Ectodermal mesenchymal stem cells (EMSCs) are cells harvested from the stem cell niche (nasal mucosa) with high therapeutic potential. To the best of our knowledge, however, the anti-inflammatory properties of these neural crest-derived EMSCs have been rarely reported. The present study aimed to explore the effects of aerosolized EMSC-Secretome (EMSC-Sec) and clarify underlying mechanisms in treating acute lung injury (ALI). EMSCs were isolated by adherent method and identified by immunofluorescence staining. EMSC-Sec was collected and evaluated using western blotting, BCA and ELISA tests. Then, mouse lung epithelial cells (MLE-12) were used to mimic inflammatory stimulation with lipopolysaccharide (LPS). After developing an ALI model through intraperitoneal injection of LPS, mice were treated with an EMSC-Sec spray. The lung in each group underwent an observation and measurement to preliminarily assess the extent of damage. H&E staining, immunohistochemical staining, immunofluorescence and western-blotting were utilized to further access the impacts of EMSC-Sec. The results showed that EMSC-Sec had great anti-inflammatory potential and was highly successful
in vitro
and
in vivo
. EMSC-Sec mitigated LPS-induced ALI with low inflammatory cell inflation and mild damage. EMSC-Sec could regulate inflammation via the NF-κB(p50/p65)/NLRP3 pathway. Overall, the present study demonstrated that EMSC-Sec regulated inflammation, hoping to provide a novel strategy for ALI treatment.