Abstract：
Purpose: to explore the anti-inflammatory effects of a nanobody (Nb) specific to β-glucan on fungal keratitis (FK). Methods: in order to verify the therapeutic and anti-inflammatory efficacy of Nb in FK, the severity of inflammation was assessed with inflammatory scores, hematoxylin-eosin (HE) staining, and myeloperoxidase (MPO) assays. In corneas of mice of FK model and human corneal epithelial cells stimulated by fungal hyphae, real-time reverse transcriptase polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines and pattern recognition receptors (PRRs). In vivo, macrophages and neutrophils infiltration in the cornea stroma was detected by immunofluorescence (IFS) staining. Results: In murine models infected with Aspergillus fumigatus (A. fumigatus), Nb treatment could reduce the inflammatory scores. HE staining and MPO results showed Nb significantly alleviated corneal edema and reduced inflammatory cell infiltration 3 days post-infection. In addition, the expression levels of LOX-1 and Dectin-1 were significantly decreased in the Nb group in vivo. The expression of chemokines CCL2 and CXCL2 also decreased in the Nb group. Compared with the PBS group, the number of macrophages and neutrophils in the Nb group was significantly decreased, which was shown in IFS results. Moreover, Nb attenuated the expression of Dectin-1, LOX-1, and inflammatory mediators, including IL-6 and IL-8 in vitro. Conclusion: our study showed that Nb could alleviate FK by downregulating the expression of PRRs and inflammatory factors as well as reducing the infiltration of macrophages and neutrophils.
摘要：
目的:探讨β-葡聚糖特异性纳米抗体(Nb)对真菌性角膜炎(FK)的抗炎作用。方法:为了验证Nb在FK中的治疗和抗炎疗效,用炎症评分评估炎症的严重程度,苏木精-伊红（HE）染色，和髓过氧化物酶(MPO)测定。在FK模型小鼠角膜和真菌菌丝刺激的人角膜上皮细胞中，实时逆转录酶聚合酶链反应，蛋白质印迹,采用酶联免疫吸附法检测炎性细胞因子和模式识别受体(PRRs)的表达水平。在体内,通过免疫荧光（IFS）染色检测角膜基质中的巨噬细胞和中性粒细胞浸润。结果：在感染烟曲霉的小鼠模型中（A.烟曲霉)，Nb处理可以降低炎症评分。HE染色和MPO结果显示，感染后3天，Nb可显着减轻角膜水肿，减轻炎症细胞浸润。此外,Nb组体内LOX-1和Dectin-1的表达水平显著降低。趋化因子CCL2和CXCL2在Nb组中的表达也降低。与PBS组相比，Nb组巨噬细胞和中性粒细胞数量显著减少,这在IFS结果显示。此外，Nb减弱了Dectin-1，LOX-1和炎症介质的表达，包括体外IL-6和IL-8。结论：我们的研究表明Nb可以通过下调PRRs和炎症因子的表达以及减少巨噬细胞和中性粒细胞的浸润来减轻FK。
