Shikonin treats Aspergillus fumigatus keratitis by activating autophagy and reducing fungal load.

The objective of this study was to explore the therapeutic potential of Shikonin (SK) in the treatment of fungal keratitis. Various tests conducted in this study were fluorescein sodium staining, Cell Counting Kit-8 (CCK-8), RT-PCR, Western blot (WB), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) methods. And the autophagic and phagocytic functions of RAW 264.7 cells were examined using transmission electron microscopy (TEM) and phagocytosis assay. The efficacy of SK in inhibiting Aspergillus fumigatus (A. fumigatus) was confirmed by determining the minimal inhibitory concentration (MIC), crystal violet staining assay, calcofluor white staining (CFW), propidium iodide absorption assay (PI), and fungal adhesion assay. Compared to the group treated with 0.1 % DMSO, the group treated with SK demonstrated a significant decrease in clinical score and the levels of IL-6, TNF-α, and IL-1β. SK also promoted the activity of autophagy proteins LC3B- II/I and Beclin-1 while reducing P62 levels. After treating with SK, there was an increase in autolysosomes in RAW 264.7 cells, accompanied by enhanced phagocytosis of fungi and conidia. Pretreatment with 3-methyladenine (3-MA) counteracted the ability of SK to reduce inflammatory markers. Furthermore, SK inhibited A. fumigatus growth, damaged cell membrane, impaired biofilms, and suppressed conidial adhesion. In conclusion, SK exhibits anti-inflammatory function by activating autophagy and anti-fungal function by reducing fungal load, highlighting its potential treatment effect for fungal keratitis.