刺芒柄花素在小鼠烟曲霉菌性角膜炎中保护作用的研究

Study on the Protective Effect of Formononetin in Mouse Aspergillus fumigates Keratitis

摘要: 刺芒柄花素(Formononetin, FMN)，又称芒柄花素、芒柄花黄素，是一种异黄酮类中药单体，主要存在于黄芪、红车轴草、葛根等豆科植物中。最近的大量研究表明，刺芒柄花素具有抗炎、抗氧化、抗凋亡、抗菌、抗肿瘤和雌激素样作用等，药用价值高。然而，刺芒柄花素在真菌性角膜炎中的作用尚未被报道。本文旨在研究刺芒柄花素在烟曲霉菌性角膜炎中的保护作用。方法：在体外使用CCK-8试剂盒检测刺芒柄花素的毒性作用。用灭活的烟曲霉菌菌丝预先刺激RAW264.7细胞1小时，随后将刺芒柄花素溶液或DMSO溶液加入共培养7小时，通过PCR检测RAW264.7细胞炎症因子的mRNA表达水平。为了进一步明确刺芒柄花素在体内对角膜炎的治疗作用及炎症因子水平的影响，我们将烟曲霉菌孢子通过角膜基质内注射建立小鼠烟曲霉菌角膜炎模型，并使用刺芒柄花素溶液或DMSO溶液局部点眼治疗，通过PCR实验检测炎症因子的表达水平。结果：刺芒柄花素对RAW264.7细胞的存活能力影响小。与DMSO处理组相比，刺芒柄花素处理可以降低灭活菌丝刺激导致的细胞中炎症因子的表达升高。此外，在体内实验中，刺芒柄花素可以减轻小鼠烟曲霉菌性角膜炎的严重程度，减低感染角膜中炎症因子的表达。结论：刺芒柄花素可以降低小鼠烟曲霉菌性角膜炎的炎症因子表达水平起到抗炎保护作用。

Abstract: Formononetin (FMN) is an isoflavone monomer commonly found in legumes such as Astragalus membranaceus, Red Axle Grass, and Pueraria lobata in traditional Chinese medicine. Numerous recent studies have demonstrated its various medicinal properties including anti-inflammatory, antioxidant, anti-apoptotic, antibacterial, antitumor, and estrogen-like effects. However, the potential role of FMN in fungal keratitis has not been extensively explored. This study aimed to investigate the protective effects of FMN in Aspergillus fumigatus (A. fumigatus) keratitis. Methods: In vitro experiments utilized the CCK-8 kit to assess the toxicity of FMN. RAW264.7 cells were pre-treated with inactivated A. fumigatus hyphae for 1 hour, followed by evaluation of mRNA expression levels of inflammatory factors after 7 hours of co-culture with FMN or DMSO. To further elucidate the therapeutic impact of FMN on A. fumigatus keratitis and inflammatory factor levels in vivo, a model of A. fumigatus keratitis was established in mice via intracorneal stromal injection. Local eye spot treatment with FMN solution or DMSO solution was administered, and inflammatory factor expression levels were assessed through PCR analysis. Results: FMN had no significant effect on the viability of RAW264.7 cells. In A. fumigatus inactivated hyphae-stimulated RAW264.7 cells, FMN treatment led to a reduction of inflammatory cytokine expression levels compared to the DMSO group. Moreover, in vivo experiments demonstrated that FMN could alleviate the severity of A. fumigatus keratitis in mice and decrease inflammatory factor expression levels in infected corneas. In conclusion, FMN exhibited anti-inflammatory protective effects by reducing inflammatory factor expression levels in A. fumigatus keratitis in mice.