Effects of NRICM102 on changes in levels of prothrombotic factor (vWF, PAI-1), NET, apoptosis and fibrotic factor (c-Kit and SCF) (at 72 hr) in S1 protein-induced lung injury in K18-hACE2 mice. Typical confocal images of lung tissue showing (A) vWF (G, green) and PAI-1 (R, red) colocalized with endothelial cell or platelet (CD31, PECAM-1) (O, orange). The arrow indicates extensive colocalization of CD31 and PAI-1. (B) S1 protein expression (O, orange) closely associated with NET formation (G, green) and leukocyte (CD11b) infiltration (R, red); the arrow indicates strong colocalization (yellow) of CD11b and NET staining (30X). (C) S1 expression (G, green) closely associated with NET formation (O, orange) and neutrophil (CD11b) infiltration (R, red); the arrow indicates strong colocalization of S1 and NET staining (100X). (D) Cleaved caspase 3 (cCasp3) (G, green) colocalized with AT1 cells (O, orange) or AT2 cells (R, red); the arrow indicates strong colocalization (yellow) of cCasp3 and AT2 or AT1. (E) c-Kit expression (G, green) colocalized with S1 protein accumulation (O, orange) and SCF (R, red); the arrow indicates strong colocalization (yellow) of S1 and c-Kit. (F) Statistical summary of positive fluorescence staining (relative stained area or cell no. counted [%]) for some selected markers. The experimental animals were divided into 3 subgroups as described in the legend for Fig. 2A. The data are the mean±SEM (n = 3 for each group). * p < 0.05 compared with the corresponding S1 +saline group by one-way ANOVA followed by the S-N-K t-test. (G) H&E staining of lung tissue. In the S1+saline group, typical leukocyte infiltration into bronchioles including neutrophils (black arrow head, possess distinctive multi-lobulated nuclei) and monocytes (red arrow head, with large round or oval cell with an eccentrically placed nucleus) infiltration, and thickened alveolar walls with interstitial edema (*) clearly observed, which less observed in the control (Ctrl) and S1+NRICM102 groups. (H) Masson's trichrome staining for typical tissue fibrosis (blue) clearly observed in the S1+saline and less observed in S1 +NRICM102.