吉林大学学报(医学版) ›› 2021, Vol. 47 ›› Issue (2): 315-322.doi: 10.13481/j.1671-587X.20210209

• 基础研究 • 上一篇    下一篇

木犀草素通过TLR4/MyD88信号通路对大鼠烟曲霉菌性角膜炎的调控作用

张淑荣(),张琦颖   

  1. 齐齐哈尔医学院附属第三医院眼科,黑龙江 齐齐哈尔 161000
  • 收稿日期:2020-05-08 出版日期:2021-03-28 发布日期:2021-03-25
  • 通讯作者: 张淑荣 E-mail:hk122221@163.com
  • 作者简介:张淑荣(1982-),女,黑龙江省齐齐哈尔市人,主治医师,医学硕士,主要从事葡萄膜视网膜疾病诊断和治疗方面的研究。
  • 基金资助:
    黑龙江省教育厅科研项目(12521625)

Regulatory effect of luteolin on Aspergillus fumigatus keratitis through TLR4/MyD88 signaling pathway in rats

Shurong ZHANG(),Qiying ZHANG   

  1. Department of Ophthalmology,Affiliated Third Hospital,Qiqihar Medical College,Qiqihar 161000,China
  • Received:2020-05-08 Online:2021-03-28 Published:2021-03-25
  • Contact: Shurong ZHANG E-mail:hk122221@163.com

摘要: 目的

探讨木犀草素通过Toll样受体4(TLR4)/髓样分化因子88(MyD88)信号通路对大鼠烟曲霉菌性角膜炎(AFK)的调控作用,阐明其抑制AFK炎症反应的机制。

方法

100只SD大鼠随机选取15只作为假手术组[未注入烟曲霉菌(AF)孢子混悬液],其余85只大鼠采用AF孢子混悬液注入法建立AFK模型。建模成功的72只大鼠随机分为模型组14只、低剂量木犀草素组14只、中剂量木犀草素组14只、高剂量木犀草素组15只和脂多糖(LPS)+木犀草素组15只。低、中和高剂量木犀草素组大鼠给予浓度为5、10和20 g·L-1木犀草素溶液局部滴眼50 μL,假手术组和模型组大鼠给予等量1% DMSO溶液局部滴眼,LPS+木犀草素组大鼠给予20 g·L-1木犀草素溶液50 μL和LPS溶液1.0 μg局部滴眼,连续7 d。观察各组大鼠角膜大体变化并评估各组大鼠角膜炎症指数;制备各组大鼠角膜组织标本,采用HE染色法观察角膜组织病理形态表现;采用酶联免疫吸附试验(ELISA)检测各组大鼠角膜组织中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和白细胞介素12(IL-12)水平,采用Western blotting法检测各组大鼠角膜组织中 TLR4、MyD88和核转录因子-κB(NF-κB)蛋白表达水平。

结果

假手术组大鼠角膜组织上皮完整,无炎性细胞浸润;模型组和LPS+木犀草素组大鼠角膜呈现白色致密溃疡灶,水肿且表面无光泽,虹膜不可见,LPS+木犀草素组溃疡灶面积更大;低、中和高剂量木犀草素组大鼠角膜溃疡灶面积逐渐变小,病变减轻。HE染色,模型组和LPS+木犀草素组大鼠角膜均可见胶原纤维肿胀、排列紊乱,大量炎性细胞浸润;低、中和高剂量木犀草素组上述变化均减轻,其中高剂量木犀草素组减轻最为明显。与模型组比较,低、中和高剂量木犀草素组大鼠角膜炎症指数和角膜组织中IL-1β、TNF-α、IL-12水平及TLR4、MyD88和NF-κB蛋白表达水平明显降低(P<0.05);LPS+木犀草素组上述指标均高于模型组和低、中及高剂量木犀草素组(P<0.05)。

结论

木犀草素对AFK大鼠具有治疗作用,可有效抑制角膜炎症反应,其作用机制可能与抑制TLR4/MyD88信号通路相关蛋白表达及其下游炎症因子有关。

关键词: 烟曲霉菌性角膜炎, 木犀草素, Toll样受体4, 髓样分化因子88, 炎症因子

Abstract:

Objective: To investigate the regulatory effects of luteolin on the Aspergillus fumigatus keratitis (AFK) through Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88(MyD88) signaling pathway in the rats, and to elucidate its mechanism of inhibiting AFK inflammatory response.

Methods

Fifteen rats were randomly selected from 100 SD rats as sham operation group [without injection of Aspergillus funmigatus (AF) spore suspension], and the other 85 rats were injected with spore suspension of AF to establish the AFK models. Seventy-two rats were successfully established and randomly divided into model group (14 rats), low dose of luteolin group (14 rats), medium dose of luteolin group (14 rats), high dose of luteolin group (15 rats), LPS + luteolin group (15 rats). The rats in low, medium and high doses of luteolin groups were given local eye drop of 50 μL luteolin with concentrations of 5, 10 and 20 g·L-1;the rats in sham operation group and model group were given the same amount of 1% DMSO solution;the rats in LPS + luteolin group were given 20 g·L-1 luteolin solution and 1.0 μg LPS solution for local eye drops;lasted for 7 d. The general changes of cornea of the rats in various groups were observed and the cornea inflammation index was evaluated. The corneal tissue samples were obtained, and the pathomorphology of cornea tissue was observed by HE staining method. The levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) in cornea tissue of the rats in various groups were detected by enzyme-linked immunosorbent assay (ELISA). The expression levels of TLR4, MyD88, and nuclear transcription factor-κB (NF-κB) proteins in cornea tissue of the rats in various groups were detected by Western blotting method.

Results

The cornea tissue epithelium of the rats in sham operation group was intact without inflammatory cell infiltration.The cornea of rats in model group and LPS + luteolin group showed white dense ulcers, edema and dull surface, the iris was not visible, and the area of ulcers in LPS + luteolin group was larger. In low, medium and high doses of luteolin groups, the areas of ulcer focus were gradually decreased and the lesions were alleviated. The HE staining results showed that the collagen fibers were swollen and disordered in model group and LPS + luteolin group, a large number of inflammatory cells were infiltrated, and the above changes were alleviated in low, middle and high doses of luteolin groups, especially in high dose of luteolin group. Compared with model group, the cornea inflammation index, the levels of IL-1β, TNF-α, IL-12 and the experssion levels of TLR4, MyD88, NF-κB proteins in corneal tissue in low, medium and high doses of luteolin groups were decreased (P<0.05).The above indexes of the rats in LPS + luteolin group were higher than those in model group and low, medium and high doses of luteolin groups (P<0.05).

Conclusion

Luteolin has a therapeutic effect in the AFK rats and can effectively inhibit corneal inflammation, and its mechanism may be ralated to inhibiting the expressions of TLR4 / MyD88 signaling pathway-related proteins and its down-stream inflammatory factors.

Key words: Aspergillus fumigatus keratitis, luteolin, Toll-like receptor 4, myeloid cell differentiation factor 88, inflammatory factor

中图分类号: 

  • R772.21