Heparin-binding protein aggravates sepsis-induced acute lung injury by inhibiting the PERK-Nrf2-HO-1 signaling pathway

Abstract

Sepsis, a systemic inflammatory response disorder triggered by infection, often leads to multiple organ dysfunction syndrome. Sepsis-induced acute lung injury (SI-ALI) is among the most common complications. Heparin-binding protein (HBP), a major inflammatory mediator, has been demonstrated to induce vascular permeability and inflammation. Nevertheless, the mechanism by which HBP functions in sepsis-induced lung injury remains unclear. This study aimed to explore the role of HBP in sepsis-induced lung injury. A sepsis mouse model was established via cecal ligation and puncture (CLP), followed by HBP blockade using heparin. Serum inflammatory cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA). Neutrophil infiltration was assessed by myeloperoxidase (MPO) activity. Lung tissue pathology was evaluated using hematoxylin-eosin (H&E) and Masson staining. Levels of ferrous iron (Fe²⁺), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase 4 (GPX4) in lung tissue lysates were quantified using commercial assay kits. Acyl-CoA synthetase long-chain family member 4 (ACSL4) expression was examined by immunohistochemistry. Reactive oxygen species (ROS) levels in lung tissues were measured using dihydroethidium (DHE) fluorescence probes. Western blot was performed to analyze the protein kinase R-like endoplasmic reticulum kinase-nuclear factor erythroid 2-related factor 2-heme oxygenase-1 (PERK-Nrf2-HO-1) signaling pathway. Additionally, an in vitro sepsis cell model was established by treating murine lung epithelial (MLE-12) cells with lipopolysaccharide (LPS) to further validate HBP’s role. HBP expression was significantly upregulated in the lung tissues of CLP mice. Pharmacological blockade of HBP reduced serum inflammatory cytokine levels, alleviated neutrophil infiltration and lung pathological injury, and attenuated ferroptosis-related alterations in septic lung tissues. HBP blockade was associated with enhanced activation of the PERK-Nrf2-HO-1 signaling pathway. Knockdown of PERK, Nrf2 or HO-1 partially abrogated the protective effects of HBP blockade against SI-ALI and ferroptosis phenotypes. Furthermore, in vitro experiments showed that HO-1 overexpression attenuated the exacerbating effect of HBP on LPS-induced ferroptosis in MLE-12 cells. The findings suggest that HBP may contribute to the progression of sepsis-induced lung injury, at least in part, by promoting ferroptosis through inhibiting the activation of the PERK-Nrf2-HO-1 signaling pathway.