p38/MAPK信号通路在高渗透压破坏角膜上皮屏障功能中的作用

目的 探讨高渗透压对角膜上皮屏障功能的影响及p38/MAPK信号通路在此过程中潜在的作用.方法 体外培养人永生化角膜上皮细胞(human corneal epithelial cells,HCEC),用不同浓度渗透压302(正常渗透压)、350、500 mOsm/L]作用于HCEC细胞24 h,CCK-8法检测细胞增殖活性;免疫荧光检测细胞紧密连接蛋白ZO-1和Claudin-1的表达和分布;p38/MAPK阻断剂预处理HCEC细胞前后,Western blot分别检测紧密连接蛋白ZO-1、Claudin-1和p38/MAPK信号通路表达及其磷酸化水平;跨膜电阻仪检测上皮细胞跨膜电阻(transepithelial electrical resistance,TEER)的变化.结果 高渗透压对HCEC细胞增殖活性没有影响;免疫荧光染色可见正常渗透压组ZO-1、Claudin-1沿着细胞膜呈线性分布,而350、500 mOsm/L高渗透压组ZO-1、Claudin-1荧光信号均减弱,且细胞间紧密连接结构破坏(P＜0.05);在高渗透压下,HCEC细胞紧密连接蛋白ZO-1、Claudin-1表达量明显下降(P＜0.05),p38/MAPK磷酸化水平增加(P＜0.05),上皮细胞跨膜电阻(TEER)降低(P＜0.05),而p38/MAPK阻断剂SB203580可抑制高渗透压引起的上述反应(P＜0.05).结论 高渗透压可以使人角膜上皮细胞屏障功能破坏,其机制为通过激活p38/MAPK信号通路导致紧密连接蛋白ZO-1、Claudin-1表达下降.

Role of p38/MAPK pathway in hyperosmosis-induced functional impairment of human corneal epithelial barrier in vitro

Objective To study the effect of hyperosmosis on corneal epithelial barrier function and explore the role of p38/MAPK pathway in this process.Methods Human corneal epithelial cells (HCECs) cultured in normal osmotic (302 mOsm/L) or in hyperosmotic (350 and 500 mOsm/L) conditions for 24 h were examined for cell proliferation using CCK-8 assay and for distribution of tight junction proteins zonula occludens 1 (ZO-1) and claudin-1 using immunofluorescence analysis.Western blotting was used to detect the expression of ZO-1,claudin-1,p38 and phosphorylation of p38 in the cells cultured in different osmotic conditions with or without prior treatment of 10 μg/L SP203580,a p38/MAPK specific inhibitor.The transepithelial electrical resistance (TEER) of the cells was measured using a transmembrane resistance meter.Results Hyperosmosis produced no obvious effect on HCEC cell proliferation.Immunofluorescence assay showed that in the cells cultured in normal osmotic condition,ZO-1 and claudin-1 emitted fluorescence in a linear pattern along the cell membrane,while the cells cultured at 350 or 500 mOsm/L exhibited a significantly lowered intensity of the fluorescence signals with obvious structural collapse of the tight junctions (P ＜ O.05).In hyperosmotic conditions,the cells expressed significantly decreased levels of tight junction proteins ZO-1 and claudin-1 (P ＜ 0.05) with significantly increased phosphorylation of p38 (P ＜ 0.05) and reduced TEER (P ＜ 0.05).Treatment of the cells with the p38 inhibitor (SB203580) prior to hyperosmotic exposures obviously suppressed these responses to hyperosmosis (P ＜ 0.05).Conclusion Hyperosmosis induces impairment of corneal epithelial barrier function by activating the p38/MAPK pathway to cause downregulation of the tight junction proteins ZO-1 and claudin-1.