丁香酚治疗小鼠真菌性角膜炎的作用机制研究

Study on the Therapeutic Mechanism of Eugenol in Mouse Fungal Keratitis

摘要: 目的：探讨丁香酚(Eugenol, EUG)在烟曲霉菌性角膜炎中的抗炎、抗氧化作用。方法：提取C57BL/6小鼠的腹腔巨噬细胞，给予丁香酚或DMSO溶液预处理后，使用灭活的烟曲霉菌菌丝刺激细胞。烟曲霉菌感染小鼠角膜，给予丁香酚或DMSO点眼处理五天。采用实时荧光定量PCR测定巨噬细胞及小鼠角膜中炎症因子(IL-1β、TNF-α、IL-10、iNOS)以及模式识别受体TLR2的mRNA表达水平。使用裂隙灯观察小鼠角膜病变情况，角膜HE染色评估炎症细胞浸润程度。采用DCFH-DA荧光探针检测巨噬细胞中活性氧的水平。结果：在小鼠腹腔原代巨噬细胞中，丁香酚预处理显著抑制了烟曲霉菌刺激引起的炎症因子及TLR2的表达升高，同时降低了细胞中活性氧水平。与DMSO对照组相比，经丁香酚处理的真菌性角膜炎小鼠的角膜更加透明、水肿减轻、溃疡面积较对照组明显减小，角膜组织中的炎症细胞浸润明显减轻。结论：丁香酚可以通过抑制炎症因子及模式识别受体TLR2的表达、减少活性氧的产生，在烟曲霉菌性角膜炎中发挥抗炎、抗氧化的作用。

Abstract: Objective: To investigate the anti-inflammatory and antioxidant effects of eugenol (EUG) on Aspergillus fumigatus (A. fumigatus) keratitis. Methods: The macrophages were collected from the abdominal cavity of C57BL/6 mice. After being pretreated with EUG or DMSO, the macrophages were stimulated by inactivated A. fumigatus mycelium. Mice corneas were infected with A. fumigatus, and then treated with EUG or DMSO spotting on the eyes for five days. Real-time PCR was performed to examine the mRNA expressions of inflammatory mediators (IL-1β, TNF-α, IL-10 and iNOS) and Toll-like receptor 2 (TLR2). The severity of keratitis was observed under a slit-lamp. HE staining was applied to evaluate the effects of EUG on inflammatory cells infiltration. DCFH-DA fluorescent probe was used to detect the expression of reactive oxygen species in macrophages. Results: EUG significantly inhibited the expressions of inflammatory factors and TLR2 stimulated by A. fumigatus, and decreased the level of reactive oxygen species in macrophages. Compared with the DMSO treatment, mice corneas treated by EUG showed increased transparency, smaller ulcers and descended edema. Corneal HE staining showed that EUG significantly reduced the infiltration of inflammatory cells in corneal tissue. Conclusion: EUG plays an anti-inflammatory and antioxidant role in A. fumigatus keratitis by inhibiting the expressions of inflammatory factors and TLR2, as well as reducing the production of reactive oxygen species.