Knockdown of Lcn2 alleviates pyroptosis in a neonatal rat model of necrotizing enterocolitis through IL-17A signaling pathway suppression.

Abstract

BACKGROUND: Necrotizing enterocolitis (NEC) is a life-threatening gastrointestinal disease primarily affecting premature infants. However, the specific regulatory molecular mechanisms focused on pyroptosis remain unclear. METHODS: Lipocalin-2 (Lcn2) was identified as a pyroptosis-associated hub gene by bioinformatics methods. Neonatal rats were fed formula milk and subjected to hypoxia (100 % N₂, 70 s) and hypothermia (4°C, 10 min). Rat intestinal epithelial cells-6 (IEC-6) and human intestinal epithelial cells-6 (HIEC-6) were stimulated with lipopolysaccharide (LPS) to mimic NEC-induced inflammation. Histological evaluation of ileal tissues was conducted using hematoxylin and eosin (HE) staining. Survival rate, body weight, and intestinal injury scores were assessed. To investigate cell viability, apoptosis, pyroptosis, and inflammatory cytokine levels, Cell Counting Kit-8 assay, flow cytometry, western blot, and enzyme-linked immunosorbent assay were conducted. RESULTS: Lcn2 expression was significantly upregulated in NEC ileal tissues and LPS-treated IEC-6 cells. Lcn2 knockdown alleviated intestinal injury, improved survival, and suppressed the expression of NLRP3, cleaved Caspase-1, GSDMD-N, and proinflammatory cytokines (IL-1β, IL-6, TNF-α, IL-18, and HMGB1) in a neonatal rat model of NEC. Lcn2 knockdown restored cell viability, reduced apoptosis, and inhibited pyroptosis in LPS-stimulated IEC-6 and HIEC cells, an effect that was notably abolished by nigericin, an NLRP3 inflammasome activator. Mechanistically, Lcn2 knockdown downregulated the expression of IL-17a and its receptor IL-17ra. Recombinant IL-17A treatment markedly reversed the protective effects of Lcn2 silencing in vitro and in vivo. CONCLUSION: Lcn2 knockdown inhibits intestinal inflammation and pyroptosis in NEC through the blockade of the IL-17A pathway, providing a promising therapeutic approach for NEC treatment.