Background: Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are critical conditions lacking effective pharmacologic therapies. Lipopolysaccharide (LPS), a bacterial endotoxin, is a well-established trigger of ALI. Emerging evidence suggests that heparin derivatives may attenuate lung injury, but their mechanisms remain unclear. Methods: This study evaluated the protective effects of 2-O, 3-O desulfated heparin (ODSH) in a murine model of LPS-induced ALI. Mice received LPS intratracheally with or without ODSH pre-treatment. Lung injury was assessed by bronchoalveolar lavage fluid (BALF) analysis, Evans blue dye albumin EBDA) extravasation, and histopathology. Results: ODSH treatment significantly reduced BALF protein concentration, inflammatory cell infiltration, and EBDA leakage. ODSH preserved endothelial barrier function in vitro, as evidenced by transendothelial electrical resistance (TER) measurements in human lung microvascular endothelial cell (HLMVEC) monolayers. Histological assessment (H&E staining) and myeloperoxidase (MPO) staining demonstrated reduced lung injury and neutrophil infiltration in the ODSH group. ODSH also downregulated pro-inflammatory mediators (NF-κB, IL-6, p38 MAPK) and upregulated the anti-inflammatory cytokine IL-10. Conclusions: ODSH mitigates LPS-induced ALI by reducing vascular permeability, neutrophilic inflammation, and pro-inflammatory signaling while enhancing IL-10 expression. These findings suggest ODSH may offer a novel therapeutic approach for treating ALI.

Bacterial lipopolysaccharide (LPS) disrupts endothelial barrier stability by inducing inflammatory events that underlie acute lung injury (ALI). Effective pharmacologic therapies capable of modifying the disease mechanisms of ALI remain limited. We previously demonstrated that 2-O, 3-O desulfated heparin (ODSH), a modified heparin with minimal anticoagulant activity and anti-inflammatory properties, attenuates LPS-induced ALI in mice and preserves human lung microvascular endothelial cell (HLMVEC) barrier integrity. In the present study, we further investigated the mechanisms of ODSH-mediated protection in mice and HLMVECs, focusing on inflammatory mediator expression and the modulation of TLR4–MyD88–NF-κB signaling. HLMVECs were pretreated with ODSH before LPS exposure, and barrier function was monitored using transendothelial electrical resistance measurements. Complementary experiments were performed in CD-1 mice receiving intratracheal LPS with or without intravenous ODSH. Across both models, ODSH attenuated LPS-induced endothelial barrier dysfunction. ODSH significantly reduced bronchoalveolar lavage fluid (BALF) protein accumulation, inflammatory cell infiltration, IL-6 and TNF-α concentrations, Evans Blue dye extravasation, and pulmonary edema. Gene expression analyses demonstrated that ODSH suppressed multiple pro-inflammatory mediators and inflammatory signaling components, including IL-1β, TNF-α, IL-6, NOS2, MAPK14, MAPK1, ICAM1, and CCL2, while enhancing IL-10 and several barrier-associated genes (CDH5, OCLN, CLDN5, and TJP1). ODSH also reduced the mRNA expression of TLR4, MYD88, and RELA. Protein analyses confirmed a reduced expression of NF-κB, MyD88, and TNF-α. Furthermore, ODSH at concentrations of 5–100 μg/mL did not significantly affect cellular metabolic activity. Collectively, these findings indicate that ODSH preserves endothelial barrier function and attenuates inflammatory responses associated with TLR4–MyD88–NF-κB signaling in experimental ALI. The findings support the further mechanistic and preclinical investigation of ODSH in inflammatory lung injury.