Reduning injection alleviated LPS-induced ARDS via multi-target regulation by modulating inflammation and immune responses based on target fishing technique and scRNA-seq analysis.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is an acute respiratory failure syndrome arising from non-cardiogenic pulmonary edema, characterized by limited therapeutic options and a poor prognosis, underscoring an urgent need for effective treatments. Reduning injection (RDN) is a traditional Chinese medicine formulation widely used in clinical practice for treating respiratory tract infections. However, its specific role and underlying mechanism in ARDS remain to be fully elucidated. PURPOSE: This study aimed to examine the multi-target protective effects of RDN against ARDS and elucidate its underlying mechanisms. STUDY DESIGN AND METHODS: A lipopolysaccharide (LPS)-induced ARDS model was established to evaluate the therapeutic potential of RDN. A multifaceted experimental approach, encompassing flow cytometry, immunofluorescence, the integration target fishing technique of thermal-solvent (TS) proteomics and affinity chromatography (AC), Western blot, real-time PCR, immunohistochemistry, scRNA-seq analysis, molecular docking, and molecular dynamics, was adopted to uncover the therapeutic mechanism of RDN in ARDS. RESULTS: RDN alleviated the progression of ARDS by improving alveolar barrier function and reducing histopathological lung damage. The underlying mechanisms involved the inhibition of the MAPK, NF‑κB, and NLRP3 pathways, which led to the decreasing of release for key inflammatory cytokines (IL‑6, IL‑1β, and TNF‑α). It also attenuated pulmonary infiltration of macrophages and neutrophils and modulated the Treg/Th17 balance, thereby maintaining pulmonary immune homeostasis. Furthermore, employing the target fishing technique, we identified folliculin (Flcn), integrin alpha-V (Itgav), and phospholipase C-β3 (Plcb3) as direct intracellular targets of RDN, which was further supported by various chemical biological methods. LC‑MS/MS analysis revealed that components of RDN entered the blood and lungs, among which isochlorogenic acid A (IAA) and genipin 1-gentiobioside (GG) were identified as bioactive components that directly bound to Flcn, Itgav, and Plcb3, respectively. CONCLUSION: RDN alleviated LPS-induced ARDS by modulating inflammatory and immune responses via multi-target regulation of Flcn, Itgav, and Plcb3, thereby inhibiting the NLRP3, NF-κB, and MAPK pathways.