摘要目的 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.

abstractsObjective 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.