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ETHNOPHARMACOLOGICAL RELEVANCE The leaves, bark, and flowers of Paulownia tomentosa Steud. have been widely used as a traditional medicine in East Asia to treat inflammatory and infectious diseases. AIM OF THE STUDY We investigated the protective effect of the methanol stem bark extract of P. tomentosa using an animal model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). MATERIALS AND METHODS The UPLC Q-TOF-MS profiles for the methanol extract of P. tomentosa stem bark showed that verbascoside and isoverbascoside were the predominant compounds. Raw 264.7 cells were used for inhibitory effects of cytokine production in vitro. C57BL/6N mice were administered intranasally with LPS (10μg/per mouse) to induce ALI. H&E staining was used to evaluate histological changes in the lung. RESULTS Treatment with P. tomentosa stem bark extract (PTBE) suppressed the production of IL-6 and TNF-α in LPS-stimulated RAW 264.7 macrophages, and the recruitment of neutrophils and macrophages in the BALF of mice with LPS-induced ALI. PTBE also decreased the levels of reactive oxygen species (ROS) and pro-inflammatory cytokines in the BALF. PTBE reduced the levels of nitric oxide (NO) in the serum and of inducible nitric oxide synthase (iNOS) in the lung of ALI mice. PTBE also attenuated the infiltration of inflammatory cells and the expression of monocyte chemoattractant protein-1 (MCP-1) in the lung. In addition, PTBE suppressed the activation of NF-κB and the reduced expression of superoxide dismutase 3 (SOD3) in the lung. CONCLUSION The results suggest that PTBE has a protective effect on LPS-induced ALI.