Glycogen synthase kinase-3 inhibition by CHIR99021 promotes alveolar epithelial cell proliferation and lung regeneration in acute lung injury

Acute respiratory distress syndrome (ARDS) is a life-threatening lung injury. After injury, severe inflammation, alveolar-capillary barrier permeability and alveolar epithelial cell (AEC) death lead to lung dysfunction. Glycogen synthase kinase-3 (GSK-3) is an important molecule in cell proliferation and differentiation. CHIR99021 (CHIR), a GSK-3 inhibitor, modulates the maturation of epithelial progenitor cells in lung 3D model. Thus, we aimed to understand the effects of CHIR on AEC regeneration using LPS-induced acute lung injury (ALI) mouse model. GSK-3 was inhibited 30min or 18h after LPS injection to investigate effects on disease progression in the early and late inflammatory response. Animals treated with CHIR for 30min were evaluated after 12h and 24h, and animals treated with CHIR for 18h were studied after 30h and 72h. We analyzed lung injury by HaE staining, barrier permeability by Evan Blue dye, inflammatory recruitment by flow cytometry, apoptosis by TUNEL and Cleaved Caspase-3 expression, proliferation by Cyclin D1 and PCNA expression. Moreover, the presence of AEC lineage was assessed by expression of HOPX (AECII-to-AECI differentiation), pro-SPC (AECII) and PDPN, AQP5 (AECI). GSK-3 inhibition reduced lung injury at 24 and 72h with no effect on inflammatory cell recruitment nor barrier permeability. CHIR enhanced cell proliferation at all times analyzed, increased AECII cell proliferation at 24h and bipotent cell proliferation at 24h and 72h. AECI-related molecules showed an increase at 24h, 30h and 72h and AECII-related markers were enhanced at 30h and 72h. In conclusion, our preliminary data suggest that CHIR promotes AEC proliferation and transdifferentiation, restores alveolar epithelial population, and thus promotes lung regeneration in LPS-induced ALI.