ETHNOPHARMACOLOGICAL RELEVANCE: Houshiheisan (HSHS) is a classical prescription remedy for cerebral small vessel disease (CSVD) in traditional Chinese medicine
however, its specific ingredients and mechanisms remain unclear. AIM OF THE STUDY: This study sought to elucidate the protective effects of HSHS on spontaneously hypertensive rats (SHR) subjected to unilateral common carotid artery occlusion (UCCAO), referred to as SHR-UCCAO, and to identify potential targets and mechanistic pathways involved in CSVD treatment using serum pharmacochemistry, network pharmacology, and experimental validation. MATERIALS AND METHODS: Serum pharmacochemistry was used to identify the components of HSHS. Network and functional enrichment analyses were performed to clarify the targets and biological mechanisms of HSHS. The CB-DOCK2 database was employed for molecular docking of the principal compounds and core target proteins. As predicted by network pharmacological analysis, the potential mechanisms of HSHS against CSVD were experimentally validated in a CSVD rat model and endothelial cells subjected to oxygen-glucose deprivation. RESULTS: A total of 50 compounds in serum samples from HSHS were identified as potential active agents. Network pharmacology and molecular docking analyses revealed that HSHS could treat CSVD via multiple components and targets. In animal experiments, HSHS ameliorated systolic and diastolic blood pressure, improved gait disturbance, and reduced the Albumin levels in the affected cortex of CSVD rats. Animal and cell experiments demonstrated that HSHS improved the endothelial barrier injury and upregulated the expression of ZO-1, Occludin, Claudin-5, and VE-Cadherin through regulation of the NF-κB/MLCK pathway. CONCLUSIONS: This study indicated that HSHS could protect the endothelial barrier in CSVD by modulating the NF-κB/MLCK signaling pathway.