脂肪间充质干细胞来源的外泌体通过抑制 NLRP3 炎性小体缓解苯扎氯铵诱导的小鼠干眼模型

Exosomes Derived from Mouse Adipose-Derived Mesenchymal Stem Cells Alleviate Benzalkonium Chloride-Induced Mouse Dry Eye Model via Inhibiting NLRP3 Inflammasome.

机构信息

Department of Ophthalmology, Loudi Central Hospital, Loudi, China.

Department of Traditional Chinese Medicine, Loudi Central Hospital, Loudi, China.

出版信息

Ophthalmic Res. 2022;65(1):40-51. doi: 10.1159/000519458. Epub 2021 Sep 16.

DOI:10.1159/000519458

PMID:34530425

Abstract

PURPOSE

The objective of the study was to investigate efficacy and mechanisms of mouse adipose-derived mesenchymal stem cell-derived exosomes (mADSC-Exos) in the benzalkonium chloride (BAC)-induced mouse dry eye model.

METHODS

Exosomes in the mADSC culture supernatant were isolated by ultracentrifugation. Western blotting, nanoparticle tracking analysis, and transmission electron microscopy were used to characterize mADSC-Exos. An experimental mouse model of dry eye was established by instillation of 0.2% BAC. mADSC-Exos were administered following BAC treatment. The positive control group was treated with commercial eye drops (0.1% pranoprofen). Corneal fluorescein staining, tear secretion, and tear film break-up time (BUT) were evaluated, and histologic analysis of the cornea and conjunctiva was performed by hematoxylin and eosin and periodic acid-Schiff staining. Apoptosis in the corneal epithelium was detected with the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and by Western blotting. Levels of pro-inflammatory cytokines in the cornea and conjunctiva were evaluated by flow cytometry, and mRNA and protein levels of NLR family pyrin domain-containing 3 (NLRP3) pathway components were assessed by quantitative real-time PCR and Western blotting, respectively.

RESULTS

mADSC-Exos were characterized as vesicles with a bilayer membrane. The particle size distribution peak was at 134 nm. mADSC-Exos specifically expressed cluster of differentiation (CD)9, CD63, and CD81. mADSC-Exos treatment repaired ocular surface damage. Additionally, mADSC-Exos inhibited cell apoptosis, decreased the levels of interleukin (IL)-1β, IL-6, IL-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, and increased levels of the anti-inflammatory cytokine IL-10. Meanwhile, NLRP3 inflammasome activation and upregulation of caspase-1, IL-1β, and IL-18 were reversed by mADSC-Exos.

CONCLUSIONS

mADSC-Exos alleviate ocular surface inflammation, suggesting that it is a promising treatment for dry eye.

摘要

目的

本研究旨在探讨小鼠脂肪间充质干细胞来源的外泌体（mADSC-Exos）在苯扎氯铵（BAC）诱导的小鼠干眼症模型中的疗效及其作用机制。

方法

通过超速离心法分离 mADSC 培养上清液中的外泌体。采用 Western blot、纳米颗粒跟踪分析和透射电子显微镜对 mADSC-Exos 进行鉴定。通过滴注 0.2% BAC 建立干眼症实验小鼠模型，在 BAC 处理后给予 mADSC-Exos 治疗。阳性对照组给予市售滴眼液（0.1%普拉洛芬）治疗。评估角膜荧光素染色、泪液分泌和泪膜破裂时间（BUT），并通过苏木精和伊红及过碘酸-希夫染色对角膜和结膜进行组织学分析。通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记法和 Western blot 检测角膜上皮细胞凋亡。通过流式细胞术评估角膜和结膜中促炎细胞因子的水平，通过定量实时 PCR 和 Western blot 分别评估 NLR 家族含吡啶结构域蛋白 3（NLRP3）通路成分的 mRNA 和蛋白水平。

结果

mADSC-Exos 呈双层膜囊泡，粒径分布峰值为 134nm。mADSC-Exos 特异性表达分化群（CD）9、CD63 和 CD81。mADSC-Exos 治疗可修复眼表损伤，同时抑制细胞凋亡，降低白细胞介素（IL）-1β、IL-6、IL-1α、干扰素（IFN）-γ和肿瘤坏死因子（TNF）-α水平，增加抗炎细胞因子 IL-10 水平。同时，mADSC-Exos 逆转了 NLRP3 炎性小体激活以及半胱天冬酶-1、IL-1β 和 IL-18 的上调。