Annexin A1 Mitigates Blood-Brain Barrier Disruption in a Sepsis-Associated Encephalopathy Model by Enhancing the Expression of Occludin and Zonula Occludens-1 (ZO-1)

Abstract

Aims: This study investigated the protective role of Annexin A1 (ANXA1) in sepsis-associated encephalopathy (SAE) by examining its effects on brain vascular endothelium and blood-brain barrier (BBB) integrity.

Methods: Mice were divided into four groups: wild type (WT), cecal ligation and puncture (CLP), ANXA1 knockout (ANXA1[-/-]), and ANXA1(-/-) with CLP. Neurobehavioral changes were assessed using the Y-maze test, while BBB integrity was evaluated through Evans blue dye (EBD) staining and permeability tests with fluorescein isothiocyanate (FITC)-dextran.

Results: Results showed that ANXA1 levels were reduced in septic mice, and its deficiency exacerbated cognitive impairment and survival rate reduction. ANXA1 deficiency also upregulated proinflammatory cytokines and adhesion molecules, worsened BBB impairment, and altered expression of tight junction proteins and VEGF-A/VEGF-R2. In vitro, ANXA1 Ac2-26 prevented LPS-induced increased permeability in bEnd.3 cells by restoring tight junction proteins and reducing VEGF-A/VEGF-R2 expression. Notably, VEGF-A negated the protective effects of ANXA1 Ac2-26.

Conclusion: The study concludes that ANXA1 reduces BBB permeability to protect against sepsis-induced brain dysfunction via VEGF-A/VEGF-R2 regulation of tight junction proteins, suggesting ANXA1 as a potential therapeutic for SAE.

Keywords: VEGF; annexin A1; blood brain barrier; occludin; sepsis‐associated encephalopathy.

FIGURE 1

Reduced expression of Annexin A1 (ANXA1) was observed in the brain cortex of septic mice. The septic mice model was established using cecal ligation and puncture (CLP). (A) ANXA1 mRNA levels in the cortex; (B) ANXA1 protein levels (***p < 0.005 vs. WT group).

FIGURE 2

Annexin A1 (ANXA1) deficiency reduced the 7‐day survival rate and exacerbated CLP‐induced neurobehavioral changes in septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) The survival rate was analyzed at day 0, 1, 2, 3, 4, 5, 6, and 7 post‐operation; (B) cognitive function at days 3, 5, and 7 after the operation was assessed using the Y‐maze test (***p < 0.005 vs. WT group; ^### p < 0.005 vs. ANXA1 (−/−) group; ^&& p < 0.01 vs. CLP group).

FIGURE 3

ANXA1 deficiency exacerbated the expression of inflammatory cytokines IL‐6, IL‐8, HMGB1, and TNF‐α in the brain cortex of septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) MRNA levels of IL‐6, IL‐8, HMGB1, and TNF‐α in the cortex; (B) protein levels of IL‐6, IL‐8, HMGB1, and TNF‐α in the cortex (***p < 0.005 vs. WT group; ^### p < 0.005 vs. ANXA1 (−/−) group; ^&& p < 0.01 vs. CLP group).

FIGURE 4

ANXA1 deficiency aggravated the expression of biomarkers of vascular endothelial dysfunction in the brain cortex of septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) MRNA levels of VCAM‐1, ICAM‐1, and MCP‐1 in the cortex; (B) protein levels of VCAM‐1, ICAM‐1, and MCP‐1 in the cortex (***p < 0.005 vs. WT group; ^### p < 0.005 vs. ANXA1 (−/−) group; ^&& p < 0.01 vs. CLP group).

FIGURE 5

ANXA1 deficiency exacerbated the dysfunction of BBB integrity and reduction of occludin and ZO‐1 in the brain cortex of septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) BBB permeability was measured using EBD staining; (B) mRNA levels of occludin and ZO‐1; (C) protein levels of occludin and ZO‐1 as measured by immunostaining. Scale bar, 100 μm (***p < 0.005 vs. WT group; ^### p < 0.005 vs. ANXA1 (−/−) group; ^&& p < 0.01 vs. CLP group).

FIGURE 6

ANXA1 deficiency aggravated the increase in the expression of VEGF‐A and VEGF‐R2 in the brain cortex of septic mice. Mice were divided into four groups: WT, CLP, ANXA1 (−/−), and ANXA1 (−/−) + CLP. (A) mRNA levels of VEGF‐A and VEGF‐R2; (B) protein levels of VEGF‐A and VEGF‐R2. Scale bar, 100 μm (***p < 0.005 vs. WT group; ^### p < 0.005 vs. ANXA1 (−/−) group; ^&& p < 0.01 vs. CLP group).

FIGURE 7

The peptide Annexin A1 (Ac2‐26) prevented LPS‐induced increased brain endothelial monolayer permeability in bEnd.3 brain microvascular endothelial cells (BVECs). Cells were stimulated with LPS (1 μg/mL) with or without ANXA1 (Ac2‐26) (0.25, 0.5 μM) for 48 h. (A) Endothelial permeability was assessed by FITC‐dextran permeation; (B) protein expression of occludin and ZO‐1 (***p < 0.005 vs. control group; ^##, ^### p < 0.01, 0.005 vs. LPS group).

FIGURE 8

Annexin A1 (Ac2‐26) reduced the expression of VEGF‐A and VEGF‐R2 in bEnd.3 BVECs. Cells were stimulated with LPS (1 μg/mL) with or without ANXA1 (Ac2‐26) (0.25, 0.5 μM) for 48 h. (A) mRNA levels of VEGF‐A and VEGF‐R2; (B) protein levels of VEGF‐A; (C) protein levels of VEGF‐R2 (***p < 0.005 vs. control group; ^##, ^### p < 0.01, 0.005 vs. LPS + group).

FIGURE 9

VEGF‐A abolished the protective effects of ANXA1 (Ac2‐26) against LPS‐induced increased brain endothelial monolayer permeability in bEnd.3 BVECs. Cells were stimulated with LPS (1 μg/mL) with or without ANXA1 (Ac2‐26) (0.5 μM) or VEGF‐A (10 ng/mL). (A) Brain endothelial permeability was assessed by FITC‐dextran permeation; (B) protein expression of occludin and ZO‐1 (***p < 0.005 vs. control group; ^## p < 0.01 vs. LPS group; ^&& p < 0.01 vs. LPS + ANXA1 group).