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

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

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 m Osm/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 m Osm/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。

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〈0.05）. 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 down-regulation of the tight junction proteins ZO-1 and claudin-1.