润目灵方激活LC3-ATG5自噬通路抑制炎症因子表达改善干眼大鼠眼表损伤的机制

doi:10.6040/j.issn.1673-3770.0.2025.217

摘要/Abstract

摘要： 目的探讨润目灵方激活LC3-ATG5自噬通路并抑制炎症因子表达改善干眼大鼠眼表损伤的作用机制。方法采用氢溴酸东莨菪碱皮下注射构建干眼模型,将60只SD大鼠随机分为空白对照、模型、润目灵方、自噬激活剂、自噬抑制剂及润目灵方联合自噬抑制剂干预组,每组各10只。通过角膜荧光素染色(corneal fluorescein staining, CFS)评分、泪膜破裂时间(tear break-up time, TBUT)、酚红棉线试验(phenol red tear test, PRTT)评估眼表功能;HE染色及透射电镜观察角膜病理与超微结构;RT-qPCR/Western blot检测自噬相关指标(LC3、ATG5、P62)及炎症因子(IL-1β、IL-18、TNF-α)的表达。统计学处理采用单因素方差分析或重复测量资料方差分析。结果模型组较空白组TBUT缩短(P<0.001),CFS评分升高(P<0.001),PRTT减少(P<0.001),角膜上皮细胞空泡化增加。润目灵方干预后显著恢复眼表稳态(TBUT,CFS和PRTT,均P<0.05),电镜显示自噬小体数量较模型组增加。分子机制显示,润目灵方显著激活LC3-ATG5自噬通路,表现为上调LC3-Ⅱ/LC3-I比值(P<0.05)及ATG5表达(P<0.05),同时显著抑制炎症因子TNF-α、IL-1β和IL-18的表达(均P<0.05)。结论润目灵方可有效改善干眼大鼠眼表炎症及损伤,其机制与激活LC3-ATG5自噬通路并抑制TNF-α、IL-1β、IL-18等炎症因子表达密切相关。

关键词: 润目灵方, 干眼, 动物模型, 自噬, 炎症因子

Abstract: Objective The objective of this study is to investigate the mechanism by which Runmu Ling Formula(RML)ameliorates ocular surface damage in rats with dry eye through activation of the LC3-ATG5 autophagy pathway and inhibition of inflammatory factor expression. Methods A dry eye model was established via subcutaneous injection of scopolamine hydrobromide. A total of 60 Sprague-Dawley(SD)rats were randomly assigned to six groups,with ten rats per group: a blank control group, a model group, an RML-treated group, an autophagy activator group, an autophagy inhibitor group, and an RML combined with autophagy inhibitor group. The ocular surface function of the subjects was assessed using a variety of methods, including corneal fluorescein staining(CFS)scores, tear break-up time(TBUT), and the phenol red thread test(PRTT). The corneas were subjected to a comprehensive evaluation that entailed the application of hematoxylin-eosin(HE)staining and transmission electron microscopy. RT-qPCR and Western blotting were employed to measure the expression of autophagy-related markers(LC3, ATG5, P62)and inflammatory factors(IL-1β, IL-18, TNF-α). Results In comparison with the blank control group, the model group demonstrated a significant reduction in TBUT(P<0.001), elevated CFS scores(P<0.001), diminished PRTT values(P<0.001), and augmented vacuolation of corneal epithelial cells. RML intervention led to a significant restoration of ocular surface homeostasis, as evidenced by substantial improvements in TBUT, CFS, and PRTT(all P<0.05). The transmission electronmicroscopy revealed an increase in the formation of autophagosomesin the RML group compared to the model group. Mechanistically, RML significantly activated the LC3-ATG5 autophagy pathway, as evidenced by increased LC3-II/LC3-I ratios(P<0.05)and elevated ATG5 expression(P<0.05). Concurrently, RML potently suppressed the expression of inflammatory factors TNF-α, IL-1β, and IL-18(all P<0.05). Conclusion The efficacy of Runmu Ling Formula in alleviating ocular surface inflammation and injury in rats with dry eye has been demonstrated by its ability to activate the LC3-ATG5 autophagy pathway and inhibit the expression of key inflammatory factors, including TNF-α, IL-1β, and IL-18.

Key words: Runmu Ling Formula, Dry eye, Animal model, Autophagy, Inflammatory factors

中图分类号:

R771

李凯,罗丹. 润目灵方激活LC3-ATG5自噬通路抑制炎症因子表达改善干眼大鼠眼表损伤的机制[J]. 山东大学耳鼻喉眼学报, 2026, 40(3): 92-101.

LI Kai, LUO Dan. Runmu ling formula ameliorates ocular surface damage in dry eye rats by activating the LC3-ATG5 autophagy pathway and inhibiting inflammatory factor expression[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2026, 40(3): 92-101.

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