Flavopiridol Protects Against Fungal Keratitis by Alleviating Inflammation Through the Promotion of Autophagy

Abstract Background: Fungal keratitis is a serious infectious keratopathy related to fungal virulence and excessive inflammatory responses. Autophagy exhibits a potent ability to resolve inflammation during fungal infection. This study aimed to investigate the protective function of flavopiridol in fungal keratitis and explore its effects on autophagy. Methods: A mouse model of fungal keratitis was established and then treated with 5 μM flavopiridol. RAW 264.7 cells were treated with 200 nM flavopiridol before fungal stimulation. The severity of corneal diseases was evaluated by slit-lamp microscopy. The expression levels of cytokines were detected by RT-PCR and ELISA. The protein levels of LC3, Beclin-1 and Atg7 were determined by western blot and immunofluorescence. A Cell Counting Kit-8 assay was used to test cell viability. Autolysosomes were detected by transmission electron microscopy (TEM). An inhibitor of autophagy, 3-methyladenine (3-MA), was used to pretreat RAW 264.7 cells. Phagocytosis of RAW 264.7 cells was evaluated by counting colony forming units. A. fumigatus was incubated with flavopiridol, and the hyphae were stained with calcofluor white. Absorbance assay, crystal violet staining and adherence assay were used to detect the antifungal activity of flavopiridol. Results: Flavopiridol treatment notably reduced corneal opacity and the clinical scores of infected corneas. Compared with DMSO treatment, flavopiridol treatment greatly downregulated IL-1β, IL-6 and TNF-a expression in infected corneas. In RAW 264.7 cells, flavopiridol treatment inhibited IL-1β, IL-6 and TNF-a expression but promoted IL-10 expression. TEM images showed that more autolysosomes were presented in infected corneas and RAW 264.7 cells after flavopiridol treatment than after DMSO treatment. Flavopiridol treatment notably upregulated the protein expression of LC3, Beclin-1 and Atg7 in infected corneas as well as in RAW 264.7 cells. 3-MA pretreatment counteracted the cytokine regulation induced by flavopiridol. Moreover, flavopiridol promoted the phagocytosis of RAW 264.7 cells. Flavopiridol also exhibited antifungal activity by restricting fungal growth and limiting fungal biofilm formation and conidial adhesion. Conclusions: Flavopiridol significantly alleviated the inflammation of fungal keratitis by activating autophagy. In addition, flavopiridol promoted the phagocytosis of RAW 264.7 cells and exhibited antifungal function, indicating the potential therapeutic role of flavopiridol in fungal keratitis.