DIA-based proteomics reveals anti-inflammatory role of DL-3-n-butylphthalide in cerebral small vessel disease-induced brain injury in hypertensive rat

Abstract Objectives Excessive neuroinflammatory responses represent a key pathological mechanism in cerebral small vessel disease (CSVD). Dl-3- n -butylphthalide (NBP), a compound previously demonstrated to possess anti-inflammatory properties in ischemic stroke, was investigated for its potential therapeutic effects in a rodent model of CSVD. This study aimed to elucidate the neuroprotective mechanisms of NBP in CSVD pathogenesis. Methods Forty-week-old spontaneously hypertensive rats were selected as a CSVD rodent model to determine the neuroprotective effects of NBP. Cognitive ability was assessed using the Morris water maze after 28 weeks of treatment. Pathological changes in the brain tissue were observed through immunohistochemistry. Data-independent acquisition (DIA) mass spectrometry was executed to identify the probable targets of NBP in CSVD. Based on the proteomics results, the expression of the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway in the rat hippocampus was evaluated by western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). Results NBP treatment ameliorated the cognitive abilities and pathological changes in CSVD. DIA proteomics revealed 262 differentially expressed hippocampal proteins, with bioinformatics analysis highlighting acute inflammatory response as a primary target. Furthermore, western blotting and qRT-PCR results confirmed these results and showed that after treatment with NBP, TLR4 regulated NF-κB pathway and inflammatory factors decreased. Conclusions Our findings demonstrated that NBP exerts neuroprotection in CSVD probably by suppressing TLR4/MyD88/NF-κB-mediated neuroinflammation. This study provides the evidence of NBP’s therapeutic mechanisms in CSVD, suggesting its potential as a targeted anti-inflammatory treatment.