Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao 266003, China.
Department of Ophthalmology, The Affiliated Hospital of Qingdao University, Qingdao 266003, China.
Int Immunopharmacol. 2022 Sep;110:108992. doi: 10.1016/j.intimp.2022.108992. Epub 2022 Jul 7.
To investigate the anti-inflammatory and antifungal role of β-ionone (BI) in fungal keratitis (FK).
In vitro antifungal activity of BI against Aspergillus fumigatus (A. fumigatus) was evaluated by using minimum inhibitory concentration (MIC), crystal violet staining, biofilm biomass measurement, propidium iodide uptake test, and adherence assay. And RT-PCR was carried out to measure the levels of RodA, RodB, Rho, FKs, CshA-D, RlmA, Cyp51A-B and Cdr1B. Network pharmacology analysis was applied to predict the relationship between BI and FK. Cell Count Kit-8 (CCK8) assay was utilized to detect the cytotoxicity of BI to RAW264.7 and immortalized human corneal epithelial cells (HCECs). The underlying mechanism of BI at regulating the level of inflammatory factors in FK was assessed by RT-PCR, ELISA and Western blot in vitro and in vivo. The therapeutic effect of BI has investigated in A. fumigatus keratitis by employing the clinical score, pathological examination, plate count, immunofluorescence and myeloperoxidase (MPO) assay. We also used the slit-lamp microscopy, clinical scores, and HE staining to assess the effect of natamycin compared with BI treatment in vivo.
BI suppressed the growth of A. fumigatus and had a significant effect on A. fumigatus biofilms and membrane permeability. RT-PCR demonstrated that exposure of A. fumigatus to BI inhibited the expression of genes that function in hydrophobin (RodA, RodB), cell wall integrity (Rho, FKs, CshA-D, RlmA), azole susceptibility (Cyp51A-B, Cdr1B). Network pharmacology showed that the effects of BI in FK implicate with C-type lectin receptor signaling pathway. In vivo, after A. fumigatus infection, BI treatment markedly reduced the severity of FK by decreasing clinical score, neutrophil recruitment, and fungal load. And BI treatment also obviously reduced the expression of inflammatory cytokines, Lectin-like oxidized LDL receptor (LOX-1), phosphorylation of p38MAPK and p-JNK versus the DMSO-treated group. BI and natamycin both significantly increased corneal transparency and decreased inflammatory cell recruitment in the FK in the mice model.
These results indicated that BI had fungicidal activities against A. fumigatus. It also ameliorated FK in mice by reducing inflammation, which was regulated by LOX-1, p-p38MAPK and p-JNK.
研究β-紫罗兰酮（BI）在真菌性角膜炎（FK）中的抗炎和抗真菌作用。
采用最低抑菌浓度（MIC）、结晶紫染色、生物膜生物量测定、碘化丙啶摄取试验和黏附试验评价 BI 对烟曲霉（A. fumigatus）的体外抗真菌活性。并采用 RT-PCR 测定 RodA、RodB、Rho、FKs、CshA-D、RlmA、Cyp51A-B 和 Cdr1B 的水平。应用网络药理学分析预测 BI 与 FK 的关系。采用细胞计数试剂盒-8（CCK8）检测 BI 对 RAW264.7 和永生化人角膜上皮细胞（HCECs）的细胞毒性。通过体外和体内 RT-PCR、ELISA 和 Western blot 评估 BI 调节 FK 中炎症因子水平的机制。采用临床评分、病理检查、平板计数、免疫荧光和髓过氧化物酶（MPO）检测评估 BI 在烟曲霉角膜炎中的治疗效果。还采用裂隙灯显微镜、临床评分和 HE 染色评估与 BI 治疗相比，体内使用那他霉素的效果。
BI 抑制烟曲霉的生长，对烟曲霉生物膜和膜通透性有显著影响。RT-PCR 表明，BI 暴露于烟曲霉可抑制参与亲水性（RodA、RodB）、细胞壁完整性（Rho、FKs、CshA-D、RlmA）、唑类敏感性（Cyp51A-B、Cdr1B）的基因表达。网络药理学显示，BI 在 FK 中的作用与 C 型凝集素受体信号通路有关。体内，烟曲霉感染后，BI 治疗通过降低临床评分、中性粒细胞募集和真菌负荷，显著减轻 FK 的严重程度。并且 BI 治疗还明显降低了炎症细胞因子、凝集素样氧化 LDL 受体（LOX-1）、p38MAPK 和 p-JNK 的磷酸化表达，与 DMSO 处理组相比。BI 和那他霉素均能显著增加 FK 模型中小鼠角膜透明度，减少炎症细胞募集。
这些结果表明 BI 对烟曲霉具有杀菌活性。它还通过降低 LOX-1、p-p38MAPK 和 p-JNK 调节的炎症，改善了 FK 模型中小鼠的 FK。
