Astragalus-Safflower Combination Promotes Vascular Neogenesis in a Rat Model of Ischemic Stroke via Inhibition of MAPK/NF-κB and Activation of VEGF/Notch1 Pathways

Introduction: The combination of Astragalus membranaceus and Safflower (AS) is known for its efficacy in benefiting Qi and activating blood circulation, making it a frequently used empirical combination in traditional Chinese medicine. Numerous reports have highlighted the interventional effect of this combination in treating ischemic stroke (IS). However, the active ingredients and potential mechanisms underlying its treatment of stroke have not been fully elucidated. Methods: Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/ MS), along with various data processing methods, were utilized to identify and assess the chemical constituents in rat serum following AS gavage administration. Chemical constituent targets were predicted using the SEA and Swiss Target Prediction databases, while IS-related targets were sourced from the GeneCards, OMIM, and TTD databases. The intersecting targets of constituents and diseases were screened, and a core target network map was constructed using the String database and Cytoscape software. KEGG pathway enrichment of core targets was analyzed using DAVID and Metascape databases. The middle cerebral artery occlusion (MCAO) rat model was established to evaluate the cerebroprotective effects of AS. The accuracy of predicted pathways was validated using immunofluorescence (IF) and Western blot (WB) analyses. Results: Thirty-five ingredients in serum were identified, and 437 targets and 3748 IS-related targets were identified, 291 of which overlapped. Protein-protein interaction (PPI) analysis predicted 15 major targets, including TNF and MAPK3. KEGG pathway analysis indicated that the MAPK/NF-κB and VEGF/Notch1 signaling pathways may play pivotal roles in the therapeutic effects of AS in IS. Moreover, AS significantly ameliorated neurological and motor function impairments, as well as brain histopathological damage, in MCAO rats. AS treatment led to reduced levels of the inflammatory cytokines IL-6 and TNF-α, inhibited astrocyte hyperactivation, decreased nuclear translocation of NF-κB p65, reduced expression of p-MAPK (Erk1/2)/ MAPK (Erk1/2) and p-NF-κB (p65)/NF-κB (p65) proteins, increased the number of CD31+/Ki67+ and VEGF+/ Ki67+-positive vessels, and upregulated the expression of VEGF, VEGFR-2, Notch1, and DLL4 proteins. Conclusion: AS may regulate MAPK/NF-κB and VEGF/Notch1 pathways to reduce inflammation and promote post-ischemic neovascularization, providing a promising method for the treatment of ischemic stroke.