多药耐药相关蛋白4过表达对脂多糖诱导血管内皮高渗透性的影响及其机制

Effect of multidrug resistant associated protein 4 overexpression on lipopolysaccharide-induced vascular endothelial hyperpermeability and its mechanism

目的 探讨多药耐药相关蛋白4（MRP4）基因过表达对脂多糖（LPS）诱导大鼠肺微血管内皮细胞（PMVECs）渗透性的影响及其分子机制.方法 体外培养PMVECs细胞,待细胞传至3~6代后分为3组：LPS组无血清培养基培养24 h后,用10μg/mL LPS刺激细胞;Ad-shRNA组用腺病毒空白载体转染细胞2 h,无血清培养基培养24 h后,用10μg/mL LPS刺激细胞;Ad-MRP4组用携带MRP4的重组腺病毒载体转染细胞2 h,无血清培养基培养24 h后,用10μg/mL LPS刺激细胞.于LPS刺激2、6、12、24 h采用Transwell小室法检测单层细胞渗透性;采用酶联免疫吸附试验（ELISA）检测细胞内环磷酸腺苷（cAMP）水平;激光共聚焦荧光显微镜下观察细胞内纤维型肌动蛋白（F-actin）形态及分布情况.于LPS刺激12 h收集细胞,采用蛋白质免疫印迹试验（Western Blot）检测PMVECs中MRP4、β-连环蛋白（β-catenin）、血管内皮-钙黏蛋白（VE-cad）和紧密连接蛋白（ZO-1）的表达水平.结果 ①LPS刺激后PMVECs细胞渗透性及细胞内cAMP水平逐渐增加,12 h达到高峰,24 h开始下降.用携带MRP4的重组腺病毒载体转染PMVECs后,细胞渗透性较LPS组及Ad-shRNA组显著增加〔12 h渗透性（A值）：1.88±0.06比1.12±0.17、1.10±0.18〕,细胞内cAMP水平显著降低〔12 h cAMP（μg/L）：2.39±0.02比2.97±0.01、3.00±0.02,均P〈0.05〕;而LPS组与Ad-shRNA组各时间点各指标差异均无统计学意义（均P〉0.05）.② 激光共聚焦荧光显微镜下显示,3组细胞均出现F-actin重构、应力纤维形成,但Ad-MRP4组细胞破坏程度较LPS组和Ad-shRNA组更加严重.③ 与LPS组和Ad-shRNA组比较,Ad-MRP4组PMVECs中MRP4蛋白表达显著上调（灰度值：0.76±0.03比0.44±0.02、0.43±0.02,均P〈0.05）,β-catenin、VE-cad和ZO-1蛋白表达显著下调〔β-catenin（灰度值）：0.14±0.03比0.23±0.04、0.23±0.03;VE-cad（灰度值）：0.21±0.01比0.34±0.02、0.35±0.04;ZO-1（灰度值）：0.14±0.02比0.37±0.06、0.33±0.07,均P〈0.05〕;而LPS组与Ad-shRNA组间各蛋白表达差异无统计学意义（均P〉0.05）.结论 MRP4基因过表达可降低细胞内cAMP水平,下调细胞间连接蛋白表达,从而增加LPS诱导的血管内皮渗透性.

Objective To investigate the effect of multidrug resistance protein 4 （MRP4） overexpression on lipopolysaccharide （LPS）-induced vascular endothelial hyperpermeability of rat pulmonary micro-vascular endothelial cells （PMVECs） and its molecule mechanism. Methods Three to six passages of PMVECs were cultured in vitro, and they were divided into three groups： the cells in LPS group were only challenged by LPS 10 μg/mL after being cultured in serum-free medium for 24 hours; the cells in Ad-shRNA and Ad-MRP4 groups were infected with the empty virus control or recombinant adenovirus expressing MRP4 for 2 hours, and then were cultured in serum-free medium for 24 hours followed by stimulation of LPS 10 μg/mL. Endothelial permeability was assayed by the Transwell chamber models at 2, 6, 12, and 24 hours after LPS stimulation. Intracellular cyclic adenosine monophosphate （cAMP） levels were detected by enzyme-linked immunosorbent assay （ELISA）. The morphological characteristics and distribution of F-actin was determined by laser confocal fluorescence microscope. The protein expressions of MRP4,β-catenin, vascular endothelium-cadherin （VE-cad） and ZO-1 were measured by Western Blot. Results ① After LPS stimulation, endothelium permeability and intracellular cAMP levels in PMVECs were significantly increased, peaked at 12 hours, and then decreased after 24 hours. Compared with LPS group and Ad-shRNA group, PMVECs of Ad-MRP4 group were exhibited a significant increase in endothelial permeability [12-hour permeability （A value）：1.88±0.06 vs. 1.12±0.17, 1.10±0.18] and a significant decrease in intracellular cAMP level [12-hour cAMP （μg/L）：2.39±0.02 vs. 2.97±0.01, 3.00±0.02, all P 〈 0.05]. There was no significant difference in endothelium permeability and intracellular cAMP levels at all time points between the LPS group and the Ad-shRNA group （all P 〉 0.05）.② Under laser confocal fluorescence microscope, after LPS stimulation, the stress fiber formation was induced in three groups. But there were pronounced irregular aggregation of fiber in PMVECs of Ad-MRP4 group. ③ Furthermore, compared with LPS group and Ad-shRNA group, protein expression of MRP4 in Ad-MRP4 group was dramatically increased （gray value： 0.76±0.03 vs. 0.44±0.02, 0.43±0.02, both P 〈 0.05）, and the protein expressions of β-catenin, VE-cad, and ZO-1 were significantly decreased [β-catenin （gray value）： 0.14±0.03 vs. 0.23±0.04, 0.23±0.03）;VE-cad （gray value）： 0.21±0.01 vs. 0.34±0.02, 0.35±0.04; ZO-1 （gray value）： 0.14±0.02 vs. 0.37±0.06, 0.33±0.07, all P 〈 0.05]. There was no significant difference in all protein expressions between the LPS group and Ad-shRNA group （all P 〉 0.05）. Conclusion MRP4 overexpression can decrease intracellular cAMP levels, reduce intercellular junction protein expression, and then exaggerate LPS-induced vascular endothelial hyperpermeability.