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. 2021 Mar 1;62(3):38. doi: 10.1167/iovs.62.3.38.
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.
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.
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.
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.
山奈酚是一种具有抗菌和抗炎特性的天然类黄酮，但它对真菌性角膜炎（FK）的作用尚不清楚。本研究旨在探讨山奈酚对烟曲霉角膜炎的抗真菌和抗炎作用。
体外通过最低抑菌浓度和细胞毒性试验在人角膜上皮细胞（HCEC）和 RAW264.7 细胞中评估山奈酚的最低有效浓度。通过扫描电子显微镜和碘化丙啶摄取试验研究抗真菌特性。通过定量实时聚合酶链反应（qRT-PCR）观察山奈酚在 HCEC 和 RAW264.7 细胞中的抗炎作用。在烟曲霉角膜炎小鼠的眼中，通过临床评分、平板计数、组织学染色和过碘酸希夫染色评估 FK 严重程度。体内通过免疫荧光染色、髓过氧化物酶测定、Western blot、酶联免疫吸附试验和 qRT-PCR 检测山奈酚的抗炎作用。
在 HCEC 和 RAW264.7 细胞中，山奈酚在 80μg/mL 时显著抑制烟曲霉分生孢子生长和菌丝活力，而不影响细胞活力。体外，山奈酚改变了烟曲霉菌丝的形态和膜完整性。在烟曲霉角膜炎小鼠模型中，山奈酚通过减少角膜真菌负荷和抑制中性粒细胞募集来减轻 FK 的严重程度。此外，在体内和体外，山奈酚治疗组 TLR-2、TLR-4、Dectin-1、IL-1β 和肿瘤坏死因子-α的 mRNA 和蛋白表达水平均显著降低。
山奈酚通过抑制烟曲霉的生长、减少中性粒细胞的募集和下调炎症因子，改善烟曲霉角膜炎小鼠的预后。
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