Fig 1.
Ulinastatin (UTI) attenuated sepsis-associated acute lung injury (SALI) and endothelial barrier dysfunction in vivo and vitro.
(A-B) Sepsis is induced by cecal ligation and puncture (CLP), treated with UTI (100 KU/kg, i.p.) or dexamethasone (DEX, 0.5 mg/kg, i.p.). The rats were harvested 24h after surgery. Representative images of H & E staining of rat lung tissue section (upper scale bar = 200μm, bottom panel = 50μm). H & E stainings indicate signs of edema (blue arrows), hemorrhage (red arrows), inflammation (black arrows), and alveolar septal thickening (yellow arrows) from the cross-section of the lung (n = 6). Quantification of lung injury scores was determined in panel B. (C-D) Lung wet-to-dry weight ratio and Evans Blue dye extravasation were determined in lung tissues (n = 6). (E-F) The levels of TNF-α and IL-6 in serum were measured by ELISA (n = 6). (G) HUVECs were exposed to UTI for 24h, and cell viability was determined by CCK8 (n = 6). (H-I) HUVECs were pretreated with UTI (10 KU/mL) or DEX (10 μM) for 1 hour before LPS stimuli for 24 h. Levels of IL-6 and TNF-α in the supernatants of HUVECs were examined by ELISA (n = 6). (J) Pattern diagram for the Transwell model. The endothelial permeability was estimated by the fluorescence signal intensity of FITC-FD40 from the Transwell system (n = 6). All data were expressed as the mean ± SD. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. ##p < 0.01, ###p < 0.001, and ####p ＜ 0.0001.
Fig 2.
UTI improved endothelial barrier function by protecting the junction proteins.
(A) Representative immunohistochemical staining images of ZO-1, VE-cadherin, and occludin in lung paraffin sections. Dark brown dots indicate positively stained cells (upper scale bar = 200μm, bottom panel = 50μm). (B) Morphometric analysis of immunostained area for each protein in relation to total area was performed quantitatively using Fiji software (n = 6). (C-D) Protein levels of ZO-1, VE-cadherin, and occludin in HUVECs were determined by western blot. The relative protein levels are expressed as the ratio of the target protein to Beta-Actin, determined in panel D. (E) Immunofluorescent staining showing the expression and distribution of intercellular junction protein (ZO-1) in LPS-challenged HUVEC. RED represents ZO-1, and blue indicates nuclei (Scale bar = 20μm). (F) Quantitative analysis of relative fluorescence intensity for ZO-1 was performed using Fiji software (n = 6). All data were expressed as the mean ± SD. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p ＜ 0.0001, &&&p < 0.001.
Fig 3.
UTI suppressed the expression of angiopoietin-2 (Ang-2) in vivo and activated the PI3K/Akt/FoxO1 signaling pathway in vitro.
(A) Representative immunohistochemical staining images of Ang-2 in lung paraffin sections. Dark brown dots indicate positively stained cells. Morphometric analysis of immunostained area for each protein in relation to the total area was performed quantitatively using Fiji software (upper scale bar = 200μm, bottom panel = 50μm) (n = 6). (B) The levels of Ang-2 in serum were measured by ELISA (n = 6). (C) Molecular docking showing the interaction between UTI and phosphatidylinositol 3-kinase (PI3K). (D-E) Protein levels of p-Akt and p-FoxO1 in HUVECs were determined by western blot (n = 3). All data were expressed as the mean ± SD. ns = no significance, ***p < 0.0001. #p < 0.05 and ####p ＜ 0.0001.
Fig 4.
The UTI-induced activation of the PI3K/AKT/FoxO1 pathway was abrogated by wortmannin.
(A-B) HUVECs were pretreated with UTI (10 KU/mL) or DEX (10 μM) for 1 h before LPS stimuli for 24 h. Immunofluorescent staining of FoxO1 showing the nuclear translocation in LPS-challenged HUVECs with UTI or DEX treatment (Scale bar = 20μm). Quantitative analysis of the ratio of nuclear area to total area in FoxO1 protein immunofluorescence staining regions was performed using Fiji software in panel B (n = 6). (C-E) HUVECs were pretreated with UTI (10 KU/mL), with or without the PI3K inhibitor wortmannin (WM, 1 μM). Protein levels of p-Akt and p-FoxO1 in HUVECs were analyzed by western blot. Quantitative data of Akt and FoxO1 phosphorylation in HUVECs in panels D, E (n = 3). (F-G) Immunofluorescent staining of FoxO1 showing the nuclear translocation in HUVECs (Scale bar = 20μm). Quantitative analysis of the ratio of nuclear area to total area in FoxO1 immunofluorescence staining regions was performed using Fiji software in panel G (n = 6). (H-J) CLP rats were intraperitoneally injected with UTI or WM (0.7 mg/kg). Representative western blots of p-Akt and p-FoxO1 in the lung tissue harvested 24 h after CLP surgery. Quantitative data of Akt and FoxO1 phosphorylation in the lung tissues in panel J (n = 3). All data were expressed as the mean ± SD. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. ##p < 0.01 and ####p ＜ 0.0001. &&&&p < 0.0001.
Fig 5.
The protective effect of UTI in SALI and endothelial dysfunction is mediated by the PI3K/Akt/FoxO1 pathway.
(A-B) CLP model rats were intraperitoneally injected with UTI or WM. H&E staining showing the tissue injury in the lung (upper scale bar = 200μm, bottom panel = 50μm). Quantification of lung injury scores was determined in panel B (n = 6). (C-D) Lung wet-to-dry weight ratio and Evans Blue dye extravasation were determined in lung tissues (n = 6). (E-F) The levels of TNF-α and IL-6 in serum were measured by ELISA (n = 6). (G-H) HUVECs were pretreated with UTI or WM before LPS stimuli for 1 hour. Levels of IL-6 and TNF-α in the supernatants of HUVECs were examined by ELISA (n = 6). (I) The fluorescence signal intensity of FITC-FD40 in HUVECs (n = 6). (J-K) Representative immunohistochemical staining images and relative analysis of Ang-2 in lung paraffin sections (upper scale bar = 200μm, bottom panel = 50μm) (n = 6). (L) The levels of Ang-2 in serum were measured by ELISA (n = 6). All data were expressed as the mean ± SD. ns = no significance, *p < 0.05 and ****p < 0.0001. #p < 0.01, ##p < 0.01, and ####p ＜ 0.0001.
Fig 6.
The protective effect of UTI on junction proteins is mediated by the PI3K/Akt/FoxO1 pathway.
(A-B) Protein levels of ZO-1, VE-cadherin, and occludin in HUVECs were analyzed by western blot. The relative protein levels are expressed in panel B (n = 3). (C) Representative immunofluorescence staining images of ZO-1 in HUVECs (n = 6). (Scale bar = 20μm). (D) Quantitative analysis of relative fluorescence intensity for ZO-1 was performed using Fiji software (n = 6). (E-F) Representative Western blots of ZO-1, VE-cadherin, and occludin protein expression in rat lung tissues. The relative protein levels are expressed in panel E (n = 3). (G-H) Immunohistochemical staining showing the levels and distribution of ZO-1, VE-cadherin, and occludin in lung tissues in panel G (upper scale bar = 200μm, bottom panel = 50μm). All data were expressed as the mean ± SD. ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. ##p < 0.01. &p < 0.01, &&p < 0.01.
Fig 7.
In sepsis, Ang-2 was activated for endothelial destabilization, leading to endothelial barrier dysfunction. UTI attenuated capillary leakage in sepsis-associated acute lung injury by activating the PI3K/Akt/FoxO1 pathway and breaking the vicious cycle of FoxO1-dependent Ang-2 production. Created in BioRender. Yating, L. (2026) https://BioRender.com/wud3kqb.