Journal: Investigative Ophthalmology & Visual Science

Article Title: Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response

doi: 10.1167/iovs.63.12.12

Figure Lengend Snippet: Antifungal effects of GA in vitro. MIC of GA at concentrations of 0, 12.5, 50, 100, 200, 400, and 800 µg/mL for 48 hours in HCECs. GA started to inhibit A. fumigatus growth at 100 µg/mL ( A ). Biofilm formation of A. fumigatus exposed to different concentrations of GA (0, 12.5, 25, 50, 100, and 200 µg/mL) for 48 hours. GA markedly inhibited biofilm formation of A. fumigatus starting from 25 µg/mL in a dose-dependent manner ( B ). H&E staining based fungal adhesion assay showed the number of A. fumigatus conidia adherent to HCECs was significantly reduced in the 100 µg/mL GA-treated group compared with PBS control (×400 magnification) ( C ). Quantitative results of H&E staining ( D ). Calcofluor white staining of A. fumigatus treated with PBS, Caspofungin acetate (positive control), or different concentrations of GA (50, 100, and 200 µg/mL) for 48 hours in vitro (×20 magnification). GA disrupted the cell wall integrity in a dose-dependent manner ( E ). PI staining of A. fumigatus treated with PBS, natamycin (positive control), or different concentrations of GA (50, 100, and 200 µg/mL) for 24 hours in vitro (×20 magnification) ( F ). All data were presented as mean ± SEM. MIC and biofilm formation results were analyzed by one-way ANOVA with Bonferroni's post hoc test, and an unpaired, two-tailed Student's t -test was used to compare fungal adhesion results.

Article Snippet: Natamycin (NAT) powder (CAS 7681-93-8; Macklin Biochemical Co. Ltd., Shanghai, China) was dissolved in PBS (Solarbio) at a storage concentration of 8 mg/mL, and further diluted to the required concentrations with DMEM (Solarbio) for RAW264.7 cells, and Sabouraud liquid medium for fungal conidia treatment.

Techniques: In Vitro, Staining, Cell Adhesion Assay, Control, Positive Control, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response

doi: 10.1167/iovs.63.12.12

Figure Lengend Snippet: GA improved A. fumigatus keratitis outcomes in mice. Representative slit-lamp photographs ( A ) and corresponding clinical scores ( B ) of A. fumigatus keratitis mice corneas with PBS, 100 µg/mL GA, and 8 µg/mL natamycin treatment at 1, 3, and 5 days p.i. ( n = 8/group/time). PAS staining (for fungal hyphae) ( C ) and H&E staining ( D ) of A. fumigatus keratitis mice eyeballs with PBS, 100 µg/mL GA, and 8 µg/mL natamycin treatment at 3 days p.i. ( n = 3/group; ×400). The clinical score in different groups was analyzed using the Mann-Whitney U test (ns: no significance).

Article Snippet: Natamycin (NAT) powder (CAS 7681-93-8; Macklin Biochemical Co. Ltd., Shanghai, China) was dissolved in PBS (Solarbio) at a storage concentration of 8 mg/mL, and further diluted to the required concentrations with DMEM (Solarbio) for RAW264.7 cells, and Sabouraud liquid medium for fungal conidia treatment.

Techniques: Staining, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response

doi: 10.1167/iovs.63.12.12

Figure Lengend Snippet: GA repressed neutrophil infiltration and MPO activity in vivo. Representative IFS photographs of A. fumigatus keratitis mice corneas treated with PBS, 100 µg/mL GA, and 8 µg/mL natamycin at 3 days p.i. ( n = 3/group). Green : NIMP-R14 labeled neutrophils; Blue : DAPI stained nucleus (×200 magnification) ( A ). IFS quantitative analysis of area fractions of neutrophils ( B ). Quantitative analysis of MPO activity levels of A. fumigatus keratitis mice corneas treated with PBS, 100 µg/mL GA, and 8 µg/mL natamycin at 3 days p.i. ( n = 5/group) ( C ). All data were mean ± SEM and analyzed by an unpaired, two-tailed Student's t -test.

Article Snippet: Natamycin (NAT) powder (CAS 7681-93-8; Macklin Biochemical Co. Ltd., Shanghai, China) was dissolved in PBS (Solarbio) at a storage concentration of 8 mg/mL, and further diluted to the required concentrations with DMEM (Solarbio) for RAW264.7 cells, and Sabouraud liquid medium for fungal conidia treatment.

Techniques: Activity Assay, In Vivo, Labeling, Staining, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response

doi: 10.1167/iovs.63.12.12

Figure Lengend Snippet: GA suppressed pro-inflammatory cytokine production in RAW264.7 cells and A. fumigatus keratitis mouse models. RAW264.7 cells were stimulated with inactivated A. fumigatus hyphae for 1 hour and treated with 100 µg/mL GA, 8 µg/mL natamycin, and PBS for 8 hours for RT-PCR and 24 hours for ELISA. RT-PCR results of the mRNA levels of IL-1β ( A ), TNF-α ( B ), COX-2 ( C ) , and LOX-1 ( D ), and ELISA results of IL-1β ( E ), TNF-α ( F ) in RAW264.7 cells. In an A. fumigatus mouse model, mice corneas were treated with 100 µg/mL GA, 8 µg/mL natamycin, and PBS eye drop 3 times/day, and the mRNA levels of IL-1β ( G ), TNF-α ( H ), COX-2 ( I ), and LOX-1 ( J ) were detected at 3 and 5 days p.i. ( n = 6/group/time). All data were mean ± SEM. RT-PCR and ELISA result from RAW264.7 cells were analyzed by one-way ANOVA with Bonferroni's post hoc test. An unpaired, two-tailed Student's t -test was used for RT-PCR results from A. fumigatus mouse models (N: normal; Af: A. fumigatus ; ns: no significance).

Article Snippet: Natamycin (NAT) powder (CAS 7681-93-8; Macklin Biochemical Co. Ltd., Shanghai, China) was dissolved in PBS (Solarbio) at a storage concentration of 8 mg/mL, and further diluted to the required concentrations with DMEM (Solarbio) for RAW264.7 cells, and Sabouraud liquid medium for fungal conidia treatment.

Techniques: Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Two Tailed Test