Integrated network pharmacology, transcriptomics and experimental validation to explore mechanisms of Wenyang Jiedu Granule on IAV-induced pneumonia

Objective: Wenyang Jiedu Granule (WYJD) is an effective traditional Chinese medicine (TCM) preparation that has been generally applied for treating respiratory infectious diseases. Clinical observations involving thousands of cases have demonstrated that WYJD could alleviate disease progression and improve symptoms in treating respiratory viral infections, including SARS-CoV-2 and influenza virus. However, the chemical basis and underlying mechanisms of WYJD against influenza A virus (IAV)-induced pneumonia remain to be elucidated. This study aimed to reveal the underlying mechanisms of WYJD in treating IAV-induced pneumonia by a combined strategy of network pharmacology, transcriptomics and experimental validation. Methods: The pneumonia model was established in BALB/c mice via infection with H1N1 IAV to evaluate the therapeutic effects of WYJD on IAV-induced pneumonia. Firstly, ultra-high performance liquid chromatography-quadrupole Exactive Orbitrap mass spectrometer/tandem mass spectrometer (UPLC-Q Exactive Orbitrap-MS/MS) was employed to analyze the main chemical components in WYJD-containing serum. Subsequently, the effects of WYJD on IAV-induced pneumonia were assessed through pathological observation, plaque forming assay, biochemical analysis, Evans blue staining assay, and immunofluorescence assay. Mechanistically, an integrated approach of network pharmacology and transcriptomics was applied to explore the potential active components, targets and related pathways of WYJD against IAV-induced pneumonia. Fluorescence TUNEL assay, quantitative real-time PCR (qRT-PCR) and Western blotting were utilized for experimental validation and mechanistic studies. Results: Using UPLC-Q Exactive Orbitrap-MS/MS, a total of 25 prototypes and 15 metabolites were identified in the serum of mice after WYJD administration. WYJD treatment showed protective effects on IAV-induced pneumonia by inhibiting inflammation and lung barrier damage in the IAV-induced pneumonia mice model. Network pharmacology combined with transcriptomics analysis indicated that WYJD exerted therapeutic effects against IAV-induced pneumonia mainly through the synergistic effects of 11 active components, which regulated ten critical signaling pathways via 86 targets. Further experimental validation demonstrated that WYJD could alleviate IAV-induced pneumonia via the IL-17 signaling pathway, Toll-like receptor 7‌ (TLR7)/Myeloid differentiation primary response dene 88 (MyD88)/mitogen-activated protein kinases (MAPKs)/activator protein 1 (AP-1) signaling pathway and apoptosis. Conclusion: This study revealed the main active components and mechanisms of WYJD against IAV-induced pneumonia through the IL-17 signaling pathway, TLR7/MyD88/MAPKs/AP-1 signaling pathway and apoptosis, which provides novel insights into the clinical application of WYJD in treating influenza and its complications.