目的:真菌性角膜炎(FK)通常由于真菌侵袭和过度的炎症反应而导致临床预后不良。为了增强纳他霉素(NAT)的治疗效果,我们使用抗炎生物多糖白名单多糖(BSP)与NAT结合制备了一种新的滴眼液-氧化白名单多糖纳他霉素(OBN)。
方法:UV-vis,FT-IR,和荧光光谱法用于鉴定OBN的合成。通过CCK-8,划痕试验确定OBN的生物相容性,和角膜毒性试验。用烟曲霉刺激RAW264.7细胞和C57BL/6小鼠并用PBS处理,OBN,或NAT。通过RT-PCR和ELISA检测OBN的抗炎活性。在患有FK的小鼠中,临床评分用于评估OBN的疗效;HE染色用于评估角膜病理变化;MPO测定和免疫荧光染色用于研究中性粒细胞浸润。
结果:OBN是通过席夫碱反应将氧化的白杨多糖(OBSP)与NAT结合合成的。OBN在HCECs中浓度为160μg/mL时不影响细胞活力,RAW264.7细胞,和老鼠角膜。OBN与NAT通过降低疾病严重程度显著改善烟曲霉角膜炎的预后,中性粒细胞浸润,体内炎症因子的表达。此外,OBN治疗下调炎症因子IL-1β的mRNA和蛋白表达水平,TNF-α,RAW264.7和小鼠模型中的IL-6。
结论:OBN是通过席夫碱反应将OBSP与NAT的亚氨基共价连接而制备的化合物。OBN治疗下调炎症并改善患有烟曲霉角膜炎的小鼠的预后。
OBJECTIVE: Fungal keratitis (FK) usually develops to a poor clinical prognosis due to the fungal invasion and excessive inflammatory reaction. In order to enhance the therapeutic effect of natamycin (NAT), we used the anti-inflammatory biological polysaccharide bletilla striata polysaccharide (BSP) combined with NAT to prepare a new eye drop -- oxidized bletilla striata polysaccharide-natamycin (OBN).
METHODS: UV-vis, FT-IR, and fluorescence spectroscopy were used to identify the synthesis of OBN. Biocompatibility of OBN was determined by CCK-8, scratch assay, and corneal toxicity test. RAW264.7 cells and C57BL/6 mice were stimulated with A. fumigatus and treated with PBS, OBN, or NAT. The anti-inflammatory activity of OBN was detected by RT-PCR and ELISA. In mice with FK, the clinical scores were used to evaluate the effect of OBN; HE staining was performed to assess the corneal pathological changes; MPO assay and immunofluorescence staining were used to investigate neutrophil infiltration.
RESULTS: OBN was synthesized by combining oxidized bletilla striata polysaccharide (OBSP) with NAT through Schiff base reaction. OBN did not affect cell viability at a concentration of 160 μg/mL in HCECs, RAW264.7 cells, and mouse corneas. OBN versus NAT significantly improved the prognosis of A. fumigatus keratitis by reducing disease severity, neutrophil infiltration, and expression of inflammatory factors in vivo. Additionally, OBN treatment down-regulated the mRNA and protein expression levels of inflammatory factors IL-1β, TNF-α, and IL-6 in RAW264.7 and mouse models.
CONCLUSIONS: OBN is a compound prepared by covalently linking OBSP to the imino group of NAT through Schiff base reaction. OBN treatment down-regulated inflammation and improved the prognosis of mice with A. fumigatus keratitis.