Utilizing network pharmacology, molecular docking, and animal models to explore the therapeutic potential of the WenYang FuYuan recipe for cerebral ischemia-reperfusion injury through AGE-RAGE and NF-κB/p38MAPK signaling pathway modulation.

BACKGROUND: Stroke is a debilitating condition with high morbidity, disability, and mortality that significantly affects the quality of life of patients. In China, the WenYang FuYuan recipe is widely used to treat ischemic stroke. However, the underlying mechanism remains unknown, so exploring the potential mechanism of action of this formula is of great practical significance for stroke treatment.

OBJECTIVE: This study employed network pharmacology, molecular docking, and in vivo experiments to clarify the active ingredients, potential targets, and molecular mechanisms of the WenYang FuYuan recipe in cerebral ischemia-reperfusion injury, with a view to providing a solid scientific foundation for the subsequent study of this recipe.

MATERIALS AND METHODS: Active ingredients of the WenYang FuYuan recipe were screened using the traditional Chinese medicine systems pharmacology database and analysis platform. Network pharmacology approaches were used to explore the potential targets and mechanisms of action of the WenYang FuYuan recipe for the treatment of cerebral ischemia-reperfusion injury. The Middle Cerebral Artery Occlusion/Reperfusion 2 h Sprague Dawley rat model was prepared, and TTC staining and modified neurological severity score were applied to examine the neurological deficits in rats. HE staining and Nissl staining were applied to examine the pathological changes in rats. Immunofluorescence labeling and Elisa assay were applied to examine the expression levels of certain proteins and associated factors, while qRT-PCR and Western blotting were applied to examine the expression levels of linked proteins and mRNAs in disease-related signaling pathways.

RESULTS: We identified 62 key active ingredients in the WenYang FuYuan recipe, with 222 highly significant I/R targets, forming 138 pairs of medication components and component-targets, with the top five being Quercetin, Kaempferol, Luteolin, β-sitosterol, and Stigmasterol. The key targets included TP53, RELA, TNF, STAT1, and MAPK14 (p38MAPK). Targets related to cerebral ischemia-reperfusion injury were enriched in chemical responses, enzyme binding, endomembrane system, while enriched pathways included lipid and atherosclerosis, fluid shear stress and atherosclerosis, AGE-RAGE signaling in diabetic complications. In addition, the main five active ingredients and targets in the WenYang FuYuan recipe showed high binding affinity (e.g. Stigmasterol and MAPK14, total energy <-10.5 Kcal/mol). In animal experiments, the WenYang FuYuan recipe reduced brain tissue damage, increased the number of surviving neurons, and down-regulated S100β and RAGE protein expression. Moreover, the relative expression levels of key targets such as TP53, RELA and p38MAPK mRNA were significantly down-regulated in the WenYang FuYuan recipe group, and serum IL-6 and TNF-a factor levels were reduced. After WenYang FuYuan recipe treatment, the AGE-RAGE signaling pathway and downstream NF-kB/p38MAPK signaling pathway-related proteins were significantly modulated.

CONCLUSION: This study utilized network pharmacology, molecular docking, and animal experiments to identify the potential mechanism of the WenYang FuYuan recipe, which may be associated with the regulation of the AGE-RAGE signaling pathway and the inhibition of target proteins and mRNAs in the downstream NF-kB/p38MAPK pathway.