System pharmacology reveals the mechanism of Puji Xiaodu Yin against Japanese encephalitis

Japanese encephalitis (JE) caused by the JE virus (JEV) lacks specific therapeutics. Puji Xiaodu Yin (PJXDY) is a classic formula used for acute and critical illnesses; however, its effects and mechanisms in the treatment of JE remain unclear. This study aimed to elucidate the pharmacological effects of PJXDY, identify its representative active components, and explore underlying mechanisms. A JE mouse model was established by subcutaneous injection of JEV into the footpad. The therapeutic effects of PJXDY were evaluated by survival rates, behavioral changes, viral load assays, inflammatory factor levels, and histopathological staining. Brain-accessible components were identified using ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry/mass spectrometry (UPLC-Q-TOF-MS/MS). System pharmacology, molecular docking, and in vitro assays were employed to screen for representative active components with the strongest activities. Drug affinity responsive target stability, bio-layer interferometry, enzyme activity assays, small interfering RNA (siRNA), Western blot, and immunofluorescence were used to explore mechanisms. PJXDY dose-dependently increased survival, improved neurological symptoms, reduced viral loads, and alleviated brain damage. Three representative active components were selected: antiviral rosmarinic acid (RA), anti-inflammatory cosmosiin (CAA), and 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) inhibiting PANoptosis-like cell death. Mechanistic studies suggested that they were associated with non-structural protein 5 methyltransferase domain (NS5 MTase), mitogen-activated protein kinase kinase 1 (MEK1), and Z-DNA binding protein 1 (ZBP1), respectively, correlating with reduced viral replication, suppressed inflammatory cytokine production, and diminished PANoptosis-related marker expression. The three-component combination showed efficacy comparable to that of PJXDY. This study lays an experimental foundation for the clinical application and modern development of PJXDY.