Bletinib ameliorates neutrophilic inflammation and lung injury by inhibiting Src family kinase phosphorylation and activity

Abstract

BACKGROUND AND PURPOSE Neutrophil overactivation is crucial in the pathogenesis of acute lung injury (ALI). Bletinib (3,3′-dihydroxy-2′,6′-bis(p-hydroxybenzyl)-5-methoxybibenzyl), a natural bibenzyl first extracted from Bletilla striata in 1983, has anti-inflammatory, antibacterial, and antimitotic potential. In this study, we evaluated the therapeutic effects of Bletinib in human neutrophilic inflammation and lipopolysaccharide (LPS)-mediated ALI. EXPERIMENTAL APPROACH We assessed integrin expression, superoxide anion production, degranulation, neutrophil extracellular trap (NET) formation, and adhesion in activated human neutrophils through flow cytometry, spectrophotometry, and immunofluorescence microscopy. Moreover, phosphorylation of Src family kinases (SFKs) and downstream proteins was evaluated through immunoblotting. Finally, a murine LPS-induced ALI model was used to investigate the potential therapeutic effects of Bletinib treatment. KEY RESULTS In activated human neutrophils, Bletinib reduced degranulation, respiratory burst, NET formation, adhesion, migration, and integrin expression; suppressed the enzymatic activity of SFKs, including Src, Lyn, Fgr, and Hck; and inhibited the phosphorylation of SFKs as well as Vav and Bruton’s tyrosine kinase (Btk). In our mice with ALI, the pulmonary sections demonstrated considerable amelioration of prominent inflammatory changes, such as haemorrhage, pulmonary oedema, and neutrophil infiltration, after Bletinib treatment. CONCLUSION AND IMPLICATIONS This is the first study to provide evidence that Bletinib regulates neutrophilic inflammation by inhibiting the SFKs–Btk–Vav pathway and that Bletinib ameliorates LPS-induced ALI in mice. Further biochemical optimisation of Bletinib may be a promising strategy for the development of novel therapeutics for inflammatory diseases.