DL-3-n-butylphthalide Protects the Blood-Brain Barrier Against Ischemia/Hypoxia Injury via Upregulation of Tight Junction Proteins
Brain ischemia is a leading cause of neurological disability, often accompanied by increased permeability of the blood-brain barrier (BBB). The BBB is a critical diffusion barrier between capillaries and neurons, regulating the passage of ions, water, solutes, and large molecular proteins. Disruption of the BBB during ischemia and reperfusion leads to an influx of vascular-derived substances into the brain, exacerbating neuronal injury. Tight junctions (TJs) are essential components of the BBB, and their alteration is closely associated with increased BBB permeability. DL-3-n-butylphthalide (NBP), a compound extracted from the seeds of Chinese celery, has demonstrated neuroprotective effects in ischemic stroke. However, the relationship between NBP and TJs remains poorly understood. This study investigates the potential effects of NBP on TJ proteins, including claudin-5, zonula occludens-1 (ZO-1), and occludin, during brain ischemia.
To explore the effects of NBP on the BBB, a chronic cerebral hypoperfusion (CCH) model was established in Sprague-Dawley rats by permanent occlusion of the bilateral common carotid arteries (BCCAO). Rats were divided into five groups: sham, model, and three NBP treatment groups (20, 40, and 80 mg/kg, administered by oral gavage once daily for 14 days). BBB permeability was assessed using the Evans blue dye leakage assay. The expressions of TJ proteins, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3b (p-GSK-3b/GSK-3b), and b-catenin/b-actin were evaluated using Western blotting and immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry, and TJ ultrastructure was observed using transmission electron microscopy.
In the CCH rat model, NBP treatment significantly reduced Evans blue dye leakage in the brain tissue, indicating decreased BBB permeability. Specifically, the Evans blue content in the NBP-M (40 mg/kg) and NBP-H (80 mg/kg) groups was 9.0 ± 0.9 mg/g and 6.7 ± 0.6 mg/g, respectively, compared to 12.3 ± 1.9 mg/g in the model group. These results suggest that NBP attenuates ischemia-induced BBB disruption in a dose-dependent manner. Furthermore, NBP treatment upregulated the expression of claudin-5 and ZO-1 in brain microvascular segments. The expression of claudin-5 in the NBP-M and NBP-H groups was 0.79 ± 0.08 and 0.97 ± 0.07, respectively, compared to 0.41 ± 0.06 in the model group. Similarly, ZO-1 expression was significantly increased in the NBP-M and NBP-H groups. However, no significant change in occludin expression was observed.
To further investigate the effects of NBP on TJs, an in vitro hypoxia model was established using primary cultured rat brain microvascular endothelial cells (BMECs). BMECs were exposed to hypoxia (1% O2 for 24 hours) with or without NBP pre-treatment (0.1 or 1.0 mmol/L). NBP treatment improved TJ ultrastructure, as observed by transmission electron microscopy, and reduced intracellular ROS levels. In the hypoxia group, ROS generation was significantly increased (73.2 ± 7.4%) compared to the control group (7.1 ± 1.7%). NBP treatment reduced ROS levels to 25.6 ± 3.0% and 17.3 ± 2.6% at concentrations of 0.1 mmol/L and 1.0 mmol/L, respectively. Additionally, NBP increased the expression of claudin-5 and ZO-1 in BMECs under hypoxic conditions. The expression of claudin-5 in the NBP-treated groups was 0.45 ± 0.06 and 0.55 ± 0.06, compared to 0.34 ± 0.05 in the hypoxia group. ZO-1 expression also increased significantly in the NBP-treated groups. In contrast, occludin expression remained unchanged in the hypoxia model but was upregulated in the oxygen-glucose deprivation/reoxygenation (OGD/R) model with NBP treatment.
The study also explored the signaling pathways involved in the protective effects of NBP on TJs. Hypoxia decreased the relative expression of p-Akt/Akt, p-GSK-3b/GSK-3b, and b-catenin/b-actin in BMECs. NBP treatment elevated the expression levels of these proteins, suggesting that NBP may preserve BBB integrity by activating the Akt/GSK-3b/b-catenin signaling pathway. Specifically, the relative expression of p-Akt/Akt in the NBP-treated groups increased to 0.36 ± 0.07 and 0.42 ± 0.08, compared to 0.22 ± 0.07 in the hypoxia group. Similarly, p-GSK-3b/GSK-3b and b-catenin/b-actin levels were significantly higher in the NBP-treated groups.
In conclusion, NBP improves the barrier function of the BBB against ischemic injury by upregulating the expression of TJ proteins, particularly claudin-5 and ZO-1. The protective effects of NBP may be mediated by reducing oxidative stress and activating the Akt/GSK-3b/b-catenin signaling pathway. These findings provide a mechanistic basis for the clinical use of NBP in treating brain ischemia and stroke.
doi.org/10.1097/CM9.0000000000000232
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