Integrated pharmacology and experimental validation reveals potential multiple mechanisms of the neutrophil elastase inhibitor sivelestat in attenuating myocarditis

Figures

• 

  FIGURE 1. Analysis of the targets associated with Sivelestat, NETs, and myocarditis. (A) Network diagram of ‘Sivelestat-target’. (B) Venn diagram of Sivelestat, NETs and myocarditis-associated targets. Includes 155 Sivelestat-related targets, 1,401 myocarditis-related targets, 5,812 NETs-related target, and 41 Sivelestat-myocarditis-NETs-related targets (center). (C) Sivelestat anti-myocarditis PPI network. A larger area indicates larger nodes, a Green color indicates higher association, lighter color less association, and the core target is the target in the inner circle. (D) Degree values of core anti-myocarditis targets in Sivelestat; the X/Y axes respectively represent the Degree value and the target name. (E) GO enrichment analysis. Fold enrichment (y-axis), term (x-axis); green, orange, and purple represent the 10 core results for BP, CC, and MF, respectively. (F) KEGG pathway enrichment analysis (DAVID). Pathways (Y-axis), FDR (X-axis), and P-values (color change). Bubble size indicates the number of genes enriched in the pathway.

• 

  FIGURE 2. The picture of molecule docking model. (A–F) Molecular docking of Sivelestat with TNF, PTPRC, PTGS2, PDGFRB, MMP9, and HSP90AA1. The hydrogen bonds were represented by yellow dotted lines, and the length was marked around the lines.

• 

  FIGURE 3. (A) Representative short-axis and long-axis M-mode echocardiographic images at day 21 post-immunization. (B–E) Quantification of left ventricular ejection fraction (LVEF) and fractional shortening (LVFS) from short-axis (B,D) and long-axis (C,E) views. Data are mean ± SEM (n = 6). (Sham vs. EAM and EAM vs. EAM + Siv). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant (one-way ANOVA with Bonferroni’s post-hoc test).

• 

  FIGURE 4. Sivelestat inhibits NETosis in EAM mice. (A) Representative immunofluorescence images of NETs (NE in green, Cit-H3 in red, DAPI in blue). Scale bar: 50 μm. (B,C) Quantification of NETs per area. (D,E) Western blot of NE and Cit-H3. (F) Densitometric analysis normalized to GAPDH. (G) Scanning electron microscopy (SEM) image showing NETs (white arrow). Data are mean ± SEM (n = 3 for Western blot). (Sham vs. EAM and EAM vs. EAM + Siv [200 mg/kg]) *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant (Kruskal-Wallis test for NETs counts; one-way ANOVA for Western blot).

• 

  FIGURE 5. Sivelestat mitigates the inflammatory response and cardiac damage in EAM mice. (A–D) Serum levels of IL-6 (A), IL-1β (B), TNF-α (C), and cTn (D) by ELISA. (E) Representative H&E-stained myocardial sections. Asterisks: necrosis; arrows: inflammatory infiltration. Scale bar: 50 μm. (F) Semiquantitative inflammatory score. Data are mean ± SEM (n = 6). (Sham vs. EAM and EAM vs. EAM + Siv). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (one-way ANOVA with Bonferroni’s post-hoc test).

• 

  FIGURE 6. Sivelestat reduces the expression of IL-6, IL-1β, and TNF-α in the myocardial tissue of EAM mice. (A) Representative immunohistochemical staining. Brown: positive signal. Arrows indicate inflammatory infiltration. Scale bar: 50 μm. (B) Quantification of positive area percentage. Data are mean ± SEM (n = 3). (Sham vs. EAM and EAM vs. EAM + Siv [200 mg/kg]). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (one-way ANOVA with Bonferroni’s post-hoc test).

• 

  FIGURE 7. Sivelestat alleviates cardiomyocyte apoptosis and activates the PI3K-Akt signaling pathway in EAM mice. (A) TUNEL staining (green apoptotic nuclei, blue DAPI). Scale bar: 1,000 μm. (B) Quantification of TUNEL-positive cells/HPF in subepicardial region. (C) Representative Western blot bands. (D–G) Quantification of p-PI3K/PI3K, p-Akt/Akt, cleaved caspase-3, and Bcl-2. Data are mean ± SEM (n = 3 for Western blot). (Sham vs. EAM; TUNEL: EAM vs. EAM + Siv 50/100/200 mg/kg; Western blot: EAM vs. EAM + Siv 200 mg/kg). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (one-way ANOVA with Bonferroni’s post-hoc test).

• 

  FIGURE 8. Sivelestat downregulates the mRNA expression of network pharmacology-predicted targets in the myocardium of EAM mice. (A–D) Relative mRNA expression of TNF (A), MMP9 (B), PTGS2 (C), and IL-17 (D) in myocardial tissues was determined by qPCR and normalized to Rplp0 (known as 36B4). Data are mean ± SEM (n = 3, each in triplicate). (Sham vs. EAM, EAM vs. EAM + Siv [200 mg/kg]). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (one-way ANOVA with Bonferroni’s post-hoc test).

• 

  FIGURE 9. Schematic summary of the multi-layered mechanisms underlying Sivelestat’s cardioprotective effects in EAM mice. Sivelestat directly inhibits NE to block NETosis, serving as a core anti-inflammatory mechanism. It activates PI3K-Akt signaling to exert anti-apoptotic effects. Additionally, Sivelestat downregulates the mRNA expression of PTGS2 and IL-17, suggesting potential involvement in broader immunomodulatory networks. Colored areas indicate mechanisms supported by direct experimental evidence; gray areas indicate mechanisms inferred from transcriptional evidence that require further functional validation. (Created with BioRender.com).