Therapeutic effect of oxidized bletilla striata polysaccharide-natamycin eye drops on fungal keratitis

Abstract

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. Conclusion: 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.