实验性干眼会刺激炎症细胞因子和基质金属蛋白酶-9的产生，并激活眼表的丝裂原活化蛋白激酶信号通路。

Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface.

作者信息

Luo Lihui, Li De-Quan, Doshi Amish, Farley William, Corrales Rosa M, Pflugfelder Stephen C

机构信息

Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, 6565 Fannin Street, Houston, TX 77030, USA.

出版信息

Invest Ophthalmol Vis Sci. 2004 Dec;45(12):4293-301. doi: 10.1167/iovs.03-1145.

DOI:10.1167/iovs.03-1145

PMID:15557435

Abstract

PURPOSE

To evaluate whether experimentally induced dry eye in mice activates mitogen-activated protein kinase (MAPK) signaling pathways, c-Jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK), and p38 and stimulates ocular surface inflammation.

METHODS

129SvEv/CD-1 mixed mice aged 6 to 8 weeks were treated with systemic scopolamine and exposure to an air draft for different lengths of time, from 4 hours to 10 days. Untreated mice were used as the control. The concentrations of IL-1beta and TNF-alpha in tear fluid washings and in corneal and conjunctival epithelia were measured by ELISA. MMP-9 in tear washings was evaluated by zymography, and gelatinase activity in the cornea and conjunctiva was determined by in situ zymography. Corneal and conjunctival epithelia were lysed in RIPA buffer for Western blot with MAPK antibodies, or they were lysed in 4 M guanidium thiocyanate solution for extraction of total RNA, which was used to determine gene expression by semiquantitative RT-PCR, real-time PCR, and gene array.

RESULTS

Compared with those in age-matched control subjects, the concentrations of IL-1beta and MMP-9 in tear fluid washings and the concentrations of IL-1beta and TNF-alpha and gelatinolytic activity in the corneal and conjunctival epithelia were significantly increased in mice receiving treatments to induce dry eye after 5 or 10 days. The expression of IL-1beta, TNF-alpha, and MMP-9 mRNA by the corneal and conjunctival epithelia was also stimulated in mice treated for 5 or 10 days. The levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia were markedly increased as early as 4 hours after treatment, and they remained elevated up to 5 days.

CONCLUSIONS

Experimental dry eye stimulates expression and production of IL-1beta, TNF-alpha, and MMP-9 and activates MAPK signaling pathways on the ocular surface. MAPKs are known to stimulate the production of inflammatory cytokines and MMPs, and they could play an important role in the induction of these factors that have been implicated in the pathogenesis of dry eye disease.

摘要

目的

评估实验诱导的小鼠干眼是否激活丝裂原活化蛋白激酶（MAPK）信号通路、c-Jun氨基末端激酶（JNK）、细胞外调节激酶（ERK）和p38，并刺激眼表炎症。

方法

对6至8周龄的129SvEv/CD-1混合小鼠全身给予东莨菪碱，并暴露于不同时长的气流中，时长从4小时至10天。未处理的小鼠作为对照。通过酶联免疫吸附测定法（ELISA）测量泪液冲洗液以及角膜和结膜上皮中白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）的浓度。通过酶谱法评估泪液冲洗液中的基质金属蛋白酶-9（MMP-9），并通过原位酶谱法测定角膜和结膜中的明胶酶活性。用含有MAPK抗体的放射免疫沉淀法（RIPA）缓冲液裂解角膜和结膜上皮用于蛋白质印迹分析，或者在4M硫氰酸胍溶液中裂解用于提取总RNA，总RNA用于通过半定量逆转录聚合酶链反应（RT-PCR）、实时PCR和基因芯片测定基因表达。

结果

与年龄匹配的对照小鼠相比，接受诱导干眼处理5天或10天后的小鼠泪液冲洗液中IL-1β和MMP-9的浓度，以及角膜和结膜上皮中IL-1β、TNF-α的浓度和明胶酶活性显著增加。处理5天或10天的小鼠角膜和结膜上皮中IL-1β、TNF-α和MMP-9 mRNA的表达也受到刺激。早在处理后4小时，角膜和结膜上皮中磷酸化的JNK1/2、ERK1/2和p38丝裂原活化蛋白激酶的水平就显著增加，并持续升高至5天。