RESUMO
BACKGROUND: Vascular endothelial dysfunction is considered a key pathophysiologic process for the development of acute lung injury. In this study, we aimed at investigating the effects of unfractionated heparin (UFH) on the lipopolysaccharide (LPS)-induced changes of vascular endothelial-cadherin (VE-cadherin) and the potential underlying mechanisms. METHODS: Male C57BL/6 J mice were randomized into three groups: vehicle, LPS, and LPSâ+âUFH groups. Intraperitoneal injection of 30 mg/kg LPS was used to induce sepsis. Mice in the LPSâ+âUFH group received subcutaneous injection of 8 U UFH 0.5 h before LPS injection. The lung tissue of the mice was collected for assessing lung injury by measuring the lung wet/dry (W/D) weight ratio and observing histological changes. Human pulmonary microvascular endothelial cells (HPMECs) were cultured and used to analyze the effects of UFH on LPS- or tumor necrosis factor-alpha (TNF-α)-induced vascular hyperpermeability, membrane expression of VE-cadherin, p120-catenin, and phosphorylated myosin light chain (p-MLC), and F-actin remodeling, and on the LPS-induced activation of the phosphatidylinositol-3 kinase (PI3K)/serine/threonine kinase (Akt)/nuclear factor kappa-B (NF-κB) signaling pathway. RESULTS: In vivo, UFH pretreatment significantly attenuated LPS-induced pulmonary histopathological changes (neutrophil infiltration and erythrocyte effusion, alveolus pulmonis collapse, and thicker septum), decreased the lung W/D, and increased protein concentration (LPS vs. LPSâ+âUFH: 0.57â±â0.04 vs. 0.32â±â0.04âmg/mL, Pâ=â0.0092), total cell count (LPS vs. LPSâ+âUFH: 9.57â±â1.23 vs. 3.65â±â0.78â×â10/mL, Pâ=â0.0155), polymorphonuclear neutrophil percentage (LPS vs. LPSâ+âUFH: 88.05%â±â2.88% vs. 22.20%â±â3.92%, Pâ=â0.0002), and TNF-α (460.33â±â23.48 vs. 189.33â±â14.19âpg/mL, Pâ=â0.0006) in the bronchoalveolar lavage fluid. In vitro, UFH pre-treatment prevented the LPS-induced decrease in the membrane expression of VE-cadherin (LPS vs. LPSâ+âUFH: 0.368â±â0.044 vs. 0.716â±â0.064, Pâ=â0.0114) and p120-catenin (LPS vs. LPSâ+âUFH: 0.208â±â0.018 vs. 0.924â±â0.092, Pâ=â0.0016), and the LPS-induced increase in the expression of p-MLC (LPS vs. LPSâ+âUFH: 0.972â±â0.092 vs. 0.293â±â0.025, Pâ=â0.0021). Furthermore, UFH attenuated LPS- and TNF-α-induced hyperpermeability of HPMECs (LPS vs. LPSâ+âUFH: 8.90â±â0.66 vs. 15.84â±â1.09 Ω·cm, Pâ=â0.0056; TNF-α vs. TNF-α + UFH: 11.28â±â0.64 vs. 18.15â±â0.98 Ω·cm, Pâ=â0.0042) and F-actin remodeling (LPS vs. LPSâ+âUFH: 56.25â±â1.51 vs. 39.70â±â1.98, Pâ=â0.0027; TNF-α vs. TNF-α + UFH: 55.42â±â1.42 vs. 36.51â±â1.20, Pâ=â0.0005) in vitro. Additionally, UFH decreased the phosphorylation of Akt (LPS vs. LPSâ+âUFH: 0.977â±â0.081 vs. 0.466â±â0.035, Pâ=â0.0045) and I kappa B Kinase (IKK) (LPS vs. LPSâ+âUFH: 1.023â±â0.070 vs. 0.578â±â0.044, Pâ=â0.0060), and the nuclear translocation of NF-κB (LPS vs. LPSâ+âUFH: 1.003â±â0.077 vs. 0.503â±â0.065, Pâ=â0.0078) in HPMECs, which was similar to the effect of the PI3K inhibitor, wortmannin. CONCLUSIONS: The protective effect of UFH against LPS-induced pulmonary endothelial barrier dysfunction involves VE-cadherin stabilization and PI3K/Akt/NF-κB signaling.
