Integrated Serum Pharmacochemistry, Network Pharmacology, and Transcriptomics Reveal the Mechanisms and Active Constituents of Qingfei Huoxue Decoction Against Bleomycin-Induced Pulmonary Fibrosis

Yule Wang,1– 3,* Juntao Wang,4,* Ruiling Lu,1,* Yan Zhu,5 Bei Li,1– 3,* Yue Gao1– 3 1The Fourth School of Clinical Medicine, Zhejiang Chinese Medical University, Hangzhou First People’s Hospital, Hangzhou, People’s Republic of China; 2Zhejiang Key Laboratory of Traditional Chinese Medicine for the Prevention and Treatment of Senile Chronic Diseases, Affiliated Hangzhou First People’s Hospital, School of Medicine, Westlake University, Hangzhou, People’s Republic of China; 3Department of Geriatrics, Affiliated Hangzhou First People’s Hospital, School of Medicine, Westlake University, Hangzhou, People’s Republic of China; 4Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, People’s Republic of China; 5State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yue Gao, Email [email protected] Yan Zhu, Email [email protected]: Pulmonary fibrosis (PF) is a progressive and fatal interstitial lung disorder with limited therapeutic options, as current drugs like pirfenidone and nintedanib offer only modest benefits and carry adverse events. Given the lung-protective and anti-inflammatory activities of Qingfei Huoxue decoction (QFHXD), its anti-PF efficacy, pharmacological mechanisms, and active constituents warrant further investigation.Methods: A bleomycin (BLM)-induced PF mouse model was used to assess QFHXD’s efficacy. The bioactive components of QFHXD and their distribution in lung tissue were identified in vivo through serum pharmacochemistry. An integrative approach combining network pharmacology, transcriptomics, and molecular validation was employed to elucidate the QFHXD’s therapeutic mechanisms. The anti-PF active substances were defined by cellular immunofluorescence and molecular docking.Results: Treatment with QFHXD effectively preserved alveolar integrity, suppressed inflammation, reduced expression of pro-fibrotic markers, and decreased extracellular matrix deposition, thereby improving histopathology and survival in PF mice. Serum pharmacochemistry identified 46 QFHXD absorbable and lung-distributed compounds. Integrated component-target-disease network and transcriptome analyses revealed a central role of anti-inflammation and anti-apoptosis for QFHXD against PF. Indeed, QFHXD inhibited the overexpression of pivotal inflammatory mediators TLR2, TLR4, NF-κB, IL-1β, IL-6, TNF-α, TGF-β 1, CCL2, SPP1, and MMP9 in fibrotic lung tissues. QFHXD also ameliorated the apoptotic phenotype and reduced the dysregulated levels of Bcl2, Bax, and Caspase-3 expression. QFHXD and several of its active components normalized pro-fibrotic markers Fibronectin and Collagen I in TGF-β 1-stimulated MRC-5 cells, an effect mediated by their multi-target binding to key PF-related proteins (TGF-β 1, TLR4, CCL2, SPP1, MMP9, and Caspase-3), as supported by molecular docking.Conclusion: Our findings demonstrated a potent anti-PF efficacy of QFHXD, attributed at least in part to its multi-targeted anti-inflammatory and anti-apoptotic activities. Loganin, baicalein, baicalin, oroxylin A, peimine, peiminine, tanshinone IIA, nobiletin, and paeoniflorin were identified as the primary active components of QFHXD against PF. Step 1: BLM-induced PF model, with an arrow to a box listing Body weight, Survival, Histopathology, Pro-fibrotic markers, Inflammatory mediator, Serum KL-6 and the caption Anti-PF efficacy of QFHXD. Step 2: Identification QFHXD components in vivo, showing a rat, blood tube, lungs, an instrument and a small plot labeled Relative abundance and m over z. Step 3: Network pharmacology, showing database and tool logos including GeneCards, DisGeNET, HERB, PubMed, SwissTargetPrediction, DrugBank, DAVID, STRING and Cytoscape. Step 4: RNA-Seq and bioinformatics analysis, showing a sequencer and multiple analysis icons. Step 5: Validation of regulatory mechanisms, listing Inflammation: TLR2, TLR4, NF-kB, IL-1β, IL-6, TNF-α, TGF-β 1, CCL2, SPP1, MMP9; Apoptosis: Bcl2, Bax, Caspase-3. Step 6: Anti-PF active constituents in QFHXD, showing a protein structure image and a list: loganin, baicalein, baicalin, oroxylin A, peimine, peiminine, tanshinone IIA, nobiletin, paeoniflorin. No decision points or conditional branches are shown; arrows indicate a single forward progression.Flowchart of six-step research workflow for Qingfei Huoxue decoction from model establishment to active constituent identification.Keywords: bleomycin-induced pulmonary fibrosis, Qingfei Huoxue decoction, serum pharmacochemistry, network pharmacology, transcriptome sequencing, inflammatory and apoptotic regulatory mechanisms, active constituents