Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China.
Department of Biochemistry, Microbiology, and Immunology, Wayne State University School of Medicine, Detroit, MI 40201, USA.
Mol Immunol. 2022 Jul;147:50-61. doi: 10.1016/j.molimm.2022.04.007. Epub 2022 Apr 30.
To explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis.
The nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators.
CS at 400 μg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1β, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells.
CS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.
探讨硫酸软骨素（CS）对烟曲霉菌角膜炎的治疗作用。
采用 Draize 眼试验和细胞计数试剂盒-8 测定 CS 的无毒浓度。细胞划痕试验和细胞增殖试验评估 CS 对人角膜上皮细胞（HCEC）增殖和迁移的影响。粘附试验和平板计数分别用于检测体内和体外的真菌负荷。临床评分和苏木精-伊红（HE）染色用于评估 CS 在烟曲霉菌角膜炎小鼠模型中的治疗效果。通过流式细胞术（FCM）、免疫荧光染色和髓过氧化物酶（MPO）测定检测中性粒细胞浸润和活性。通过免疫荧光染色检测 RAW 264.7 细胞和小鼠角膜中 Toll 样受体 4（TLR-4）的表达。实时 PCR（RT-PCR）、western blot 和酶联免疫吸附试验（ELISA）用于检测炎症介质的表达。
400μg/mL（非细胞毒性）的 CS 可显著促进 HCEC 的增殖和迁移。在烟曲霉菌角膜炎小鼠模型中，CS 治疗通过降低角膜真菌负荷和抑制中性粒细胞浸润来减轻真菌性角膜炎（FK）的严重程度。在 RAW 264.7 细胞中，siTLR-4 处理组 TLR-4、磷酸化核因子（NF）-κB（p-NF-κB）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、环氧化酶 2（COX-2）和巨噬细胞炎症蛋白-2（MIP-2）的 mRNA 和蛋白水平明显低于相应对照组，rTLR-4 处理组则高于相应对照组。CS 治疗抑制 rTLR-4 诱导的 RAW 细胞 TLR-4、p-NF-κB、IL-1β、IL-6、COX-2、TNF-α和 MIP-2 的 mRNA 和蛋白表达。
CS 通过促进角膜上皮细胞增殖、抑制中性粒细胞募集和活性以及通过下调 TLR-4/NF-κB 信号通路抑制炎症反应，可能改善烟曲霉菌角膜炎的预后。
