GDF15 nanotherapy ameliorates NLRP3-associated redox imbalance and cardiac injury in sepsis

Figures

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  Fig. 1. Multifaceted validation of GDF15 changes in serum from SICM patients and their clinical associations. (A) GDF15 levels were quantified using the Luminex platform. (B) A volcano plot illustrated the gene expression distribution of GDF15 among differentially expressed genes (DEGs) in whole blood. (C) A heatmap displayed the expression profiles of GDF15 and inflammatory cytokines. (D) Serum GDF15 levels in patients. (E) Pearson correlation analysis demonstrated the association between GDF15 and SOFA score, as well as EF. (F) ROC curves were plotted to assess the diagnostic accuracy of GDF15 and SOFA score in identifying SICM. (G) Multivariate logistic regression analysis was performed to identify independent risk factors for the development of SICM in septic patients. ∗p < 0.05 indicates significant differences; ns: no significant differences.

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  Fig. 2. Upregulation of GDF15 in the SICM model. (A) A schematic workflow for the establishment of the SICM model in C57BL/6J mice via intraperitoneal injection of LPS or saline. (B) Cardiac contractile function parameters, including EF and FS. (C) Serum levels of GDF15 and IL-6. (D) Histopathological analysis of heart tissue, H&E staining (left) and immunohistochemical staining for Ly6G and CD68 (right). Black arrows indicate inflammatory cell infiltration; scale bar: 50 μm. (E) Western blot analysis of GDF15 protein expression in heart tissue. n = 4. (F) qPCR analysis of Gdf15, Bnp, Il-1β, Il-6, Icam-1 and Vcam-1 mRNA levels in heart tissue. (G) Identification of GDF15-positive cells in single-cell RNA-sequencing dataset (GSE190856). (H) qPCR analysis of Gdf15 and Il-1β, Il-6, Nos2, Ptgs2 mRNA expression in BMDM after LPS stimulation. ∗p < 0.05 indicates significant differences; n = 6 per group.

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  Fig. 3. GDF15 deficiency exacerbates LPS-induced SICM in mice. (A) Schematic workflow for the establishment of the SICM model in Gdf15−/− mice. Gdf15−/− mice were intraperitoneally injected with LPS or saline to induce SICM, with tissue samples collected 24 h post-injection for further analysis. (B) Echocardiographic assessment of EF and FS. (C) H&E staining of heart tissue, black arrows indicate inflammatory cell infiltration. scale bar: 50 μm. (D) CD68 immunofluorescence staining of heart tissue. Blue staining highlights nuclei, red staining identifies CD68+ macrophages; scale bar: 20 μm. (E) qPCR analysis of mRNA expression levels of Bnp, Il-1β, Il-6, and Mcp-1 in heart tissue. n = 6 per group.

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  Fig. 4. Fabrication and characterization of macrophage membrane-coated rhGDF15 nanoparticles. (A) Preparation process of MGP. (B) Hydrodynamic diameter and zeta potential of GP and MGP. (C) Western blot analysis of macrophage membrane protein expression on GP and MGP. (D) TEM images of GP and MGP. Scale bar: 100 nm. (E) Hemolysis assay results and absorbance quantification at 540 nm for GP and MGP. (F) Cell viability assessment after 24-h incubation with varying concentrations of GP or MGP. (G–H) Fluorescence imaging of macrophage uptake of DiD-labeled MGP, (G) Representative fluorescence images of MGP uptake after LPS stimulation. (H) Time-dependent changes in MGP uptake were observed at 4 and 8 h. Red staining indicates DiD-labeled MGP, green staining identifies actin filaments, and blue staining highlights nuclei. Scale bar: 50 μm. n = 6 per group. ∗p < 0.05 indicates significant differences; ns: no significant differences.

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  Fig. 5. MGP ameliorates LPS-induced SICM in mice. (A) Schematic diagram of the experimental protocol: C57BL/6J mice were administered saline or MGP via tail vein injection (1 time/day for 3 consecutive days), followed by intraperitoneal injection of LPS. (B) Echocardiographic assessment of EF and FS. (C) qPCR analysis of mRNA expression levels of Bnp, Il-1β, Il-6, and Mcp-1 in heart tissue. (D) IVIS images and fluorescence signals accumulated in the hearts of SICM mice. n = 3. (E) Fluorescence imaging of myocardial tissue: blue staining for nuclei, red staining for DiD, green staining for CD68. Scale bar: 50 μm. n = 6 per group.

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  Fig. 6. MGP suppresses myocardial injury by inhibiting the NLRP3 pathway. (A) KEGG pathway enrichment analysis and GSEA. (B) qPCR analysis of mRNA expression levels of Il-1β, Il-6, Mcp-1, and Ptgs2 in BMDM treated with LPS and MGP for 24 h. (C) DHE staining images: blue staining for nuclei, red staining for DHE signals. Scale bar: 20 μm. (D) Western blot detection of protein expression levels of NLRP3, ASC, and IL-1β/pro-IL-1β in BMDM treated with LPS and MGP. (E) qPCR analysis of mRNA expression levels of Nlrp3 and Asc in BMDM treated with LPS and MGP. (F) Co-culture system: LPS or PBS-treated BMDM were collected for 24 h and then incubated with H9c2 cells (n = 3). (G) Western blot detection of NLRP3 protein expression in H9c2 cells treated with LPS, MGP, and supernatant from LPS/PBS-treated BMDM. n = 6 per group.

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  Fig. 7. MGP exerts anti-inflammatory effects via the MYPT1/AKT/YBX-1 signaling pathway. (A) IP-MS of BMDM to identify the interaction with GDF15. MYPT1 is marked in red. (B) Z-DOCK predicted the interaction domain between GDF15 and MYPT1. Pink represents GDF15, green represents MYPT1, and the boxed region indicates the binding domain. (C) Co-IP combined with Western blot analysis of GDF-15 and MYPT1 binding in macrophages after LPS treatment. (n = 3). (D) Immunofluorescence detection of co-localization between GDF15 (green) and MYPT1 (red), with blue staining for nuclei. Scale bar: 20 μm. (E) Protein expression levels of p-YBX-1, YBX-1, and p-AKT, AKT in BMDM after LPS and/or MGP treatment, with gray-scale intensity analysis of relative expression differences. (F) Representative immunofluorescence images of YBX-1 staining in BMDM after LPS and/or MGP treatment. Blue staining highlights nuclei, and red staining identifies YBX-1. Scale bar: 20 μm ∗p < 0.05, significantly different from control group. #p < 0.05, significantly different from LPS group. n = 6 per group.

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  Fig. 8. YBX-1 mediates GDF15-mediated transcriptional regulation of the NLRP3 pathway. (A) qPCR analysis of mRNA expression levels of Nlrp3, Asc, and Il-1β in LPS-stimulated BMDM after Si-Ybx-1. (B) Western blot analysis of protein expression levels of NLRP3 and IL-1β in LPS-stimulated BMDM after YBX-1 knockdown. (C) qPCR analysis of mRNA expression levels of Nlrp3 and Il-1β in LPS and MGP-treated BMDM after YBX-1 knockdown. (D) Schematic diagram of the luciferase reporter plasmid for the Nlrp3 promoter. (E) Luciferase activity of pcDNA3.1-YBX-1 or empty vector-transfected cells. (F) Luciferase activity after LPS and MGP treatment. n = 6 per group.

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  Fig. 9. Mechanism of action of macrophage-biomimetic nanocarriers delivering GDF15 to target the YBX-1-NLRP3 axis in SICM. Macrophage-biomimetic nanocarriers loaded with rhGDF15 are targeted to inflammatory sites in the heart, enhancing local drug accumulation, while GDF15 binds to MYPT1 to inhibit YBX-1 phosphorylation and block its nuclear translocation, leading to reduced nuclear YBX-1 expression and decreased transcriptional activity of the Nlrp3 promoter, which suppresses NLRP3 inflammasome assembly and pro-inflammatory cytokine release such as IL-1β, ultimately alleviating macrophage inflammatory responses, myocardial cell injury, and improving cardiac function in SICM.