Integrative network pharmacology and experimental validation of multi-target synergy against multidrug-resistant klebsiella pneumoniae via PI3K/AKT-MAPK pathway disruption.

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) infections present a global health crisis, with escalating resistance to last-line antibiotics. Traditional Chinese medicines (TCMs) demonstrate multi-component synergy against resistant pathogens. This study deciphered the antibacterial mechanisms of bioactive TCM components targeting MDR-KP through integrative pharmacology and experimental validation. Four core TCMs (Scutellaria baicalensis, Forsythia suspensa, Lonicera japonica, Aloe vera) were screened via Chinese Pharmacopoeia and TCMSP database. Network pharmacology identified core components and enriched pathways. Clinically isolated MDR-KP strains (resistant to meropenem, polymyxin E, and tigecycline) were characterized. Synergistic effects were evaluated using checkerboard assays, biofilm quantification, and membrane permeability assays. Molecular docking and RT-qPCR analyzed target interactions, while ELISA and CCK-8 assessed anti-inflammatory activity and cytotoxicity. Consequently, luteolin, wogonin, and kaempferol were identified as key components targeting PI3K/AKT and MAPK pathways. Their triple combination (2/4/8 μg/mL) exhibited potent synergy (FIC = 0.38), reducing biofilm biomass by 68% (p < 0.001) and extracellular polysaccharide thickness from 15.1 μm to 3.1 μm (p < 0.001). Flow cytometry confirmed a 144.8% increase in membrane permeability. Molecular docking revealed high-affinity binding to PI3K (- 6.78 kcal/mol), AKT1 (- 6.60 kcal/mol), and MAPK1 (- 7.24 kcal/mol), with RT-qPCR showing significant downregulation of these genes (p < 0.001). The combination suppressed LPS-induced TNF-α and IL-6 release by 72% and 65%, respectively, while maintaining 95.5% cell viability at therapeutic concentrations. In Conclusion, this study unveils a triple-action mechanism of luteolin-wogonin-kaempferol against MDR-KP, simultaneously disrupting resistance pathways, biofilms, and hyperinflammation. The regimen offers a safe, multi-target therapeutic strategy for combating drug-resistant infections.