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

BackgroundMyocarditis 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.ObjectiveTo elucidate the cardioprotective mechanisms of Sivelestat, a neutrophil elastase inhibitor, in experimental autoimmune myocarditis (EAM).MethodsNetwork 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.ResultsComputational 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.ConclusionSivelestat 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.