Integrating Machine Learning and Pharmacology Reveals NF-κB-Mediated Inflammatory Regulation by Shuanghuanglian Injection in Acute Lung Injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuanghuanglian Injection (SHLI) is a traditional Chinese medicine formulation derived from Lonicerae Japonicae Flos, Scutellariae Radix, and Forsythiae Fructus and developed based on the TCM principle of heat-clearing and detoxifying. It has been widely used for respiratory infections and inflammatory disorders, but its protective mechanisms in acute lung injury (ALI) warrant further investigation. AIM OF THE STUDY: To characterize the chemical profile of SHLI and delineate its pharmacological mechanisms in ALI, with particular attention to inflammatory regulation and the NF-κB signaling cascade. MATERIALS AND METHODS: UPLC-MS/MS profiling defined the chemical components of SHLI. Candidate targets were retrieved from TCMSP, GeneCards, DisGeNET, and the Open Targets Platform, then organized into a protein-protein interaction network with subsequent enrichment analyses. Transcriptomic data from GEO provided differentially expressed genes, which were further refined using a combination of machine learning approaches (LASSO, random forest, SVM-RFE, and XGBoost) to prioritize key targets. Molecular docking assessed the binding affinity between representative compounds and targets. An LPS-induced ALI rat model was used to verify therapeutic effects through hematological indices, histopathology, RT-qPCR, ELISA, and Western blot assays. RESULTS: 10 principal components were identified and quantified in SHLI, predominantly flavonoids and phenolic acids. Integrating network pharmacology with machine learning highlighted three genes (IL-1α, IL-1β and TNFAIP6) as central nodes. Docking analysis suggested that quercetin interacts stably with these key targets. In vivo, SHLI mitigated lung injury in a dose-dependent manner and partially restored disordered leukocyte responses while lowering pro-inflammatory cytokine levels. At the molecular level, SHLI modulated the transcription of IL-1α, IL-1β and TNFAIP6 and reduced the secretion of TNF-α, IL-1β and TNFAIP6. Concurrently, SHLI dampened NF-κB activation by limiting IκBα phosphorylation and restraining p65 activation. CONCLUSION: SHLI protects against ALI through coordinated multi-component, multi-target interactions, with its core mechanism associated with the suppression of NF-κB mediated inflammatory responses.