Barrier-preserving effects of 2-O, 3-O desulfated heparin via TLR4–MyD88–NF-κB modulation in LPS-induced acute lung injury

Bacterial metabolite lipopolysaccharide (LPS) triggers acute lung injury (ALI), by disrupting endothelial barrier integrity and a surge in pro-inflammatory signaling. To date, no promising therapeutic options are available to combat ALI. Accounting to this need, we hypothesized to test 2-O, 3-O desulfated heparin (ODSH), to attenuate LPS-induced endothelial dysfunction and inflammatory gene expression in human lung microvascular endothelial cells (HLMVECs) and CD-1 mice. Recent literature has demonstrated that ODSH, a low-anticoagulant heparin derivative, exhibits protective effects in models of cystic fibrosis and thrombin-induced permeability via modulating HMGB1 release and inflammatory cascades. In the present study, confluent HLMVEC monolayers were pretreated with 50 µg/mL ODSH for 40 min, followed by 100 eu/mL LPS exposure. Transendothelial electrical resistance (TER) was monitored for 18 h using ECIS. Parallel in vivo studies were conducted in CD-1 mice subjected to intratracheal LPS with or without intravenous ODSH. Gene expression profiling (IL-1β, TNF-α, IL-6, TLR4, MyD88, NF-κB, RAGE, iNOS, p38, MAPK1, ICAM1, CCL2, IL-10, SOCS3, CDH5, OCLN, CLDN5, and ZO1) was performed on HLMVECs and lung tissues. Western blot was performed for NF-κB, phospho-p38, MyD88, and IL-6. Apoptosis was evaluated by flow cytometry using an Annexin V detection kit. The results indicate that ODSH pretreatment significantly preserved TER in LPS-challenged HLMVECs, indicating enhanced barrier function. In both HLMVECs and murine lungs, ODSH regulated pro-inflammatory mediators (IL-1β, TNF-α, IL-6, NF-κB, iNOS, p38, ICAM1, and CCL2), anti-inflammatory signaling (IL-10) and barrier-stabilizing/protective genes (SOCS3, CDH5, OCLN, CLDN5, and ZO1) in response to LPS challenge. ODSH treatment also suppressed upstream receptor-driven signaling, with reduced expression of TLR4, MyD88, RAGE, and MAPK1. Western blot analysis demonstrated that ODSH attenuates LPS-induced upregulation of NF-κB, phospho-p38, MyD88, and IL-6 protein expression. ODSH also prevented the cell death against LPS challenge. These effects align with our prior findings and data from literature. In conclusion, ODSH mitigates LPS-induced endothelial hyperpermeability and inflammatory gene activation in vitro and in vivo. Specifically, ODSH may inhibit inflammatory events by targeting TLR4-MyD88-NF-κB axis and restore endothelial function. Its dual action-enhancing barrier integrity and modulating cytokine signaling-supports its potential as a therapeutic agent for ALI and ARDS. These findings reinforce the translational relevance of ODSH in pulmonary vascular inflammation and barrier regulation. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.