Dectin-1和TLR4在烟曲霉菌性角膜炎大鼠角膜组织中的表达及其在角膜炎发病中的作用

Expressions of TLR4 and Dectin-1 in cornea tissue of rats with Aspergillus fungal keratitis and their effects in pathogenesis of keratitis

目的:探讨烟曲霉菌感染大鼠角膜后Dectin-1和Toll样受体-4(TLR4)表达及炎症因子的分泌特点,阐明其在大鼠角膜炎发病中的作用。方法:40只SD大鼠随机分为对照组(n=10)和烟曲霉菌性角膜炎组(n=30),对照组不予任何干预,烟曲霉菌性角膜炎组给予双眼烟曲霉菌感染造模,分别于感染12h(n=10,12h组)、24h(n=10,24h组)和48h(n=10,48h组)后取角膜组织,ELISA法检测IL-1、TNF-α、IL-6、IL-10和NF-κB水平,免疫组织化学法、RT-PCR法及Western blotting法测定角膜组织中Dectin-1和TLR4mRNA及蛋白表达。结果:大鼠造模均成功,眼角膜裂隙灯检测观察可见对照组眼睛清澈,感染烟曲霉菌后,12h组眼睛表面有明显的一层薄膜,薄膜与健康的角膜边界清晰;24h组可见眼部有一层白色的浸润灶形成,且表面粗糙;48h组角膜白色浸润灶进一步加深,且范围明显扩大,基本覆盖了整个眼球。烟曲霉菌感染后,12h组、24h组和48h组角膜组织中炎症因子IL-1、IL-6、IL-10、TNF-α和NF-κB水平明显高于空白对照组(P<0.05),其中24h组和48h组明显高于12h组(P<0.05),48h组明显高于24h组(P<0.05);免疫组织化学检测,大鼠角膜组织中Dectin-1和TLR4表达水平随着感染时间的增加逐渐增加,RT-PCR法和Western blotting法检测,大鼠眼角膜组织烟曲霉菌性角膜炎组(12h组、24h组和48h组)Dectin-1和TLR4蛋白及mRNA表达水平明显高于对照组(P<0.05),其中24h组和48h组明显高于12h组(P<0.05),48h组明显高于24h组(P<0.05)。结论:Dectin-1和TLR4在受到烟曲霉菌感染后可过量分泌,进而造成大鼠炎性相关因子表达,最终促使角膜炎的发生。

Objective To explore the expressions of Dectin-1 and Toll-like receptor 4 （TLR4）and the secretory charateristics of inflammatory factors of the Aspergillus fumigatus infected cornea of the rats,and to clarify theirs role in the pathogenesis of rat keratitis.Methods 40 SD rats were separated into control group （n = 10）and aspergillus keratitis group （n=30）.The rats in aspergillus keratitis group were infected by Aspergillus fumigatus in two eyes for 12,24 and 48 h,and the cornea tissue was took and ELISA kit was used to detect the level of inflammatory cytokines IL-1,TNF-a,IL- 6,IL-10,and NF-κB;immunohistochemistry,Western blotting and RT-PCR methods were used to determine the Dectin-1 and TLR4 protein and mRNA expressions.Results The rat models were successfully established.The eyes of the rats in control group were limpid tested by corneal slit lamp, however,there existed a clear boundary film on the surface of eyes of the rats in aspergillus keratitis group infected for 12 h,white infiltrates and bloodshot in the eyes in 24 h group and the white infiltrates were further strengthened and almost covered the entire eyes in 48 h group. The order of inflammatory cytokines IL-1,TNF-α,IL-6,IL-10, NF-κB was：48 h group〉 24 h group〉 12 h group〉 control group （P 〈 0.05）,so did the expression levels of Dectin-1 and TLR4 protein and mRNA in various groups tested by Western blotting and RT-PCR methods.The expression levels of Dectin-1 and TLR4 were increased with the prolongation of time investigated by immunohistochemical staining.Conclusion Dectin-1 and TLR4 are secreted in excess after the eyes are infected with fumigatus,which promotes the expressions of related inflammatory cytokines and results in the occurrence of keratitis.