The mechanism of Bovis Culus Sativus protecting BBB damage in stroke: Insights from network pharmacology,...

Bovis calculus (BC) has a medicinal history of over 2000 years in treating stroke in China. Bovis Culus Sativus (BCS) has similar pharmacological effects to BC. Due to the scarcity of BC, BCS is often used as a substitute for BC in clinical practice for treating stroke in traditional Chinese medicine.

This study aims to investigate the inhibitory effect of BCS on blood-brain barrier (BBB) damage following stroke, and to elucidate the molecular basis of BCS neuroprotection through network pharmacology and bioinformatics.

The contents of bilirubin and bile acids in BCS were quantified using HPLC. A cerebral ischemia-reperfusion injury (CIRI) rat model was established to assess neurological function, cerebral infarction, pathological damage, and Evans Blue staining. R language was used to analyze GEO public data to identify therapeutic targets for ischemic stroke. Public databases and literature were utilized to screen for active components of BCS, and the Swiss Target Prediction database was used to predict the active drug targets. Network pharmacology analysis was conducted on drug and disease targets, followed by immune infiltration and molecular docking of key targets. Finally, ELISA, RT-PCR, Western blot, IHC, and TEM were employed to validate the effectiveness of the targets.

The content of bile acids and bilirubin in the tested BCS was 6.9% and 37.89%, respectively. The study showed that BCS reduced neurological function scores and cerebral infarction rates in stroke rats, prevented Evans Blue leakage, and mitigated histopathological damage in the ischemic brain region. Additionally, BCS improved the structural and functional integrity of the BBB, enhancing the expression of Occludin, ZO-1, and Claudin-5 while downregulating the expression of MDR1, aquaporin-4, MMP-9, and MMP2. Bioinformatics and network pharmacology analyses indicated that the therapeutic effects of BCS in stroke are primarily associated with the inhibition of inflammatory pathways, including TNF, NFKB, and MAPK. ELISA, RT-PCR, and Western blot results further confirmed that BCS significantly suppressed neuroinflammation in stroke rats.

BCS shows promising efficacy against ischemic stroke, maintaining the function and structural integrity of the BBB. Its protective effect on the BBB may be related to the inhibition of the TNF-NFκB-MAPK signaling pathways.