Silicate-based therapy for inflammatory dilated cardiomyopathy by inhibiting the vicious cycle of immune inflammation via FOXO signaling

Figures

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  Fig. 1.. Two distinct CS-based treatment approaches for iDCM. (A) Systemic delivery of CS ion (SiO32−) solution through intravenous injection to treat iDCM. (B) Local delivery of SiO32− ions through MN-CS to treat iDCM.

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  Fig. 2.. Evaluation of cardiac function in iDCM mice after MN-CS or CS ion solution treatment. (A to G) Effects of MN-CS on iDCM. (A) Schematic diagram illustrating the establishment of the iDCM model and treatment with MN or MN-CS. (B and C) Representative M-mode echocardiography diagrams and quantification of cardiac function. (D) Representative images of H&E staining, Masson’s trichrome staining, and periostin immunohistochemistry staining of iDCM cardiac tissue. Scale bars, 50 μm. (E to G) Quantification of histological grades, blue-stained collagen fiber area, and periostin-positive area. (H to N) Effects of CS ion solution on iDCM. (H) Schematic diagram illustrating the establishment of the iDCM model and treatment with PBS or CS ion solution. (I and J) Representative M-mode echocardiography diagrams and quantification of cardiac function. (K) Representative images of H&E staining, Masson’s trichrome staining, and periostin immunohistochemistry staining of iDCM cardiac tissue. Scale bars, 50 μm. (L to N) Quantification of histological grades, blue-stained collagen fiber area, and periostin-positive area. n = 10, #P < 0.05, ##P < 0.01, ###P < 0.001 versus iDCM 60 day; *P < 0.05, **P…

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  Fig. 3.. Evaluation of the therapeutic effects of CS ion solution on myocarditis (21 days) and the prevention effects of CS ion solution on the progression of myocarditis to iDCM (42 days). (A) Schematic diagram of myocarditis (21 days) and iDCM (42 days) mouse model establishment and PBS or CS ion solution treatment. (B and C) Therapeutic effects of CS ion solution on myocarditis (21 days). (B) Representative H&E staining images of cardiac tissues (21 days). Scale bars, 50 μm. (C) Quantification of the histological grades based on H&E staining images (21 days). n = 6, ###P < 0.001 versus control; n = 6, *P < 0.05, **P < 0.01, ***P < 0.01 versus myocarditis. (D to J) Prevention effects of CS ion solution on myocarditis to iDCM (42 days). (D) Representative M-mode echocardiography diagrams (42 days). (E and F) Quantification of EF, FS, LVIDd, and LVIDs according to the M-mode echocardiography diagrams (42 days). (G) Representative images of H&E staining, Masson’s trichrome staining, and periostin staining of cardiac tissues (42 days). Scale bars, 50 μm. (H to J) Quantification of histological grades based on H&E staining, blue-stained collagen fiber area based on Masson’s trichrome…

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  Fig. 4.. Sequencing analysis of iDCM myocardial tissue gene (day 42) changes with or without CS solution treatment and verification of the differential signaling pathways in vitro. (A and B) Cluster heatmap and volcano map of differential genes between samples (iDCM myocardial tissue with or without CS solution treatment). Red represents significantly up-regulated genes, and blue represents significantly down-regulated genes. (C) KEGG pathway analysis of regulated genes in iDCM myocardial tissue after the treatment with CS solution. (D) Western blot analysis demonstrating the activation of FOXO1 by CS in TH17 cells. (E to G) Quantitative analysis of p-FOXO1 and t-FOXO1 protein expression levels as well as p-FOXO1/t-FOXO1 ratio based on Western blot. (H and I) Flow cytometric analysis of CD4+/IL-17A+ cells to evaluate the percentage of TH17 cells. (J) Measurement of TH17-related cytokines (IL-17A) in CD4+ T cells using ELISA. n = 6, ###P < 0.001 versus control; *P < 0.05, ***P < 0.001 versus TH17; &P < 0.05, &&P < 0.01 versus TH17 + CS + inhibitor (control: CD4+ T cells not initiating TH17 cell differentiation; TH17: CD4+ T cells induced to TH17 differentiation; TH17 + CS: CD4+ T cells…

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  Fig. 5.. Effects of CS ion solution on modulation of the formation of NETs when FOXO3 was inhibited. (A) Western blot analysis demonstrating the activation of FOXO3 by CS in NETs. (B to D) Quantitative analysis of p-FOXO3 and t-FOXO3 protein expression levels as well as p-FOXO3/t-FOXO3 ratio based on Western blot. (E) The NET markers, including MPO-DNA, NE-DNA, cfDNA, and IL-1β, were measured in the medium after using CS ion solution and siFOXO3 by ELISA. (F) NETs treated with CS ion solution and siFOXO3 were stained with MPO (red), NE (green), and DAPI for visualization. Scale bars, 20 μm. n = 6, ###P < 0.001 versus control; *P < 0.05, **P < 0.01, ***P < 0.001 versus NETs; &P < 0.05, &&P < 0.01 versus NETs + CS + siFOXO3 (control: neutrophils not initiating NET differentiation; PMA: neutrophils initiating NET differentiation; PMA + CS: neutrophils initiating NET differentiation cultured with CS solution; PMA + siFOXO3: neutrophils initiating NET differentiation cultured with siFOXO3; PMA + CS + siFOXO3: neutrophils initiating NET differentiation cultured with CS ion solution and siFOXO3). The positive expression of NE or MPO and net structure DNA indicates that the neutrophils have…

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  Fig. 6.. Effects of CS ion solution on TGF-β–induced myofibroblast formation when FOXO3 was inhibited. (A) Western blot analysis demonstrating the activation of FOXO3 by CS in TGF-β–induced myofibroblasts. (B to D) Quantitative analysis of p-FOXO3 and t-FOXO3 protein expression levels as well as p-FOXO3/t-FOXO3 ratio based on Western blot. (E to G) Measurement of myofibroblast markers (α-SMA, Col1a, and FN) in TGF-β–induced myofibroblasts impacted by CS ion solution and siFOXO3 using qRT-PCR. (H) Representative immunofluorescence for α-SMA (green) and F-actin (red) of cardiac fibroblasts treated with TGF-β, CS ion solution, and siFOXO3. Scale bars, 20 μm. n = 6, ###P < 0.001 versus control; *P < 0.05, **P < 0.01, ***P < 0.001 versus NETs; &P < 0.05, &&P < 0.01 versus NETs + CS + siFOXO3 (control: normal cardiac fibroblasts; TGF-β: cardiac fibroblasts cultured with TGF-β; TGF-β + CS: cardiac fibroblasts cultured with TGF-β and CS ion solution; TGF-β + siFOXO3: cardiac fibroblasts cultured with TGF-β and siFOXO3; TGF-β + CS + siFOXO3: cardiac fibroblasts cultured with TGF-β, CS ion solution, and siFOXO3).

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  Fig. 7.. Effect of the FOXO inhibitor JY-2 on cardiac function and FOXO expression of CS-treated iDCM mice on day 42. (A) Representative M-mode echocardiography diagrams and quantification of EF and FS according to the M-mode echocardiography diagrams. (B) Western blot analysis of the expression of FOXO1 in CD4+ T cells isolated from iDCM mice treated with JY-2 and CS. (C) Western blot analysis of the expression of FOXO3 in neutrophils isolated from iDCM mice treated with JY-2 and CS. Results are presented as the means ± SD. n = 3 for (A) and (B), n = 6 for (C), *P < 0.05, **P < 0.01 versus iDCM; ##P < 0.01 versus iDCM + CS; ###P < 0.001 versus iDCM + CS (iDCM: iDCM mice on day 42; iDCM + CS: iDCM mice treated with CS ion solution and analyzed on day 42; iDCM + CS: iDCM mice treated with JY-2 ion solution and analyzed on day 42; iDCM + CS + JY-2: iDCM mice treated with CS ion solution as well as JY-2 and analyzed on day 42).

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  Fig. 8.. Effects of CS on the interaction between CD4+ T cells and neutrophils. (A and B) Effects of CS on regulating CD4+ T cells to induce the formation of NETs. (A) Measurement of supernatant NET markers (MPO-DNA, NE-DNA, and cfDNA) and related inflammation cytokines (IL-1β) in neutrophils using ELISA. (B) NET marker staining [MPO (red) and NE (green)] for observing NET formation from neutrophils. Scale bars, 20 μm (control: neutrophils not initiating NET differentiation; PMA: neutrophils initiating NET differentiation; PMA + TH17CM: neutrophils initiating NET differentiation cultured with conditioned medium collected from TH17 cells; PMA + CS-TH17CM: neutrophils initiating NET differentiation cultured with conditioned medium collected from TH17 cells stimulated by CS). (C to F) Effects of CS on regulating neutrophils to induce the differentiation of TH17 cells. (C and D) Flow cytometric analysis of CD4+/IL-17A+ cells to evaluate the percentage of TH17 cells. (E) Measurement of supernatant IL-17A levels in CD4+ T cells using ELISA. (F) Measurement of TH17 markers (IL-17A and RORγt) mRNA expression in CD4+ T cells using qRT-PCR. n = 6, ###P < 0.001 versus TH17; *P < 0.05, **P < 0.01,…

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  Fig. 9.. Effects of CS on cardiac fibroblasts affected by CD4+ T/TH17 cells. (A) Schematic diagram illustrating cardiac fibroblasts cocultured with CD4+ T/TH17 cells isolated from different mice. (B) Fibroblasts (α-SMA, red) cocultured with CD4+ T cells (CD4, green) from different mice directly to evaluate the adhesion of CD4+ T cells. Scale bars, 20 μm. (C) Number of CD4+ T/TH17 cells adhered to myofibroblasts. (D) Fibroblasts cocultured with CD4+ T/TH17 cells from different mice indirectly (used Transwell) to evaluate the conversion of fibroblasts (F-actin, red) to myofibroblasts (α-SMA, green). Scale bars, 20 μm. (E to G) Measurement of myofibroblast markers (α-SMA, Col1a, and FN) in cardiac fibroblasts affected by CD4+ T/TH17 cells using qRT-PCR. (H and I) Representative immunofluorescence staining with β-catenin (red), Smad3 (red), and α-SMA (green) in cardiac fibroblasts in indicated groups (the arrows represent the nuclear translocations of β-catenin and Smad3). Scale bars, 20 μm. n = 6, ##P < 0.01, ###P < 0.001 versus control; *P < 0.05, **P < 0.01 versus iDCM + PBS (control: cardiac fibroblasts cocultured with CD4+ T/TH17 cells isolated from normal mice; iDCM + PBS: cardiac…

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  Fig. 10.. Biological mechanism of SiO32− treating iDCM. SiO32− modulated multicellular differentiation and interactions via the FOXO signaling pathway. SiO32− activated the FOXO1 signaling pathway in CD4+ T cells, leading to a significant reduction in their differentiation into TH17 cells. Concurrently, CS promotes FOXO3 expression in neutrophils and fibroblasts, thereby inhibiting their differentiation into NETs and myofibroblasts, respectively. This comprehensive, multicellular regulatory mechanism disrupts the harmful interactions between CD4+ T/TH17 cells and neutrophils/fibroblasts, breaking the vicious cycle of myocardial inflammation and blocking the progression of fibrotic lesions post-iDCM. This figure was created with FigDraw.