Journal: iScience

Article Title: Exercise is associated with attenuated aging-related osteoporosis through TMAO alpha Klotho inflammasome signaling

doi: 10.1016/j.isci.2026.117170

Figure Lengend Snippet: Higher habitual physical activity is associated with higher hip BMD, lower TMAO, a formation-favorable remodeling profile, and reduced inflammation in older adults (A) Flowchart of the clinical study design, illustrating participant enrollment, exclusion criteria, grouping according to habitual physical activity level, and outcome assessments. Participants were categorized into a higher activity group and a lower activity group based on self-reported habitual physical activity. (B–D) Comparison of bone mineral density T-scores at the (B) lumbar spine (L1–L4), (C) femoral neck, and (D) total hip. (E and F) TMAO concentrations measured in (E) feces and (F) serum. (G and H) Serum bone turnover markers showing (G) PINP and (H) CTX levels. (I–L) Inflammatory cytokine levels, including (I) serum IL-18, (J) fecal IL-18, (K) serum IL-1β, and (L) fecal IL-1β. Data are presented as mean ± SD; dots represent individual participants. n = 10 participants per group. For (B)–(L), between-group comparisons were performed using an unpaired two-tailed Student’s t test for normally distributed data or the Mann-Whitney U test otherwise, as specified in the . No post hoc test was applied for two-group comparisons. p < 0.05; ns, not significant. Abbreviations: BMD, bone mineral density; CTX, C-terminal telopeptide of type I collagen; PINP, procollagen type I N-terminal propeptide; TMAO, trimethylamine N-oxide.

Article Snippet: Trimethylamine N-oxide (TMAO) , MedChemExpress , Cat# HY-108915; Trimethylamine N-oxide dihydrate.

Techniques: Activity Assay, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Exercise is associated with attenuated aging-related osteoporosis through TMAO alpha Klotho inflammasome signaling

doi: 10.1016/j.isci.2026.117170

Figure Lengend Snippet: Exercise mitigates TMAO-induced loss of bone strength and mass in aged rats (A) Representative photograph of the three-point bending setup used for femoral biomechanical testing. (B–F) Mechanical properties of femora from control (C), model (M), exercise (E), TMAO (T), and TMAO plus exercise (T + E) groups, including (B) maximum load, (C) maximum deformation, (D) bending stiffness, (E) elastic modulus, and (F) bending stress. TMAO reduced maximum load compared with the model group, whereas exercise improved load-bearing capacity and partially restored maximum load in TMAO-exposed rats. No significant differences were observed in maximum deformation, bending stiffness, elastic modulus, or bending stress among groups. (G) Representative micro-CT reconstructions of the distal femoral metaphysis showing trabecular architecture across groups. Scale bars, 1,000 μm. (H–K) Quantitative micro-CT parameters, including (H) bone volume fraction (BV/TV), (I) trabecular thickness (Tb.Th), (J) trabecular number (Tb.N), and (K) trabecular separation (Tb.Sp). Exercise preserved BV/TV and Tb.N and reduced Tb.Sp, whereas TMAO aggravated trabecular deterioration. (L) Representative H&E staining of the distal femoral metaphysis. The upper part of the images show low-magnification images, and the lower part of the images show high-magnification trabecular regions. Scale bars, 1,000 μm in upper images and 100 μm in lower images. Data are presented as mean ± SD; dots represent individual animals. n = 3 rats per group. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. ∗ p < 0.05; ns, not significant. Group labels: C, control; M, model; E, exercise; T, TMAO; T + E, TMAO plus exercise.

Article Snippet: Trimethylamine N-oxide (TMAO) , MedChemExpress , Cat# HY-108915; Trimethylamine N-oxide dihydrate.

Techniques: Control, Micro-CT, Staining

Journal: iScience

Article Title: Exercise is associated with attenuated aging-related osteoporosis through TMAO alpha Klotho inflammasome signaling

doi: 10.1016/j.isci.2026.117170

Figure Lengend Snippet: Exercise suppresses TMAO-induced bone resorption and systemic inflammation in aged rats (A and B) (A) Femoral marrow TMAO (ng/g) and (B) plasma TMAO (nmol/L) quantified by ultra‑high‑performance liquid chromatography‑mass spectrometry (UHPLC-MS)/MS. Exercise lowered TMAO relative to the model (E < M), whereas TMAO loading increased TMAO (T > M) and was partially rescued by exercise (T + E < T). (C) Serum CTX (pg/mL) was elevated by TMAO and reduced by exercise, consistent with attenuation of bone resorption. (D) Serum PINP (ng/mL) showed the opposite pattern—elevated with exercise, reduced by TMAO, and restored in T + E. (E) Volcano plot of differentially expressed circulating cytokines between model (M) and TMAO (T) groups; significantly altered proteins are highlighted. (F) Heatmap of selected cytokines across individual animals (M-1–M-3, T-1–T-3) showing higher IL-6, CINC-3, GM-CSF, and related mediators in T. (G) Volcano plot comparing T vs. T + E demonstrates cytokine shifts with exercise. (H) Heatmap illustrates a reduction in the pro-inflammatory cytokine IL-6 and an increase in the anti-inflammatory cytokine IL-10 in T + E relative to T. Bars indicate mean ± SD; dots are individual animals ( n per group in ). n = 3 rats per group for (A)–(D). Statistical comparisons for (A)–(D) were performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. For cytokine antibody array analyses in (E)–(H), n = 3 rats per group; normalized signal intensities were used for two-group screening of differentially expressed cytokines, and heatmaps display normalized relative intensities. ∗ p < 0.05; ns, not significant. Group labels: C, control; M, model; E, exercise; T, TMAO; T + E, TMAO plus exercise.

Article Snippet: Trimethylamine N-oxide (TMAO) , MedChemExpress , Cat# HY-108915; Trimethylamine N-oxide dihydrate.

Techniques: Clinical Proteomics, Tandem Mass Spectroscopy, Ab Array, Control

Journal: iScience

Article Title: Exercise is associated with attenuated aging-related osteoporosis through TMAO alpha Klotho inflammasome signaling

doi: 10.1016/j.isci.2026.117170

Figure Lengend Snippet: TMAO accelerates osteoblast senescence and suppresses osteogenic function in vitro (A and B) Optimization of the senescence model in rat osteoblasts using D-galactose (0, 50, 75, and 100 mM for 24–72 h). Cell viability (CCK-8, OD450) declined and SA-β-gal-positive cells increased in a dose- and time-dependent manner; 100 mM for 72 h was chosen for subsequent assays. (C) Senescence burden (SA-β-gal-positive rate) in non-aged control (Ctrl), D-gal-aged + vehicle (NS), D-gal-aged + TMAO (TMAO), and D-gal-aged + DMB treatment (TMAO Inh) groups. TMAO treatment further increased senescence positivity compared to the NS group, whereas DMB treatment mitigated this effect. (D) Representative immunoblots for the osteogenic marker PINP with GAPDH as the loading control. (E) Densitometric quantification (relative expression of PINP/GAPDH) shows that TMAO exposure suppresses PINP expression, while DMB treatment partially restores it. (F) ELISA-derived RANKL/OPG ratio in conditioned supernatants indicates that TMAO shifted osteoblast-derived signaling toward a more pro-osteoclastogenic state, whereas DMB treatment reduced this shift. Data are presented as mean ± SD. For (A) and (B), n = 3 independent experiments per concentration/time condition, and statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. For (C) and (F), n = 3 independent biological replicates per group, and comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. For (E), n = 3 independent western blot experiments and comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant. Abbreviations: DMB, 3,3-dimethyl-1-butanol; PINP, procollagen type I N-terminal propeptide; RANKL, receptor activator of nuclear factor-kappa B ligand; OPG, osteoprotegerin.

Article Snippet: Trimethylamine N-oxide (TMAO) , MedChemExpress , Cat# HY-108915; Trimethylamine N-oxide dihydrate.

Techniques: In Vitro, CCK-8 Assay, Control, Western Blot, Marker, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Concentration Assay

Journal: iScience

Article Title: Exercise is associated with attenuated aging-related osteoporosis through TMAO alpha Klotho inflammasome signaling

doi: 10.1016/j.isci.2026.117170

Figure Lengend Snippet: TMAO activates the TXNIP/α-Klotho/NLRP3 axis and upregulates inflammasome components in aged osteoblasts, which is reversed by DMB treatment Rat osteoblasts were subjected to D-gal-induced aging and treated with vehicle (NS), DMB (TMAO Inh), or TMAO, with a non-aged control (Ctrl). (A) RT-qPCR analysis of α-Klotho, TXNIP, NLRP3, ASC, and caspase-1 mRNA levels normalized to GAPDH shows that TMAO increases TXNIP, NLRP3, ASC, and caspase-1 while decreasing α-Klotho; TMAO inhibition mitigates these changes. (B) Representative immunoblots for the indicated proteins with GAPDH as the loading control. (C–G) Densitometric quantification of TXNIP, α-Klotho, NLRP3, ASC, and caspase-1 protein abundance relative to GAPDH confirms the transcriptional trends. (H and I) ELISA of culture supernatants shows that TMAO increased the secretion of (H) IL-1β and (I) IL-18, whereas DMB treatment attenuated these increases. Bars represent mean ± SD. For (A), n = 3 independent biological replicates per group. For (C)–(G), n = 3 independent western blot experiments. For (H) and (I), n = 3 independent biological replicates per group. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns, not significant. Abbreviations: DMB, 3,3-dimethyl-1-butanol; TXNIP, thioredoxin-interacting protein; ASC, apoptosis-associated speck-like protein containing a CARD.

Article Snippet: Trimethylamine N-oxide (TMAO) , MedChemExpress , Cat# HY-108915; Trimethylamine N-oxide dihydrate.

Techniques: Control, Quantitative RT-PCR, Inhibition, Western Blot, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Exercise is associated with attenuated aging-related osteoporosis through TMAO alpha Klotho inflammasome signaling

doi: 10.1016/j.isci.2026.117170

Figure Lengend Snippet: TMAO promotes NLRP3 inflammasome assembly and colocalization of inflammasome components in senescent osteoblast-like cells Rat osteoblast-like cells were subjected to D-galactose-induced aging and treated with vehicle (NS), DMB (TMAO Inh), or TMAO, with non-aged cells used as the control (Ctrl). (A) Co-immunoprecipitation (coIP) with anti-NLRP3 or anti-ASC antibodies showing the interaction between NLRP3 and ASC. Representative input and immunoprecipitated protein blots are shown. Molecular weight markers are indicated in kDa. (B and C) Densitometric quantification of (B) ASC/NLRP3 in NLRP3 immunoprecipitates and (C) NLRP3/ASC in ASC immunoprecipitates. (D) coIP with anti-NLRP3 or anti-caspase-1 antibodies showing the interaction between NLRP3 and caspase-1. Representative input and immunoprecipitated protein blots are shown. Molecular weight markers are indicated in kDa. (E and F) Densitometric quantification of (E) caspase-1/NLRP3 in NLRP3 immunoprecipitates and (F) NLRP3/caspase-1 in caspase-1 immunoprecipitates. TMAO increased NLRP3 inflammasome complex formation, whereas DMB treatment reduced these interactions. (G) Representative double immunofluorescence images showing NLRP3, ASC, DAPI nuclear staining, and merged NLRP3/ASC signals. (H) Representative double immunofluorescence images showing NLRP3, caspase-1, DAPI nuclear staining, and merged NLRP3/caspase-1 signals. TMAO increased punctate colocalization signals, whereas DMB attenuated this pattern. Scale bars, 50 μm. Data are presented as mean ± SD. For (B), (C), (E), and (F), n = 3 independent coIP experiments per group. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple-comparisons post hoc test. ∗ p < 0.05; ∗∗ p < 0.01; ns, not significant. Unedited representative immunofluorescence images corresponding to (G) and (H) are provided in the . Abbreviations: ASC, apoptosis-associated speck-like protein containing a CARD; coIP, co-immunoprecipitation; DAPI, 4′,6-diamidino-2-phenylindole; DMB, 3,3-dimethyl-1-butanol; IP, immunoprecipitation; NLRP3, NOD-like receptor family pyrin domain-containing 3; TMAO, trimethylamine N-oxide.

Article Snippet: Trimethylamine N-oxide (TMAO) , MedChemExpress , Cat# HY-108915; Trimethylamine N-oxide dihydrate.

Techniques: Control, Immunoprecipitation, Molecular Weight, Immunofluorescence, Staining

Journal: iScience

Article Title: Exercise is associated with attenuated aging-related osteoporosis through TMAO alpha Klotho inflammasome signaling

doi: 10.1016/j.isci.2026.117170

Figure Lengend Snippet:

Article Snippet: Trimethylamine N-oxide (TMAO) , MedChemExpress , Cat# HY-108915; Trimethylamine N-oxide dihydrate.

Techniques: Immunofluorescence, Immunoprecipitation, Clinical Proteomics, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, CCK-8 Assay, Ab Array, Control, Negative Control, Sequencing, Plasmid Preparation, Clone Assay, Expressing, Software, Micro-CT, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy