Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China.
Department of Biochemistry, Microbiology and Immunology, Wayne State University School of Medicine, Detroit, Michigan, United States.
Invest Ophthalmol Vis Sci. 2022 Nov 1;63(12):12. doi: 10.1167/iovs.63.12.12.
The purpose of this study was to explore the antifungal and anti-inflammatory effects of gallic acid (GA) on Aspergillus fumigatus (A. fumigatus) keratitis.
CCK-8 assay and Draize eye test were used to determine the non-cytotoxic concentration of GA in RAW264.7 cells and an A. fumigatus keratitis mouse model. The antifungal effects of GA were analyzed using minimal inhibitory concentration (MIC), biofilm formation test, fungal adherence assay, calcofluor white staining, and propidium iodide staining. The therapeutic effects of GA were estimated by slit lamp photographs, clinical score, hematoxylin and eosin (H&E) staining, and Periodic acid-Schiff staining in vivo. Immunofluorescence staining and myeloperoxidase assay were conducted to identify neutrophil infiltration and activity. RT-PCR, ELISA, and Western blot were performed to detect the expression of pro-inflammatory cytokines and Nrf2/HO-1.
In HCECs and A. fumigatus keratitis mouse model, GA at 100 µg/mL did not affect cell viability, thus this concentration was applied to subsequent experiments. In vitro, GA significantly inhibited A. fumigatus growth, biofilm formation, and adhesion. In vivo, 100 µg/mL GA alleviated the severity of fungal keratitis (FK) by repressing fungal load, reducing neutrophil infiltration, and lowering MPO activity. Besides, the expression of IL-1β, TNF-α, LOX-1, and COX-2 was inhibited, whereas Nrf2 and HO-1 expression was enhanced at both mRNA and protein levels in the 100 µg/mL GA treated group in comparison to PBS control.
GA ameliorates FK severity through inhibiting A. fumigatus load, reducing neutrophils infiltration, downregulating the expression of pro-inflammatory cytokines, and enhancing the Nrf2/HO-1 pathway, which provides new insight into A. fumigatus keratitis treatment.
本研究旨在探讨没食子酸（GA）对烟曲霉（A. fumigatus）角膜炎的抗真菌和抗炎作用。
CCK-8 法和 Draize 眼试验用于确定 RAW264.7 细胞和烟曲霉角膜炎小鼠模型中 GA 的非细胞毒性浓度。通过最小抑菌浓度（MIC）、生物膜形成试验、真菌黏附试验、钙荧光白染色和碘化丙啶染色分析 GA 的抗真菌作用。通过裂隙灯照相、临床评分、苏木精和伊红（H&E）染色和过碘酸希夫（PAS）染色在体内评估 GA 的治疗效果。免疫荧光染色和髓过氧化物酶（MPO）测定用于鉴定中性粒细胞浸润和活性。通过 RT-PCR、ELISA 和 Western blot 检测促炎细胞因子和 Nrf2/HO-1 的表达。
在 HCECs 和烟曲霉角膜炎小鼠模型中，100μg/mL 的 GA 不影响细胞活力，因此将该浓度应用于后续实验。在体外，GA 显著抑制 A. fumigatus 的生长、生物膜形成和黏附。在体内，100μg/mL GA 通过抑制真菌负荷、减少中性粒细胞浸润和降低 MPO 活性来缓解真菌性角膜炎（FK）的严重程度。此外，与 PBS 对照组相比，100μg/mL GA 处理组的 IL-1β、TNF-α、LOX-1 和 COX-2 的表达降低，而 Nrf2 和 HO-1 的表达在 mRNA 和蛋白水平上均增强。
GA 通过抑制 A. fumigatus 负荷、减少中性粒细胞浸润、下调促炎细胞因子的表达以及增强 Nrf2/HO-1 通路来改善 FK 的严重程度，为烟曲霉角膜炎的治疗提供了新的思路。
