Capsaicin diet drives gut inflammation and exosomal miR-17-3p elevation in idiopathic short stature

Figures

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  Fig. 1. Plasma exosomes from ISS children inhibit normal human chondrocyte proliferation and bone formation in vitro. a Morphological analysis of the extracted material was conducted using transmission electron microscopy (n = 3 biological replicates). b Nano-Sight analysis confirmed that the particles had an average size of around 100 nm. c Western blotting demonstrated the presence of exosome markers CD63, CD9, and CD81 in the samples. (n = 3 biological replicates per group) e Successful uptake of FAM-labeled exosomes (indicated by green fluorescence) by chondrocytes (blue fluorescence) was verified. d, f, g CCK8 (d) and EdU (f, g) staining results indicated significant suppression of chondrocyte proliferation following co-culture with ISS plasma exosomes (n = 3 biological replicates per group). h, i Flow cytometry analysis showed a cell cycle arrest at the G0/G1 phase (n = 3 biological replicates per group). j–l Western blot and RT-qPCR results revealed a notable downregulation of genes associated with chondrocyte differentiation (COL10A1, RUNX2) and osteogenic marker gene (OPN, OCN) after co-culture with ISS plasma exosomes (n = 3 biological replicates per group). m The activity of…

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  Fig. 2. miR-17-3p as a Key Mediator in ISS Exosome-Induced Inhibition of Chondrocyte Proliferation and Bone Formation. a Experimental Grouping: Group A (Gray) consisted of chondrocytes co-cultured with NC-Exosomes; Group B (Pine) included chondrocytes co-cultured with ISS-Exosomes; Group C (Blue) involved chondrocytes co-cultured with ISS-Exosomes and siRNA to silence miR-17-3p expression. b The expression of miR-17-3p was significantly elevated in Group B compared to Group A, while siRNA effectively silenced miR-17-3p expression following co-culture with ISS-Exosomes(n = 3 biological replicates per group). c, f, g CCK8 (c) and EdU (f, g) assays demonstrated a marked improvement in chondrocyte proliferation upon silencing of miR-17-3p(n = 3 biological replicates per group). d, e Flow cytometric analysis indicated a restoration of the cell cycle to the G2/M phase following downregulation of miR-17-3p(n = 3 biological replicates per group). h–j RT-qPCR and Western blot analyses revealed significant upregulation of marker genes associated with chondrocyte hypertrophic differentiation (COL10A1, RUNX2) and osteogenic marker genes (OCN, OPN) after the downregulation of miR-17-3p(n = 3…

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  Fig. 3. miR-17-3p inhibits growth plate chondrocyte proliferation and bone formation in vivo, leading to a short-stature phenotype. a Experimental Grouping: Group A (Gray) consisted of normal control rats (WT); Group B (Pine) comprised rats with exosomal overexpression of miR-17-3p; Group C (Blue) included rats with exosomal overexpression of miR-17-3p alongside overexpression of Znf148; Group D (Green) included rats with exosomal overexpression of miR-17-3p alongside overexpression of Sos1. b–e Although body height and femur and tibia lengths were significantly shorter in Group B compared to Group A, no significant differences were observed in body height or bone lengths between Group A and Group C or D, indicating that overexpression of Znf148 or Sos1 counteracted the inhibitory effects of miR-17-3p on growth plate proliferation and bone formation(n = 5 biological replicates per group). f, h Calcein staining demonstrated that exosomal miR-17-3p overexpression inhibited new bone formation (area between the two red lines), as observed in Group B. However, overexpression of Znf148 or Sos1 significantly restored bone formation, as shown in Groups C and D(n = 3 biological replicates per…

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  Fig. 4. miR-17-3p inhibits growth plate chondrocyte proliferation and bone formation by regulating Erk and Pi3k pathways in vivo, leading to a short-stature phenotype. a The overexpression of exosomal miR-17-3p did not suppress the expression of key genes in the Erk and Pi3k pathways—Erk1/2, Pi3k, and Akt. Instead, it reduced Erk, Pi3k, and Akt protein phosphorylation levels. This suggests that exosomal miR-17-3p affects the Erk and Pi3k pathways by modulating the phosphorylation of Erk1/2, Pi3k, and Akt proteins. Data are presented as mean ± SD (n = 6 biological replicates per group). Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001 compared to the control group.

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  Fig. 5. Capsaicin-Diet Induced Short-Stature Phenotype. a, c, d, e Capsaicin diet led to the shortened femur, tibia, and overall stunted growth in rats, compared to the wild-type (WT) control group (n = 6 biological replicates per group). b, f Safranin O/Fast Green staining showed reduced growth plate size in capsaicin-fed rats compared to the WT group (n = 3 biological replicates per group). g, h Calcein staining indicated inhibited new bone formation in the growth plates of capsaicin-fed rats (n = 3 biological replicates per group). i–m Immunofluorescence staining, RT-qPCR, and Western blot analyses demonstrated suppressed expression of hypertrophic chondrocyte differentiation markers (Col10a1, Runx2) and osteogenic markers (Ocn, Opn) in the capsaicin group (n = 3 biological replicates per group). As a result, capsaicin-fed rats exhibited a short-stature phenotype. Data are presented as mean ± SD. Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001 compared to the control group.

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  Fig. 6. Construction of Exosomes Targeting Chondrocytes and Loaded with siRNA and Growth Hormone (ET-Exo-si-miR-17-3p-oeGH, abbreviated as ET-Exo) Derived from Chondrocytes. a–d Transmission electron microscopy, Nano-Sight analysis, zeta potential, and Western blot analyses confirmed the successful extraction of exosomes derived from chondrocytes (n = 3 biological replicates per group). e–j RT-qPCR, Western blot, confocal microscopy, and immunofluorescence analyses demonstrated the expression of exosomal membrane fusion proteins. Additionally, siRNA and growth hormone were successfully enriched within the exosomes (n = 3 biological replicates per group). l The targeted exosomes (ET-Exo) efficiently entered chondrocytes and rats’ growth plate cartilage. k Compared to control exosomes, ET-Exo exhibited bone-targeting solid capability. Data are presented as mean ± SD. Statistical significance was assessed using a one-way ANOVA and a two-tailed Welch’s t-test. Results were considered not significant (ns) unless *P < 0.05, **P < 0.01, ***P < 0.001, or ****P < 0.0001 compared to the control group.

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  Fig. 7. ET-Exo-si-miR-17-3p-oeGH antagonizes miR-17-3p-induced inhibition of chondrocyte proliferation and bone formation in Human Chondrocyte. a Group A (Gray): Chondrocytes overexpressing the mock vector (Mock); Group B (Pine): Overexpression of miR-17-3p followed by treatment with cartilage-targeted exosomes loaded with siRNA and growth hormone (GH), labeled as OE-miR-17-3p + ET-Exo (si-miR-17-3p + GH); Group C (Blue): Overexpression of miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with siRNA and GH, labeled as OE-miR-17-3p + Exo (si-miR-17-3p + GH); Group D (Green): Overexpression of miR-17-3p only; Group E (Yellow): Overexpression of miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with siRNA, labeled as OE-miR-17-3p + Exo (si-miR-17-3p); Group F (Purple): Overexpression of miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with GH, labeled as OE-miR-17-3p + Exo (GH). b, c, e CCK8 (b) and EdU assays (c, e) showed that while miR-17-3p overexpression significantly inhibited chondrocyte proliferation, this inhibitory effect was restored upon treatment with ET-Exo (si-miR-17-3p + GH), as demonstrated in…

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  Fig. 8. ET-Exo (si-miR-17-3p + Gh) Antagonizes miR-17-3p-Induced Inhibition of Chondrocyte Proliferation and Bone Formation in Rat Growth Plate Cartilage, Reversing the Short Stature Phenotype. a Group A (Gray): Wild-type control rats (WT); Group B (Pine): Rats overexpressing miR-17-3p followed by treatment with cartilage-targeted exosomes loaded with siRNA and growth hormone (Gh), labeled as OE-miR-17-3p + ET-Exo (si-miR-17-3p + Gh); Group C (Blue): Rats overexpressing miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with siRNA and Gh, labeled as OE-miR-17-3p + Exo (si-miR-17-3p + Gh); Group D (Green): Rats overexpressing miR-17-3p only; Group E (Yellow): Rats overexpressing miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with siRNA, labeled as OE-miR-17-3p + Exo (si-miR-17-3p); Group F (Purple): Rats overexpressing miR-17-3p followed by treatment with non-cartilage-targeted exosomes loaded with GH, labeled as OE-miR-17-3p + Exo (GH). b–e Overexpression of miR-17-3p significantly inhibited rat growth, leading to shorter femurs, tibias, and reduced height (Group D). However, as demonstrated in Group B, this inhibitory effect was…