Potential mechanisms of baicalin-geniposide combination against cerebral ischemia: An integrated study of network pharmacology and experimental validation

To explore the potential mechanisms of a baicalin-geniposide combination against cerebral ischemia using a network pharmacology strategy. We used network pharmacology integrating drug-target-disease interactions to identify key pathways which were validated in a rat middle cerebral artery occlusion model treated with baicalin (55 mg/kg), geniposide (5 mg/kg), or their 11:1 combination. Therapeutic efficacy and mechanistic insights were evaluated using triphenyltetrazolium chloride staining, Evans blue assay, enzyme-linked immunosorbent assay, and western blot. The results revealed that the nuclear factor-kappa B (NF-κB) signaling pathway is inhibited in combination treatment of cerebral ischemia. Ten targets were identified as key nodes in the protein-protein interaction network: interleukin 6 (IL-6), interleukin-1β (IL-1β), interleukin 18 (IL-18), C-C motif ligand 2 (CCL2), C-C motif ligand 4 (CCL4), interleukin 10 (IL-10), interferon-γ-inducible protein 10 (CXCL10), C-C motif ligand 3 (CCL3), tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α). The baicalin-geniposide combination significantly reduced infarct volume, improved neurological deficits, and alleviated brain edema/blood-brain barrier leakage compared with monotherapy. Additionally, it significantly inhibited toll-like receptor 4 (TLR4)/NF-κB signaling and downregulated pro-inflammatory cytokines TNF-α and IL-6 levels. The baicalin-geniposide combination alleviated cerebral ischemia-reperfusion injury by synergistically suppressing the TLR4/NF-κB pathway and its downstream inflammatory factors.