Hydroxysafflor yellow A mitigates lipopolysaccharide-induced acute lung injury by enhancing pulmonary microvascular endothelial barrier function via Calpain-1/HIF-1α inhibition.

Abstract

<p>OBJECTIVE: Pulmonary microvascular endothelial cell (PMEC) barrier dysfunction is a critical pathological hallmark of acute lung injury (ALI). Hydroxysafflor yellow A (HSYA), a major active component of safflower, exhibits potential protective effects against lung injury. The current study aimed to investigate whether HSYA preserves PMEC barrier integrity in lipopolysaccharide (LPS)-induced ALI by inhibiting the Calpain-1/hypoxia-inducible factor-1α (HIF-1α) pathway. METHODS: In vivo, mice with LPS-induced ALI were pretreated with HSYA or a Calpain-1 overexpression lentivirus. In vitro, LPS-stimulated PMECs were treated with HSYA, Calpain-1 siRNA, or HIF-1α overexpression plasmids. Lung injury, cellular apoptosis, endothelial barrier integrity, and related molecular expressions were assessed using histological staining, wet/dry ratio, flow cytometry, transendothelial electrical resistance (TEER), ELISA, RT-qPCR, Western blotting, and immunofluorescence. Furthermore, direct HSYA-protein interactions were analyzed via molecular docking and surface plasmon resonance (SPR). RESULTS: HSYA significantly ameliorated LPS-induced lung damage, inflammatory responses, oxidative stress, and pulmonary microvascular endothelial barrier dysfunction in vivo, while reducing apoptosis and restoring barrier integrity in PMECs in vitro. Mechanistically, molecular docking and SPR analyses confirmed that HSYA directly binds to Calpain-1 with high affinity, thereby inhibiting its activity and subsequently downregulating HIF-1α expression. Furthermore, silencing Calpain-1 provided the protective effects against LPS-induced PMEC injury, whereas HIF-1α overexpression partially abolished these benefits. Moreover, Calpain-1 overexpression partly reversed the protective effects of HSYA on PMEC barrier dysfunction and ALI in mice. CONCLUSIONS: HSYA mitigates LPS-induced ALI by preserving pulmonary microvascular barrier function via direct inhibition of the Calpain-1/HIF-1α pathway.</p>