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

Background – Myocarditis is an inflammatory cardiomyopathy characterized by high level of inflammatory cell infiltration and progressive cardiac dysfunction. The underlying pathogenesis involves direct pathogen damage and inflammation-mediated tissue destruction. Previous studies showed the therapeutic potential of Sivelestat (the neutrophil elastase inhibitor) in mice models, but the underlying mechanisms remain elusive. Objective – To elucidate the cardioprotective mechanisms of Sivelestat, a neutrophil elastase inhibitor, in experimental autoimmune myocarditis (EAM). Methods – Network pharmacology identified shared targets between Sivelestat and myocarditis. Molecular docking validated binding affinities. EAM was induced in male BALB/c mice (n = 6/group) using α-myosin heavy chain peptide. Interventions included Sivelestat sodium (50/100/200 mg/kg/day i. p., 14 days). Cardiac function (echocardiography), inflammation (serum IL-1β/IL-6/TNF-α/cTn; histopathology; immunohistochemistry for myocardial IL-6, IL-1β, and TNF-α), NETosis (Cit-H3/NE immunofluorescence/Western blot/SEM), apoptosis (TUNEL), and PI3K-Akt signaling (Western blot) were assessed. Results – Computational analysis identified 41 potential targets, highlighting the PI3K-Akt and IL-17 signaling pathways as top candidates. High-affinity binding was confirmed for key targets (e.g., PTGS2: −9.0 kcal/mol). In vivo administration of Sivelestat (200 mg/kg) significantly improved left ventricular function and fractional shortening, and reduced serum levels of inflammatory cytokines (IL-1β, IL-6, TNF-α) and cardiac troponin (cTn). Immunohistochemical analysis confirmed that Sivelestat significantly reduced the myocardial expression of IL-6, IL-1β, and TNF-α consistent with the serum findings and further demonstrating its local anti-inflammatory effects. Moreover, qPCR validation demonstrated that Sivelestat significantly downregulated the mRNA expression of the network pharmacology-predicted targets TNF, MMP9, PTGS2, and IL-17 in myocardial tissue, providing transcriptional evidence supporting the multi-target mechanism. Additionally, Sivelestat decreased cardiomyocyte apoptosis through activation of the PI3K-Akt pathway, as demonstrated by increased p-Akt and Bcl-2, and decreased cleaved caspase-3. Conclusion – Sivelestat mitigated myocarditis through multiple mechanisms, including immunomodulatory effects (NETosis inhibition and PTGS2 inhibition mediated IL-17A Pathway modulation) and PI3K-Akt-mediated anti-apoptosis. Due to the multi-target action and established clinical safety profile, Sivelestat had the potential for rapid clinical translation.