This study aimed to investigate the pharmacodynamic material basis and potential mechanism of Qingre Huatan Tongluo Formula (QHTF) in treating ischemic stroke (IS) based on network pharmacology and experimental validation. A IS rat model was established using the filament occlusion method. The therapeutic effects of QHTF were evaluated by observing post-modeling behavioral changes, examining cerebral histopathology via HE staining, and detecting alterations in serum inflammatory cytokine levels using ELISA. Active components of QHTF absorbed into the rat bloodstream and cerebrospinal fluid were analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Core targets of the brain-entering QHTF components were identified through network pharmacology and bioinformatics analysis, followed by verification via molecular docking. An in vitro inflammatory model was established using lipopolysaccharide (LPS)-induced BV2 microglial cells. After treatment with QHTF-containing serum, the release of nitric oxide (NO) was measured by the Griess method, cell viability was assessed by the CCK-8 assay, and levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 in the cell supernatant were quantified using ELISA kits. The protein expression of the epidermal growth factor receptor (EGFR) and key proteins in the phosphatidylinositol 3-kinase/protein kinase B (PI3K-AKT) pathway was detected by immunofluorescence. Animal experiments demonstrated that QHTF significantly ameliorated pathological injury in IS model rats and markedly reduced serum levels of TNF-α, IL-1β, and IL-6. Following oral administration of QHTF decoction, UPLC-Q-TOF-MS/MS analysis identified 31 components that entered the bloodstream, among which 11 were detected in the brain. Network pharmacology results indicated that baicalein, apigenin, chrysophanol, senkyunolide F, and naringenin were the core components. The core targets for QHTF against IS were identified as tumor protein 53 (TP53), EGFR, AKT1, heat shock protein 90 alpha family class A member 1 (HSP90AA1), proto-oncogene tyrosine-protein kinase Src (SRC), and B-cell lymphoma 2 (BCL-2). QHTF is suggested to exert therapeutic effects against IS primarily by modulating the EGFR, PI3K-AKT, and VEGF signaling pathways. Molecular docking results showed favorable binding interactions between the core components and the core targets. In vitro cell experiments revealed that QHTF-containing serum reduced the levels of inflammatory factors in LPS-induced BV2 cells, downregulated EGFR expression, and promoted the phosphorylation of PI3K and AKT. In conclusion, QHTF may exert therapeutic effects against ischemic stroke potentially by inhibiting EGFR expression and activating the PI3K-AKT signaling pathway.

Qingre Huatan Tongluo Formula, component analysis, network pharmacology, ischemic stroke; EGFR/PI3K-AKT signaling pathway