Crocin inhibits neutrophil migration and activation to treat hypoxic pulmonary hypertension through targeting HCK

Background: Crocin (CRO) holds promise as a treatment for hypoxic pulmonary hypertension (HPH); however, its pharmacological mechanism remains poorly understood.

Objectives: We investigated how CRO improves HPH by acting on multiple cell types and pathological pathways, using single-cell RNA Sequencing (scRNA-seq).

Materials and methods: We first established a hypoxia-induced HPH rat model to evaluate the therapeutic effects of CRO. We then performed scRNA-seq to analyze how CRO alters cell populations and gene expression. Then, the effects of CRO on neutrophil migration and activation were investigated. Furthermore, molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) were used to validate the direct target of CRO. Finally, a co-culture system of neutrophils and pulmonary arterial smooth muscle cells (PASMCs) was used to confirm the link between CRO-mediated inhibition of PASMCs proliferation and neutrophil migration and activation.

Results: In HPH rat model, CRO treatment significantly improved hemodynamic parameters and alleviated pathological changes in lung. scRNA-seq analysis revealed a marked reduction in the proportions of neutrophils and PASMCs. KEGG analysis of differentially expressed genes (DEGs) in neutrophils showed significant enrichment in cell migration and activation-related pathways. In vitro, CRO inhibited fMLP-induced neutrophils migration and PMA-induced neutrophil extracellular traps (NETs) formation. Molecular docking, CETSA, and DARTS analyses identified Hck as a primary binding target of CRO. Subsequent experiments using an Hck Inhibitor confirmed that CRO inhibited neutrophils migration and activation through Hck. Furthermore, neutrophil-PASMCs co-culture experimental system confirmed that the inhibitory effects of CRO on PASMCs were associated with the inhibition of neutrophils activation.

Conclusion: CRO targets Hck to inhibit neutrophils migration and activation, and subsequently preventing neutrophil-induced PASMCs proliferation. These results highlight a novel therapeutic target for HPH and provide a scientific basis for the potential clinical application of CRO.

Crocin; HCK; Hypoxic pulmonary hypertension; Neutrophils; Pulmonary artery smooth muscle cells; scRNA-seq.