Therapeutic Delivery of Bone Marrow Mesenchymal Stem Cell-Derived Exosomal miR-143-3p Inhibits Myocardial and Systemic Inflammation and Attenuates Sepsis-Related Myocardial Injury

Figures

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  Fig. 1. Characterization of BMSCs and BMSC-Exos. A Flow cytometric analysis of BMSC surface markers (CD29, CD44, CD73, CD90, CD105, CD34, CD45, CD11b). B Nanoparticle tracking analysis (NTA) showing size distribution of BMSC-Exos (mean diameter ~ 86.3 nm). C Representative TEM image of BMSC-Exos (cup-shaped vesicles). Scale bar: 100 nm. D Western blot detection of exosomal markers CD9, CD63, CD81, ALIX, TSG101, and HSP70. Data are mean ± SD (n = 3). **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 2. BMSC-Exos promote M2 polarization of LPS-stimulated macrophages in vitro. A Immunofluorescence showing uptake of PKH67-labeled BMSC-Exos (green) by RAW 264.7 macrophages (CD86, red). Merged images indicate co-localization. Scale bar: 20 μm. B Representative flow cytometry plots of M1 (iNOS⁺, TNF-α⁺, CXCL1⁺) and M2 (Arg1⁺, Chi3l3⁺) expression in RAW 264.7 macrophages under the indicated treatments. C Quantification of M1 (iNOS⁺, TNF-α⁺, CXCL1⁺) and M2 (Arg1⁺, Chi3l3⁺) cell percentages from three independent experiments. D ELISA of cytokines (IFN-β, TNF-α, IL-6, IL-10) in culture supernatants. E qRT-PCR analysis of M1 (iNOS, TNF-α, CXCL1) and M2 (Arg1, Chi3l3) marker transcripts. Data are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 3. BMSC-Exos attenuate LPS-induced myocardial injury and promote cardiac M2 polarization in vivo. A Kaplan–Meier survival curves (n = 6 per group). LPS: 41.7%; LPS+BMSC-Exo: 83.3%. B Immunofluorescence staining of cardiac macrophages for iNOS (M1, red) and Arg1 (M2, red), with DAPI nuclear counterstain (blue). Scale bar: 20 μm. C Representative flow cytometry plots of M1 and M2 makers expression in cardiac CD11b⁺ macrophages. D Quantification of M1 (iNOS⁺/TNF-α⁺/CXCL1⁺) and M2 (Arg1⁺/Chi3l3⁺) macrophage percentages. E H&E staining of myocardial sections (100×) with corresponding 200× magnified insets showing interstitial edema. F Masson’s trichrome staining of myocardial sections and quantitative analysis of collagen-positive area. Scale bar: 100 μm. G Serum myocardial injury markers (cTnT, CK-MB, myoglobin). H Serum cytokines (IFN-β, TNF-α, IL-6, IL-10). I Echocardiographic assessment of LVEF and LVFS with representative M-mode images. All analyses in this figure were performed at 72 h post-LPS (48 h after BMSC-Exo administration). Data are mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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  Fig. 4. Identification of miR-143-3p as a key exosomal miRNA associated with inflammatory pathways. A Pearson correlation heatmap of miRNA-seq replicates. B Density plot of miRNA expression distribution across groups. C Volcano plot of differentially expressed miRNAs (BMSC vs. BMSC-Exo). miR-143-3p is highlighted. D Hierarchical clustering heatmap of differentially expressed miRNAs. E GO enrichment analysis (Biological Process, Cellular Component, Molecular Function) of predicted miRNA targets. F KEGG pathway classification of target genes. G KEGG enrichment scatter plot (dot size: gene count; color: significance). H qRT-PCR validation of miR-143-3p, miR-155-5p, and miR-1246 in BMSC-Exos under LPS stimulation. Data in (H) are mean ± SD (n = 3). ***P < 0.001; ns, not significant

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  Fig. 5. MiR-143-3p regulates macrophage polarization and inflammatory cytokine secretion in vitro. A qRT-PCR confirmation of miR-143-3p overexpression/inhibition. B Immunofluorescence for CD86 (M1 marker). miR-143-3p mimic reduces CD86 signal; inhibitor enhances it. Scale bar: 20 μm. C Flow cytometry of M1 (CXCL1, iNOS, TNF-α) and M2 (Arg1, Chi3l3) markers. D qRT-PCR analysis of M1/M2 transcripts following miR-143-3p modulation. E ELISA of cytokines (IFN-β, TNF-α, IL-6, IL-10). miR-143-3p mimic suppresses pro-inflammatory cytokines and promotes IL-10; inhibitor reverses BMSC-Exo effects. Data are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant

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  Fig. 6. In vivo modulation of miR-143-3p affects myocardial injury and cardiac macrophage polarization. A qRT-PCR of myocardial miR-143-3p after in vivo delivery. B Kaplan–Meier survival curves (n = 6). Mimic improves survival; inhibitor does not. C Echocardiography: LVEF and LVFS. Mimic restores function; inhibitor worsens it. D Immunofluorescence of cardiac iNOS (M1) and Arg1 (M2). Scale bar: 20 μm. E Flow cytometry of cardiac macrophage subsets. F H&E staining of myocardial sections (100×) with corresponding 200× magnified insets showing interstitial edema and inflammatory infiltration. G Masson's trichrome staining of myocardial sections and quantitative analysis of collagen-positive area. Scale bar: 100 μm. H Serum myocardial injury biomarkers (cTnT, CK-MB, myoglobin). I Serum cytokines (IFN-β, TNF-α, IL-6, IL-10). All analyses were performed at 72 h post-LPS. Data are mean ± SD(n=3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant

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  Fig. 7. MiR-143-3p directly targets TLR4, and TLR4 knockdown promotes M2 polarization in vitro. A Dual-luciferase reporter assay confirming miR-143-3p binding to TLR4 3′-UTR. B Western blot analysis showing miR-143-3p negatively regulates TLR4 protein expression in macrophages. Left: representative blot; right: densitometric quantification. C Western blot analysis showing miR-143-3p negatively regulates TLR4 protein expression in C57BL/6 mouse myocardium. Left: representative blot; right: densitometric quantification. D Immunofluorescence for CD86 in sh-TLR4 macrophages. Scale bar: 20 μm. E Flow cytometry of M1/M2 markers in sh-TLR4 cells. F qRT-PCR of M1/M2 transcripts in sh-TLR4 cells. G ELISA of cytokines in sh-TLR4 cell supernatants. H Western blot validation of TLR4 knockdown efficiency and MyD88/NF-κB pathway suppression in RAW 264.7 cells. β-actin served as loading control. Data are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant (one-way ANOVA with Tukey’s post hoc test)

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  Fig. 8. In vivo TLR4 knockdown induces M2 polarization and alleviates sepsis-related myocardial injury. A Kaplan–Meier survival curves (n = 6). sh-TLR4 improves survival. B Echocardiography showing improved LVEF and LVFS in sh-TLR4 mice. C Immunofluorescence staining of cardiac macrophages for iNOS (M1, red) and Arg1 (M2, red), with DAPI nuclear counterstain (blue). Scale bar: 20 μm. D Flow cytometry of cardiac macrophage subsets. E Serum cytokines (IFN-β, TNF-α, IL-6, IL-10). F H&E staining of myocardial sections (100×) with corresponding 200× magnified insets showing inflammatory infiltration and tissue architecture. G Masson’s trichrome staining of myocardial sections and quantitative analysis of collagen-positive area. Scale bar: 100 μm. H Serum myocardial injury markers (cTnT, CK-MB, myoglobin). I Western blot of TLR4/MyD88/NF-κB pathway in cardiac tissue. J Schematic of the BMSC-Exo/miR-143-3p/TLR4/NF-κB axis in SRMI. BMSC-Exos deliver miR-143-3p to macrophages, targeting TLR4 3’-UTR, suppressing TLR4/MyD88/NF-κB signaling, and promoting M2 polarization to confer cardioprotection. All analyses were performed at 72 h post-LPS. Data are mean ± SD(n = 3). *P < 0.05, **P < 0.01, ***P <…