百里醌对烟曲霉菌角膜炎的抗炎作用及其机制

Anti-inflammatory effects of thymoquinone against Aspergillus fumigatus keratitis and its mechanism of action

目的 探讨百里醌(TQ)在体内、体外真菌性角膜炎模型中的抗炎作用及其机制。方法 采用细胞计数试剂盒(CCK-8)和小鼠体内Draize试验筛选出TQ的安全抗炎浓度。建立小鼠真菌性角膜炎模型,给予小鼠角膜3.75 mg/L TQ(TQ组)或体积分数0.001二甲基亚砜(DMSO组)结膜下注射。显微镜下观察两组小鼠角膜炎症情况并进行临床炎症评分;采用苏木精-伊红(HE)染色法和髓过氧化物酶(MPO)实验观察TQ对小鼠角膜中性粒细胞募集的影响;应用实时荧光定量聚合酶链反应(qRT-PCR)和酶联免疫吸附试验(ELISA),检测各组人角膜上皮细胞(HCECs)环氧化酶2(COX-2)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)蛋白和mRNA的表达水平。结果 当TQ浓度低于3.75 mg/L时,HCECs存活率≥95%,健康小鼠角膜未见荧光素钠染色,HCECs和小鼠角膜均不受TQ影响。与DMSO组相比,TQ组小鼠角膜炎症减轻,临床评分降低(F3 d=2.01,P<0.01;F5 d=3.00,P<0.01),中性粒细胞的募集和活性降低(t=6.55,P<0.001)。PCR和ELISA检测结果显示,与DMSO组相比较,TQ组HCECs中COX-2、IL-1β及IL-6 mRNA和蛋白的表达均明显降低(t=6.12~15.06,P<0.001)。结论 TQ可以通过减少中性粒细胞的聚集、降低炎症因子的表达,在真菌性角膜炎中发挥抗炎作用。

Objective To investigate the anti-inflammatory effects of thymoquinone(TQ)in in-vivo and in-vitro models of fungal keratitis and the associated mechanisms.Methods The safe anti-inflammatory concentration of TQ was determined using Cell Counting Kit-8 and the in-vivo Draize test with mice.A mouse model of fungal keratitis was established.A subconjunctival injection of either 3.75 mg/L TQ or 0.001 dimethyl sulfoxide(DMSO)was given into the mouse cornea.The mouse cornea was exa-mined forevaluating the clinical inflammation score under a microscope.The effect of TQ on neutrophil recruitment in the mouse cornea was examined with hematoxylin-eosin staining and the myeloperoxidase test.The mRNA and protein expression levels of cyclooxygenase-2(COX-2),interleukin(IL)-1β,and IL-6 in human corneal endothelial cells(HCECs)were measured by quantitative real-time polymerase chain reaction(qRT-PCR)and enzyme-linked immunosorbent assay(ELISA).Results When the concentration of TQ was below 3.75 mg/L,the survival rate of HCECs was≥95%,and sodium fluorescein staining was negative in the cornea of healthy mice,indicating that it was not toxic to HCECs and the mouse cornea.Compared with the DMSO group,the mice in the TQ group showed significantly reduced clinical inflammation scores(F 3 d=2.01,P<0.01;F 5 d=3.00,P<0.01)and significantly reduced recruitment and activity of neutrophils(t=6.55,P<0.001).The qRT-PCR and ELISA results showed that compared with the DMSO group,HCECs in the TQ group had significantly lower mRNA and protein expression levels of COX-2,IL-1β,and IL-6(t=6.12-15.06,P<0.001).Conclusion TQ has anti-inflammatory effects against fungal keratitis by reducing neutrophil aggregation and decreasing the expression of inflammatory factors.