LCN2 knockout protects endothelial barrier function in mice with sepsis-induced acute lung injury by regulating the NF-κB signaling pathway

doi:10.12122/j.issn.1673-4254.2026.08.06

Abstract

Abstract:

Objective To investigate the protective effect of lipocalin-2 (LCN2) gene knockout against sepsis-induced acute lung injury (ALI) and vascular endothelial dysfunction in mice and the mediating role of the nuclear factor-κB (NF-κB) signaling pathway. Methods Wild-type (WT) C57BL/6 mice receiving sham operation or cecal ligation and puncture (CLP) to induce sepsis were randomized into 3 subgroups for intraperitoneal injections of saline, PDTC, or Bay 11-7082 (n=10). Twenty LCN2 knockout (LCN2KO) mice were randomized for sham operation or CLP modeling. Pulmonary histopathological changes, lung wet-to-dry (W/D) ratio, lung index, Evans blue extravasation, and serum TNF-α and IL-6 levels in the mice were assessed. The colocalization of NF-κB, ICAM-1, and VCAM-1 with CD31 were analyzed using immunofluorescence staining, and endothelial cell apoptosis was examined with TUNEL/CD31 double staining. Western blotting and co-immunoprecipitation assay were performed to analyze the protein expressions of LCN2, p-P65/P65, ICAM-1, VCAM-1 and VE-cadherin and the interaction between LCN2 and P65. Results The WT mice receiving CLP showed severe lung structural damage with obvious edema and inflammation. In contrast, both the LCN2KO mouse and PDTC-treated WT mouse models of CLP showed milder lung injury with lower W/D ratio, lung index, Evans blue leakage, serum TNF‑α and IL-6 levels and markedly reduced colocalization signals of NF-κB, ICAM-1, and VCAM-1 with CD31. CLP resulted in significantly increased expression levels of LCN2, p-P65/P65, ICAM-1, and VCAM-1, lowered VE-cadherin levels, and enhanced endothelial cell apoptosis, and all these changes were significantly ameliorated in LCN2KO mice and WT mice with NF‑κB inhibitor treatment. Co-immunoprecipitation results suggested the interaction between lung LCN2 and P65. Bay 11-7082 significantly reduced inflammatory cytokine production, downregulated p-P65 expression and its downstream adhesion molecules, and restored VE-cadherin expression. Conclusion LCN2 knockout alleviates sepsis-induced ALI and vascular endothelial dysfunction in mice by inhibiting NF-κB signaling.

Key words: lipocalin-2, sepsis-induced acute lung injury, endothelial dysfunction, nuclear factor-κB signaling pathway

Mengjie YU, Anbo ZHAO, Yixuan HAO, Yiren WANG, Yuexian LIU, Hongwei YE, Qin GAO. LCN2 knockout protects endothelial barrier function in mice with sepsis-induced acute lung injury by regulating the NF-κB signaling pathway[J]. Journal of Southern Medical University, 2026, 46(8): 1779-1789.

Figures/Tables 6

Fig.1 HE staining for pathological analysis of mouse lung tissues indifferent groups (A) and lung injury scores (B; Mean±SD, n=4). ###P<0.001 vs WT-Sham group; *P<0.05, **P<0.01 vs WT-CLP group.

Fig.2 Lung index (A) and wet-to-dry (W/D) ratio (B) of the mice (Mean±SD, n=6). ##P<0.01 vs WT-Sham group; **P<0.01, ***P<0.001 vs WT-CLP group.

Fig.4 Changes of serum TNF-α (A) and IL-6 (B) levels in different groups (Mean±SD, n=6). ##P<0.01,###P<0.001 vs WT-Sham group; **P<0.01, ***P<0.001 vs WT-CLP group.

Fig.7 Proteins expression levels in mouse lung tissues from different groups. A: Western blots of the proteins in the lung tissues. B: Quantitative analysis of the protein expression levels (Mean±SD, n=4). ##P<0.01, ###P<0.001 vs WT-Sham group; *P<0.05, **P<0.01, ***P<0.001 vs WT-CLP group.

Fig.8 Co-immunoprecipitation analysis of the interaction between LCN2 and P65 in mouse lung tissue.

Fig.9 Changes of proteins expression levels in mouse lung tissues and serum inflammatory cytokine levels in different groups. A: Western blots of the proteins in lung tissues. B: Quantitative analysis of protein expression levels (Mean±SD, n=4). C: Serum TNF-α and IL-6 levels measured by ELISA (Mean±SD, n=6). ###P<0.001 vs WT-Sham group; *P<0.05, **P<0.01, ***P<0.001 vs WT-CLP group.

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