. 1C).

Bone staining revealed distinct outcomes. OVX mice showed increased bone cavities, while Hematoxylin and Eosin (H&E) staining indicated enhanced new bone trabeculae formation in irisin-treated mice (Fig. 1D). MASSON staining showed a more developed collagen matrix and newly formed bone tissue in the irisin-treated group (Fig. 1E), and Goldner staining revealed increased osteoblast activity within the bone tissue (Fig. 1F). Compared to Sham mice, whose cavities were filled with mature bone tissue, irisin treatment promoted new bone formation, indicating its role in bone regeneration.

Analysis of bone metabolic markers showed increased levels of bone resorption factors CTX-1 and TRACP-5b in OVX mice, as well as decreased levels of bone-forming factors ALP and BGP, and a bone protective protein OPG. Irisin treatment tended to restore these markers toward Sham group levels, supporting its bone-protective effects (Fig. 1G, H).

Irisin regulates the intestinal barrier and reduces inflammation in ovariectomized mice

H&E staining of small intestinal tissues revealed a reduction in mucosal tissue on the surface of the intestinal folds in the OVX group, which was partially restored in the irisin-treated group (Fig. 2A). Intestinal epithelial cells appeared enlarged in both the OVX and irisin groups, although the enlargement was less pronounced in the latter.

Fig. 2
figure 2

Irisin regulates the intestinal barrier and reduces inflammation in ovariectomized mice. (A) H&E staining of intestinal tissues showing reduced mucosal tissue in the OVX group, which was partially restored in the irisin-treated group. Enlarged intestinal epithelial cells were observed in both OVX and r-irisin groups, with less pronounced enlargement in the latter. B–E Immunohistochemical staining and quantitative average optical density (E) of tight junction proteins Occludin (B), ZO-1 (C), and Claudin-1 (D) showing reduced expression in the OVX group compared to the Sham group, and partial restoration with irisin treatment. F Levels of inflammatory markers IL-1β, IL-6, and TNF-α were elevated in the OVX group and significantly reduced in the irisin-treated group. Data are presented as mean ± standard deviation (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001 versus OVX group. Sham: Sham-operated mice; OVX: Ovariectomized mice; r-Irisin: Irisin-treated OVX mice

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Immunohistochemical analysis showed that the expression levels of the tight junction proteins Occludin (Fig. 2B), ZO-1 (Fig. 2C), and Claudin-1 (Fig. 2D) was highest in the Sham group, significantly reduced in the OVX group, and partially restored with irisin treatment (Fig. 2E). Structural observations further indicated that intestines in the OVX group were larger, with looser cellular organization and diminished villi structure. These structural changes were partially reversed following irisin administration.

Analysis of inflammatory markers revealed IL-1β, IL-6, and TNF-α levels in the OVX group were 1.3-, 1.4-, and 2.1-fold higher than Sham (P < 0.05), respectively. Irisin treatment significantly reduced these levels to 1.0-, 1.1-, and 1.4-fold of Sham values (P < 0.05 vs. OVX), suggesting its role in mitigating inflammation (Fig. 2F).

Effect of irisin on gut microbiota in ovariectomized mice and correlation with inflammatory factors

The 16S rDNA sequencing analysis revealed alterations in the microbial community among the groups. Specifically, differences in the Chao1 index indicated variations in alpha diversity between the OVX and irisin-treated groups (Fig. 3A). Additionally, PCoA analysis demonstrated distinct clustering patterns among the groups (Fig. 3B). At the phylum level, irisin treatment significantly reduced Firmicutes abundance by 14% compared to OVX (P < 0.05, Kruskal–Wallis test) and restored Verrucomicrobiota to 28% of Sham levels (P < 0.05), counteracting OVX-induced shifts (Fig. 3C). However, the upregulated Bacteroidota remained elevated even with irisin treatment, suggesting a context-dependent role in polysaccharide use of irisin (Fig. 3C) [48]. LEfSe analysis further compared the bacterial community characteristics across the different groups (Fig. 3D). At the genus level, Desulfovibrio, Bifidobacterium, unclassified Clostridia_UCG-014, Turicibacter, and Alloprevotella were identified as the most differentially associated taxa among the three groups (Fig. 3D).

Fig. 3
figure 3

Irisin altered microbiota composition in ovariectomized mice. A Alpha‐diversity analysis based on numbers of features. B Beta-diversity index analysis based on Jaccard dissimilarities (PCoA). C Relative abundances of gut bacteria at the phylum level. D LEfSe analysis showed comparison of bacterial community characteristics among different groups. E PICRUSt2 findings with significant differences among different groups. F Spearman correlation analysis between different gut microbiota (at the genus) and inflammatory factors. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Sham: Sham-operated mice; OVX: Ovariectomized mice; r-Irisin: Irisin-treated OVX mice

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PICRUSt2 analysis highlighted pathways potentially linked to systemic inflammation and metabolic regulation, which are known to intersect with bone health. These included niacin/niacinamide metabolism (implicated in energy homeostasis and osteoblast function [49]), cofactor/vitamin metabolism (e.g., vitamin K, critical for osteocalcin carboxylation [50]), and ascorbate metabolism (required for collagen biosynthesis [51]). While mismatch repair and Vibrio cholerae pathogenicity pathways are not directly associated with bone, their enrichment may reflect microbial responses to host inflammatory stress or DNA damage, which could indirectly influence bone remodeling [52]. These findings suggest gut microbiota may modulate osteoporosis via interconnected metabolic and inflammatory axes (Fig. 3E). 

Spearman correlation analysis was conducted to examine the relationship between different gut microbiota (at the genus level) and inflammatory factors (Fig. 3F). Notably, changes in the levels of inflammatory factors TNF-α, IL-6, and IL-1β were positively correlated with Bifidobacterium, Candidatus Saccharimionas, and unclassified Erysipelatoclostridiaceae. In contrast, the level of IL-6 was negatively correlated with Turicibacter. These findings underscore the role of gut microbiota in modulating intestinal inflammation.

Effect of irisin on fecal metabolites in ovariectomized mice and association with gut microbiota

To elucidate the effects of irisin-induced gut microbiome changes on bone health in ovariectomized mice, we examined fecal metabolites using liquid chromatography-tandem mass spectrometry. Metabolic profiling of mice feces was conducted using PCA and Partial Least Squares Discriminant Analysis (PLS-DA), which uncovered distinct metabolite profiles across different groups (FIG. 4A-B). Compared to the OVX group, the irisin-treated group displayed 173 increased and 46 decreased metabolites (Fig. 4C). Among them, the top 25 differentially expressed metabolites included 12,13-EpOME, isopilocarpine, dipentyl phthalate, and enterodiol (Fig. 4D). Functional annotation highlighted key compounds linked to bone metabolism and inflammation: 12,13-EpOME (an anti-inflammatory eicosanoid [53]), enterodiol (a lignan metabolite that enhances osteoblast differentiation [54]), and dipentyl phthalate (a member of phthalate ester that relate to systemic inflammation [55, 56]).

Fig. 4
figure 4

Effect of irisin on fecal metabolites in ovariectomized mice and association with gut microbiota. (A) PCA. (B) PLS-DA. C Volcano plot of fecal metabolites between OVX and irisin-treated mice. D Heatmap displaying the top 25 fecal metabolites differently expressed among groups. Different colors indicate different metabolite expressions. E Association between markedly distinct gut microbiome (at genus) and significantly modified fecal metabolites. (*P < 0.05, **P < 0.01, ***P < 0.001). Sham: Sham-operated mice; OVX: Ovariectomized mice; r-Irisin: Irisin-treated OVX mice

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To explore the relationship between specific microbes and differential metabolites, we conducted correlation analyses to identify coordinated changes between differentially expressed microbiomes and metabolites. With the criteria of |r|> 0.50 and P < 0.05, we detected 125 pairs of significantly correlated microbiota-metabolite pairs (P < 0.05), of which 76 pairs had more significant correlations (P < 0.01), and 48 pairs had correlations with a significance level of P < 0.001 (Fig. 4E). Notably, the irisin treatment-induced specific enterobacterial genera Bifidobacterium, Candidatus Saccharimonas, and unclassified Erysipelatoclostridiaceae were closely correlated with most of the differential metabolites (Fig. 4E).

Based on these results, we hypothesize that the modulation of key metabolite levels by specific gut microbiota may influence relevant metabolic pathways, potentially elucidating the therapeutic mechanism underlying irisin’s action.

Correlative analysis of bone metabolic factors with differential microbiology and metabolites

As Fig. 5A shows, bone protective factors (OPG, BGP, ALP, BV/TV, BMD, Tb.Th, Tb.N) were negatively correlated with differential microbiota, particularly Bifidobacterium (Spearman’s r = -0.74 to -0.60, P < 0.05), Candidatus Saccharimonas (r = -0.57 to -0.80, P < 0.05), and unclassified Erysipelatoclostridiaceae (r = -0.50 to -0.73, P < 0.05). Conversely, bone resorption factors (CTX-1, TRACP-5b) and Tb.Sp were positively correlated with these taxa (r = 0.50 to 0.81, P < 0.05).

Fig. 5
figure 5

Correlative analysis of bone factors with differential microbiology and metabolites. A The correlation between differential gut microbiota (at the genus) and bone factors. B The correlation between significantly altered fecal metabolites and bone factors. C The correlation between significantly altered fecal metabolites and inflammatory factors. *P < 0.05, **P < 0.01, ***P < 0.001

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Most differential fecal metabolites (e.g., korseveriline, dipentyl phthalate, isopilocarpine) were negatively correlated with bone protective factors (r = -0.52 to -0.89, P < 0.05) and positively correlated with bone resorption markers (r = 0.50 to 0.85, P < 0.05; Fig. 5B). Inflammatory cytokines (TNF-α, IL-6, IL-1β) showed positive correlations with metabolites like isopilocarpine (r = 0.64 to 0.79, P < 0.01) and atractylon (r = 0.58 to 0.79, P < 0.05), while negatively correlating with enterodiol (r = -0.63 to -0.43, P < 0.05; Fig. 5C).

These results indicate that irisin-induced gut microbiota and metabolite alterations are closely linked to bone defect repair.

Discussion

This study demonstrates that irisin administration effectively mitigates ovariectomy (OVX)-induced bone loss and enhances bone formation through a multifaceted mechanism involving the gut-bone axis. These findings provide valuable insights into irisin’s therapeutic potential for osteoporosis.

Irisin treatment was associated with improvements in BMD, BV/TV, Tb.Th, and Tb.N, suggesting anabolic effects on bone. The concurrent reduction in Tb.Sp further supports its role in preserving trabecular architecture. These results align with prior studies demonstrating irisin’s capacity to promote osteoblast differentiation and inhibit osteoclastogenesis [30, 32, 57]. While histological evidence of enhanced collagen deposition and osteoblast activity reinforces its bone-protective potential, the direct mechanisms (e.g., receptor interactions, downstream signaling) remain to be fully elucidated in this model.

Beyond its direct effects on bone, irisin’s regulation of the gut microbiota emerges as a critical mechanism underlying its beneficial effects in this model. The reversal of OVX-induced dysbiosis, particularly the normalization of Firmicutes and Verrucomicrobiota abundances, suggests that irisin restores microbial balance. Furthermore, the correlation between specific bacterial genera which involve in bone metabolism, such as Bifidobacterium and Turicibacter, and improved bone parameters highlights the gut microbiota’s role in mediating irisin’s effects [58,59,60]. Differentially expressed metabolites identified in the fecal metabolome analysis further support the hypothesis of a microbiota-metabolite-bone axis. Metabolites such as atractylon and enterodiol, which are implicated in inflammatory regulation and/or bone metabolism, were significantly modulated by irisin treatment [61,62,63]. The irisin-induced increase in Verrucomicrobiota and reduction in 12,13-EpOME may synergistically suppress NF-κB signaling, thereby reducing osteoclastogenesis. Future studies using knockout models or in vitro assays could validate potential pathways.

While our study identifies correlations between irisin-induced changes in Bifidobacterium and Turicibacter and improved bone parameters, the exact mechanisms warrant further investigation. Bifidobacterium species are known producers of short-chain fatty acids (SCFAs), such as acetate and butyrate, which enhance intestinal barrier function and suppress osteoclastogenesis by downregulating pro-inflammatory cytokines, such as TNF-α and IL-6 [58, 64]. Regarding Turicibacter, our findings show that its abundance was significantly downregulated in the OVX group compared to the sham group, consistent with previous research demonstrating associations between reduced Turicibacter levels and osteoporotic conditions in ovariectomized mouse models [60]. Notably, irisin treatment led to a slight but measurable recovery of Turicibacter levels toward baseline, coinciding with improved bone parameters. This pattern suggests a complex relationship between Turicibacter and bone health that may be context-dependent. Some studies have reported positive correlations between Turicibacter abundance and bone mineral density following certain treatments or in specific microbiome configurations[65, 66]. These seemingly contradictory findings may reflect the complex interplay between Turicibacter, host metabolism, and immune regulation in different physiological states. Future studies using germ-free models or fecal microbiota transplantation could directly test the causative role of these taxa in irisin’s bone-protective effects.

The restoration of tight junction protein expression (Occludin, ZO-1, and Claudin-1) and the reduction of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) indicate that irisin enhances intestinal barrier integrity while mitigating systemic inflammation. Given the known links between chronic inflammation, intestinal permeability, and bone loss, these findings suggest that irisin’s anti-inflammatory effects contribute significantly to its bone-protective properties [67,68,69,70]. Irisin’s restoration of intestinal barrier integrity may reduce systemic endotoxin exposure, such as lipopolysaccharide (LPS), which is known to trigger bone resorption via TLR4/NF-κB signaling [71]. By upregulating tight junction proteins (Occludin, ZO-1, Claudin-1), irisin could limit LPS translocation, thereby suppressing osteoclast activation. Additionally, fecal metabolomic changes, such as increased enterodiol (a phytoestrogen metabolite with anti-osteoporotic properties [72]), may reflect microbiota-driven shifts in bioactive compounds that directly target bone cells. The interplay between irisin, gut-derived metabolites, and osteoimmune regulation represents a promising area for future research. By improving intestinal barrier function, irisin may prevent the translocation of endotoxins that trigger systemic inflammation, thereby breaking the cycle of inflammation-induced bone resorption.

Unlike previous studies focused on irisin’s direct effects on bone cells or systemic inflammation [30], our work uncovers its role in modulating gut microbiota diversity and restoring intestinal barrier function. These findings establish irisin as a multi-target agent bridging gut health and bone metabolism, offering a novel therapeutic paradigm for osteoporosis. However, several limitations warrant consideration. While our sample size (n = 6) met statistical power requirements, future studies with larger cohorts may further validate these findings. The study’s focus on short-term effects necessitates long-term investigations to evaluate the sustainability of irisin’s benefits and safety. The study lacks a sham-irisin control group, precluding differentiation between surgical stress and irisin-specific effects. Additionally, the specific receptors mediating irisin’s effects and its safety profile in clinical settings remain to be elucidated. Future research should explore irisin’s interactions with existing osteoporosis therapies and its potential in personalized medicine approaches targeting the gut-bone axis.

Conclusions

These findings suggest that irisin may mitigate OVX-induced bone defects by enhancing bone formation, restoring intestinal barrier integrity, and modulating gut microbiota composition. The observed suppression of inflammatory cytokines (IL-1β, IL-6, TNF-α) and restoration of bone anabolic markers (ALP, BGP) highlight a dual role for irisin, though direct mechanistic links remain to be established. Notably, this study identifies the gut-bone axis as a critical mediator of irisin’s effects, offering a novel framework for osteoporosis intervention. However, further studies are required to confirm causal relationships between specific microbial taxa, metabolites, and bone outcomes. Long-term preclinical and clinical trials are also needed to validate irisin’s therapeutic potential and safety profile in humans. By targeting interconnected pathways of the gut-bone axis, irisin may represent a mechanistic foundation for future holistic approaches to managing osteoporotic bone defects and associated systemic effects.

Data availability

The 16S rDNA sequencing data generated in this study have been deposited in the SRA database of National Center for Biotechnology Information (NCBI) under the accession number PRJNA1210833. The fecal metabolomics sequencing data are available in the Metabolights repository with the accession number MTBLS11968. All other raw data are available from the corresponding author on reasonable request.

Abbreviations

ALP:

: Alkaline phosphatase

BGP:

: Bone Gla protein

BMD:

: Bone mineral density

BV/TV:

: Bone volume/tissue volume ratio

CTX-1:

: C-terminal telopeptide of type I collagen

IL-1β:

: Interleukin-1β

IL-6:

: Interleukin-6

NF-κB:

: Nuclear factor kappa-light-chain-enhancer of activated B cells

OPG:

: Osteoprotegerin

OVX:

: Ovariectomized

PCoA:

: Principal coordinates analysis

PCA:

: Principal component analysis

PLS-DA:

: Partial Least Squares Discriminant Analysis

Tb.N:

: Trabecular number

Tb.Sp:

: Trabecular separation

Tb.Th:

: Trabecular thickness

TNF-α:

: Tumor necrosis factor-alpha

TRACP-5b:

: Tartrate-resistant acid phosphatase 5b

ZO-1:

: Tight junction protein 1

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  1. Yiran Wang and Huimin Deng contributed equally to this work.

Authors and Affiliations

  1. Department of Traumatology and Orthopaedic Surgery, Orthopaedic Institute, Huizhou Central People’s Hospital, Huizhou, Guangdong, 516001, China

    Yiran Wang, Zhihui Zhang, Hongbo Wu & Xiaofeng Wang

  2. Department of Gastroenterology, Huizhou Central People’s Hospital, Huizhou, Guangdong, 516001, China

    Huimin Deng

  3. Department of Traumatology and Orthopaedic Surgery, Orthopaedic Institute, Huizhou Central People’s Hospital, No. 60, East Second Ring South Road, Huizhou, Guangdong, 516001, China

    Zhiwen Zhang

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Contributions

Y.W. and H.D. designed, performed the experiments, and drafted the manuscript. Z.H.Z. and H.W. performed data analysis. X.W. carried out visualization. Z.W.Z. designed the study and revised the manuscript. All authors have read and approved the manuscript.

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Correspondence to Zhiwen Zhang.

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Wang, Y., Deng, H., Zhang, Z. et al. Irisin mitigates osteoporotic-associated bone loss and gut dysbiosis in ovariectomized mice by modulating microbiota, metabolites, and intestinal barrier integrity. BMC Musculoskelet Disord 26, 374 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12891-025-08622-y

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Keywords

BMC Musculoskeletal Disorders

ISSN: 1471-2474

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