Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load, Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines

Abstract

Purpose: Isorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis.

Methods: In vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR.

Results: In HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1β, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro.

Conclusions: Isorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.

Figure 1.

Isorhamnetin antifungal activity and host cell cytotoxicity. A. Fumigatus conidia were cultured with 0.5% DMSO and isorhamnetin at different concentrations (5, 10, 20, 40, 80, 160, and 320 µg/mL) for 48 hours (MIC₉₀ = 80 µg/mL) (A). Isorhamnetin showed antifungal effect at 20 µg/mL and obviously inhibited hyphae viability at 80 µg/mL (B). HCECs and RAW264.7 cells were cultured with isorhamnetin at different concentrations for 24 hours. Cell viability of HCECs (C) and RAW264.7 (D) at different concentrations (0, 5, 10, 20, 40, 80, 160, and 320 µg/mL) of isorhamnetin. All data were mean ± SEM. MIC was analyzed by an unpaired, two-tailed Student's t-test, and CCK-8 and fungal viability assay were tested by one-way analysis of variance with post hoc analysis.

Figure 2.

Isorhamnetin alters A. fumigatus hyphal morphology and membrane integrity. Representative SEM images of A. fumigatus hyphae treated by 0.5% DMSO, magnification × 2000 (A) and × 5000 (B), and 80 µg/mL isorhamnetin, magnification × 2000 (C) and × 5000 (D) for 24 hours. Representative fluorescence images of PI uptake test showed A. fumigatus hyphae treated by 0.5% DMSO (E) and isorhamnetin at 40 µg/mL (F), 80 µg/mL (G), and 160 µg/mL (H) for 24 hours (magnification × 200; scale bar: 100 µm).

Figure 3.

FK severity after A. fumigatus infection followed by DMSO or isorhamnetin treatment. Representative slit lamp photographs of DMSO or isorhamnetin treated A. fumigatus keratitis mice at days 1, 3, and 5 (A). Clinical scores of DMSO or isorhamnetin treated mice cornea (n = 5 mice/group) (B). Representative plates of DMSO (C) or isorhamnetin (D) treated A. fumigatus–infected mouse cornea at five days p.i., and quantitative diagram (E). PAS staining of corneal tissue sections (magnification × 400) of DMSO (F) or isorhamnetin (G) treated A. fumigatus keratitis mice at three days p.i. (n = 3 mice/group). Clinical scores were analyzed using a nonparametric Mann-Whitney U test. Horizontal lines indicate the median values. Plate count results were analyzed by an unpaired, two-tailed Student's t-test.

Figure 4.

Effects of isorhamnetin on neutrophil infiltration in A. fumigatus keratitis mouse model. Mice with FK treated with isorhamnetin (80 µg/mL) or 0.5% DMSO, and stained with NIMP-R14-FITC (neutrophils, green) and DAPI (nucleus, blue) (n = 3 mice/group). Representative IFS images of mice corneas (magnification × 400) (A) and quantitative analysis (B). MPO activity in mice with FK treated by isorhamnetin (80 µg/mL) or 0.5% DMSO (n = 5 mice/group) (C). H&E staining of corneal tissue sections (magnification × 400) of DMSO (D) or isorhamnetin (E) treated A. fumigatus keratitis mice at day 3 (n = 3 mice/group). All data were mean ± SEM and analyzed by an unpaired, two-tailed Student's t-test.

Figure 5.

Effects of isorhamnetin on the inflammatory response in HCECs and RAW264.7 cells. After stimulating the cells with A. fumigatus (60 µL, 3 × 10⁸ CFU/mL) for two hours, the cells were cultured with isorhamnetin (80 µg/mL) or 0.5% DMSO for eight hours, and only A. fumigatus was added to the culture medium of positive control group. The negative control groups were set as uninfected cells treated with or without isorhamnetin and DMSO. The qRT-PCR was used to detect the mRNA level of each group. Compared with the control groups, isorhamnetin (80 µg/mL) significantly inhibited the mRNA expression of proinflammatory factors including TLR-2 (A), TLR-4 (B), Dectin-1 (C), IL-1β (D), TNF-α (E) in HCECs, and TLR-2 (F), TLR-4 (G), Dectin-1 (H), IL-1β (I), TNF-α (J) in RAW264.7 cells. All data were mean ± SEM and analyzed using an unpaired, two-tailed Student's t-test.

Figure 6.

Effects of isorhamnetin on the inflammatory response in A. fumigatus infected mouse cornea. qRT-PCR results for TLR-2 (A), TLR-4 (B), Dectin-1 (C) TNF-α (J) and IL-1β (K) at 1,3, and 5 days p. i. Western blot results and grayscale analysis of TLR-2 (D, G), TLR-4 (E, H), Dectin-1 (F, I) at 3 days p. i. ELISA results of TNF-α (L) and IL-1β (M) at 3 and 5 days p. i. (n = 6 mice/group). All data were mean ± SEM and analyzed by an unpaired, two-tailed Student's t-test.