Protective effects of 4-HBd on blood-brain barrier integrity in MCAO/R model rats based on brain pharmacokinetic characteristics

Jin Feng Qian Yang Ming Chen Yan Wang Dan Luo Dongxiong Hu Jianjun Cheng Xuelan Song Xiaonan Zhou Qingting Meng Qing Lin ^* Fangyan He ^*

• Yunnan University of Traditional Chinese Medicine, Kunming, China

Objectives: This study explored the brain-targeting properties and mechanisms of 4hydroxybenzaldehyde (4-HBd), the primary active component of Gastrodia elata, in mitigating ischemic stroke (IS)-induced injury by preserving blood-brain barrier (BBB) integrity, based on brain pharmacokinetic characteristics. Methods: The anti-IS effects of Gastrodia elata extract were assessed using a rat middle cerebral artery occlusion/reperfusion (MCAO/R) model, leading to the identification of 4-HBd as the principal active ingredient. BBB protection was evaluated through neurological scoring, Evans Blue (EB) extravasation, cerebral infarct volume, and ultrastructural integrity. Oxidative stress markers, including superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO), and inducible nitric oxide synthase (iNOS), were quantified in ischemic brain tissue via biochemical assays. The expression levels of tight junction (TJ) proteins claudin-5 and occludin, as well as matrix metalloproteinase MMP-2/9 and aquaporin-4 (AQP-4), were analyzed by western blotting. Microdialysis, combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), was employed to determine the temporal distribution of 4-HBd in the brains of both normal and MCAO/R model rats. The ability of 4-HBd to scavenge intracellular reactive oxygen species (ROS) in brain endothelial cells (bEnd.3) was evaluated using a single-cell biochemical analyzer. Results: Gastrodia elata ethanol extract exhibited significant anti-IS effects. Compared with the model group, 4-HBd treatment markedly alleviated BBB disruption and neurological deficits, suppressed oxidative stress in ischemic brain tissue, reduced MDA and NO levels, and enhanced SOD activity. The expression of claudin-5, occludin, MMP-2/9, and AQP-4 was significantly up-regulated in the 4-HBd group relative to the model group. Additionally, 4-HBd selectively eliminated nuclear-derived ROS. Pharmacokinetic analysis demonstrated that 4-HBd preferentially accumulated in the striatum and cortex of both normal and MCAO/R model rats. Under ischemic conditions, 4-HBd exhibited accelerated cortical penetration, increased exposure, and prolonged retention. Conclusions: These findings indicate that 4-HBd exerts a pronounced brain-targeting effect and preserves BBB integrity via the RNS/ROS-MMP-TJ signaling pathway, highlighting its potential as a therapeutic agent for IS.