Therapeutic Effect and Mechanism Study of Auranofin in Fungal Keratitis

目的：探索金诺芬(Auranofin)在小鼠烟曲霉菌感染角膜炎模型中的抗炎作用。方法：采用细胞计数试剂盒(CCK-8)及Calcein/PI细胞活性与细胞毒性检测试剂盒检测金诺芬的细胞毒性，采用RT-PCR方法检测人角膜上皮细胞(Hcecs)中肿瘤坏死因子α (TNF-α)、白细胞介素1β (IL-1β)、白细胞介素6 (IL-6)及趋化因子2 (CCL-2)的mRNA的表达水平；采用ELISA方法检测Hcecs细胞中的TNF-α、IL-1β、IL-6和CCL2蛋白表达水平；采用RT-PCR方法检测金诺芬处理后Hcecs细胞中Nrf2、HO-1的mRNA表达水平；给予Brusatol (简称BT，Nrf2抑制剂)、Znpp (HO-1抑制剂)预处理Hcecs细胞，检测金诺芬对炎症因子表达情况的影响。结果：1 µg/mL的金诺芬对Hcecs细胞活力无影响，PCR结果显示金诺芬可降低真菌感染角膜细胞中TNF-α、IL-1β、IL-6和CCL-2的mRNA及蛋白水平，差异具有统计学意义；金诺芬可显著激活Nrf2/HO-1通路，逆转真菌刺激对炎症因子产生的促进作用。结论：金诺芬通过抑制炎症因子产生并激活Nrf2/HO-1信号通路在烟曲霉菌感染中发挥抗炎作用。Purpose: To explore the anti-inflammatory effects of Auranofin in a mouse model of Aspergillus fumigatus-infected keratitis. Methods: The cytotoxicity of Auranofin was detected using a cell counting kit (CCK-8) and a Calcein/PI cell activity and cytotoxicity kit; mRNA expression levels of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6) and chemokine 2 (CCL-2) were detected by RT-PCR in human corneal epithelial cells (Hcecs); TNF-α, IL-1β, IL-6 and CCL2 protein expression levels in Hcecs cells were detected by ELISA; mRNA expression levels of Nrf2 and HO-1 were detected by RT-PCR in Hcecs cells treated with Auranofin; administration of Brusatol (abbreviated as BT, the Nrf2 inhibitor) and Znpp (HO-1 inhibitor) were given to pretreat Hcecs cells, and the effect of Auranofin on the expression of inflammatory factors was detected. Results: 1 µg/mL of Auranofin had no effect on Hcecs cell activity. PCR and ELISA results showed that Auranofin reduced the mRNA and protein levels of TNF-α, IL-1β, IL-6, and CCL-2 in fungal-infected corneal cells, with significant differences; Auranofin significantly activated the Nrf2/HO-1 pathway, reversing the promotion of inflammatory factor production by fungal stimulation. Conclusion: Auranofin exerts anti-inflammatory effects in Aspergillus fumigatus infection by inhibiting inflammatory factor production and activating the Nrf2/HO-1 signaling pathway.