Preclinical study on cynaroside as a selective granzyme B inhibitor in the treatment of atopic dermatitis

Granzyme B (GrB) is a serine protease primarily involved in cytotoxic T lymphocyte (CTL)-mediated apoptosis through caspase activation. However, emerging evidence suggests that GrB also plays a role in inflammation and extracellular matrix (ECM) degradation, contributing to skin disorders such as atopic dermatitis (AD). GrB is expressed by various immune and non-immune cells, including mast cells, macrophages, and keratinocytes, where it functions independently of perforin to degrade ECM components like fibronectin and decorin. In AD, elevated GrB levels correlate with disease severity, and GrB-deficient mice exhibit reduced AD symptoms, suggesting that inhibiting GrB could be a promising therapeutic approach. Despite its pathological significance, selective GrB inhibitors remain scarce. In this study, we identified cynaroside, a flavonoid compound, as a novel natural inhibitor of GrB. Virtual screening and molecular docking analysis revealed that cynaroside interacts with the Asp88 residue of GrB, forming stable hydrogen bonds that interfere with its catalytic activity. Molecular dynamics (MD) simulations confirmed that this interaction remains stable over 200 ns. Additionally, cynaroside inhibited GrB activity in vitro and significantly reduced fibronectin cleavage. Immunohistochemical analysis further demonstrated decreased GrB expression and preserved fibronectin levels in cynaroside-treated AD mice. Moreover, cynaroside treatment led to a reduction in epidermal thickening and immune cell infiltration, including mast cells and Th2 cells-key indicators of AD severity. These findings highlight cynaroside's potential as a targeted GrB inhibitor for inflammatory skin diseases.