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

Runmuling formula ameliorates ocular surface damage in dry eye rats by activating the LC3-ATG5 autophagy pathway and inhibiting inflammatory factor expression

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。

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 electron microscopy revealed an increase in the formation of autophagosomes in 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.

关键词

润目灵方 / 干眼 / 动物模型 / 自噬 / 炎症因子

Key words

Runmu Ling Formula / Dry eye / Animal model / Autophagy / Inflammatory factors

参考文献

[1] Morthen MK , Magno MS , Utheim TP , et al. The physical and mental burden of dry eye disease: a large population-based study investigating the relationship with health-related quality of life and its determinants[J]. Ocul Surf, 2021, 21: 107-117. doi: 10.1016/j.jtos.2021.05.006

[2] 王佳慧, 刘学勤 . 全球近10年干眼相关生活质量研究: 基于VOSviewer和CiteSpace的文献计量学及可视化分析[J]. 山东大学耳鼻喉眼学报, 2024, 38(2): 61-72. doi: 10.6040/j.issn.1673-3770.0.2023.035

[3] WANG Jiahui , LIU Xueqin . A global study on quality of life related to dry eye in the last ten years: bibliometrics and visualization analysis through VOSviewer and CiteSpace[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2024, 38(2): 61-72. doi: 10.6040/j.issn.1673-3770.0.2023.035

[4] Zemanová M . Dry eyes disease. a review[J]. Czech Slovak Ophthalmol, 2021, 77(3): 107-119. doi: 10.31348/2020/29

[5] 中华医学会眼科学分会角膜病学组 . 我国角膜上皮损伤临床诊治专家共识(2016年)[J]. 中华眼科杂志, 2016, 52(9): 644-648. doi: 10.3760/cma.j.issn.0412-4081.2016.09.002

[6] Tsai CY , Jiesisibieke ZL , Tung TH . Association between dry eye disease and depression: an umbrella review[J]. Front Public Health, 2022, 10: 910608. doi: 10.3389/fpubh.2022.910608

[7] Greco G , Pistilli M , Asbell PA , et al. Association of severity of dry eye disease with work productivity and activity impairment in the dry eye assessment and management study[J]. Ophthalmology, 2021, 128(6): 850-856. doi: 10.1016/j.ophtha.2020.10.015

[8] Mohamed Z , Alrasheed S , Abdu M , et al. Dry eye disease prevalence and associated risk factors among the middle east population: a systematic review and meta-analysis[J]. Cureus, 2024, 16(9): e70522. doi: 10.7759/cureus.70522

[9] Mohamed HB , Abd El-Hamid BN , Fathalla D , et al. Current trends in pharmaceutical treatment of dry eye disease: a review[J]. Eur J Pharm Sci, 2022, 175: 106206. doi: 10.1016/j.ejps.2022.106206

[10] Li S , Lu ZY , Huang Y , et al. Anti-oxidative and anti-inflammatory micelles: break the dry eye vicious cycle[J]. Adv Sci, 2022, 9(17): 2200435. doi: 10.1002/advs.202200435

[11] Liu Z , Chen D , Chen X , et al. Autophagy activation protects ocular surface from inflammation in a dry eye model in vitro[J]. Int J Mol Sci, 2020, 21(23): 8966. doi: 10.3390/ijms21238966

[12] 王明武, 于莉 . 干眼药物研究的沿革与展望[J]. 中华实验眼科杂志, 2021, 39(2): 97-101. doi: 10.3760/cma.j.cn115989-20200204-00049

[13] WANG Mingwu , YU Li . Evolution and outlook of dry eye pharmatheutical research[J]. Chinese Journal of Experimental Ophthalmology, 2021, 39(2): 97-101. doi: 10.3760/cma.j.cn115989-20200204-00049

[14] 狄宇, 李莹 . 干眼炎症反应机制及抗炎治疗的研究进展[J]. 山东大学耳鼻喉眼学报, 2022, 36(2): 144-150. doi: 10.6040/j.issn.1673-3770.0.2020.484

[15] DI Yu , LI Ying . Research progress in the inflammatory reaction and anti-inflammatory treatments in dry eye[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2022, 36(2): 144-150. doi: 10.6040/j.issn.1673-3770.0.2020.484

[16] 庞莹, 张永刚, 南雨杰, 等 . 滋阴养血和解汤治疗干眼的疗效观察[J]. 中国中医眼科杂志, 2025, 35(1): 30-35. doi: 10.13444/j.cnki.zgzyykzz.2025.01.005

[17] PANG Ying , ZHANG Yonggang , NAN Yujie , et al. Observation on the therapeutic effect of Ziyin Yangxue hejie decoction in the treatment of dry eye[J]. China Journal of Chinese Ophthalmology, 2025, 35(1): 30-35. doi: 10.13444/j.cnki.zgzyykzz.2025.01.005

[18] 李卓, 亢泽峰, 袁慧艳, 等 . 养血润目方治疗蒸发过强型干眼的疗效观察[J]. 中国中医眼科杂志, 2025, 35(2): 127-131. doi: 10.13444/j.cnki.zgzyykzz.2025.02.005

[19] LI Zhuo , KANG Zefeng , YUAN Huiyan , et al. Observation on the therapeutic effect of Yangxue runmu prescription in the treatment of evaporative dry eye[J]. China Journal of Chinese Ophthalmology, 2025, 35(2): 127-131. doi: 10.13444/j.cnki.zgzyykzz.2025.02.005

[20] 杭丽, 张传伟, 李凯 . 中药润目灵颗粒联合0.1%玻璃酸钠滴眼液治疗高度近视飞秒激光微小切口基质透镜取出术(SMILE)术后患者干眼的疗效分析[J]. 临床医药文献电子杂志, 2017, 4(A1): 19869-19870. doi: 10.16281/j.cnki.jocml.2017.a1.047

[21] HANG Li , ZHANG Chuanwei , LI Kai . Efficacy of Runmuling Granule combined with 0.1% sodium hyaluronate eye drops in the treatment of dry eyes of patients with high myopia after femtosecond laser micro-incision stromal lens extraction (SMILE)[J]. Electronic Journal of Clinical Medical Literature, 2017, 4(A1): 19869-19870. doi: 10.16281/j.cnki.jocml.2017.a1.047

[22] 杨继红, 李凯, 王育良, 等 . 鬼针草水提物对雄激素缺乏性干眼大鼠泪液分泌和泪腺炎症的影响[J]. 中国中医眼科杂志, 2017, 27(4): 211-217. doi: 10.13444/j.cnki.zgzyykzz.2017.04.001

[23] YANG Jihong , LI Kai , WANG Yuliang , et al. Effects of aqueous extract of Bidens pilosa L. on tear secretion and lacrimal gland inflammation of dry eye due to androgen deficiency in rats[J]. China Journal of Chinese Ophthalmology, 2017, 27(4): 211-217. doi: 10.13444/j.cnki.zgzyykzz.2017.04.001

[24] Luo D , Ji HJ , Yan XQ , et al. Run-mu-Ling granules mitigate ocular surface inflammatory injury associated with dry eye by suppressing the NLRP3/GSDMD-mediated pyroptosis pathway[J]. J Inflamm Res, 2024, 17: 10769-10784. doi: 10.2147/JIR.S496231

[25] Chaudhari P , Satarker S , Thomas R , et al. Rodent models for dry eye syndrome: Standardization using benzalkonium chloride and scopolamine hydrobromide[J]. Life Sci, 2023, 317: 121463. doi: 10.1016/j.lfs.2023.121463

[26] Liu P , Jiang PF , Yu YF , et al. Modified Danzhi Xiaoyao Powder (MDXP) improves the corneal damage in dry eye disease (DED) mice through phagocytosis[J]. J Ethnopharmacol, 2024, 321: 117544. doi: 10.1016/j.jep.2023.117544

[27] Gui JX , Liu J , Wang LM , et al. Autophagy alleviates hippocampal neuroinflammation by inhibiting the NLRP3 inflammasome in a juvenile rat model exposed particulate matter[J]. Toxicology, 2024, 502: 153730. doi: 10.1016/j.tox.2024.153730

[28] Rolando M , Barabino S . The subtle role of para-inflammation in modulating the progression of dry eye disease[J]. Ocul Immunol Inflamm, 2021, 29(4): 811-816. doi: 10.1080/09273948.2021.1906908

[29] Al Saihati HA , Badr OA , Dessouky AA , et al. Exploring the cytoprotective role of mesenchymal stem Cell-Derived exosomes in chronic liver Fibrosis: Insights into the Nrf2/Keap1/p62 signaling pathway[J]. Int Immunopharmacol, 2024, 141: 112934. doi: 10.1016/j.intimp.2024.112934

[30] Klionsky DJ , Abdel-Aziz AK , Abdelfatah S , et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)(1)[J]. Autophagy, 2021, 17(1): 1-382. doi: 10.1080/15548627.2020.1797280

[31] Orioli E , De Marchi E , Giuliani AL , et al. P2X7 receptor orchestrates multiple signalling pathways triggering inflammation, autophagy and metabolic/trophic responses[J]. Curr Med Chem, 2017, 24(21): 2261-2275. doi: 10.2174/0929867324666170303161659

[32] Rivera M , Ramos Y , Rodríquez-Valentín M , et al. Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells[J]. PLoS One, 2017, 12(6): e0179587. doi: 10.1371/journal.pone.0179587

[33] Yang Q , Wan Q , Wang Z . Curcumin mitigates polycystic ovary syndrome in mice by suppressing TLR4/MyD88/NF-κB signaling pathway activation and reducing intestinal mucosal permeability[J]. Sci Rep, 2024, 14(1): 29848. doi: 10.1038/s41598-024-81034-5

[34] Sun Q , Du M , Kang YF , et al. Prebiotic effects of goji berry in protection against inflammatory bowel disease[J]. Crit Rev Food Sci Nutr, 2023, 63(21): 5206-5230. doi: 10.1080/10408398.2021.2015680

基金资助

江苏省中医药科技发展计划重点项目(ZD202102)

全国名老中医药专家传承工作室建设项目(2024YL03204)