纤溶酶原激活物抑制因子1在腹膜透析患者中的表达变化及对基质金属蛋白酶-2/血管内皮生长因子/细胞外调节蛋白激酶1/2信号通路的调控作用

梁雪艳; 苏瑞; 李铭; 李婷; 卢家美; 邹晓荣

doi:10.3969/j.issn.1671-2390.2022.03.007

纤溶酶原激活物抑制因子1在腹膜透析患者中的表达变化及对基质金属蛋白酶-2/血管内皮生长因子/细胞外调节蛋白激酶1/2信号通路的调控作用

Expression of plasminogen activator inhibitor 1 in peritoneal dialysis patients and its regulation of matrix metalloproteinase 2/vascular endothelial growth factor/extracellular regulated protein kinases 1/2 signaling pathway

摘要

摘要: 目的揭示纤溶酶原激活物抑制因子1（plasminogen activator inhibitor 1，PAI-1）在腹膜透析患者中的表达及其功能。方法选择100例腹膜透析患者作为研究对象，透析7个月后，根据腹膜平衡试验结果将患者分为高转运组和低转运组。透析1个月时和7个月时，使用酶联免疫吸附测定试剂盒检测所有患者透析液中PAI-1、基质金属蛋白酶-2（matrix metalloproteinase 2，MMP-2）和血管内皮生长因子（vascular endothelial growth factor，VEGF）水平。对C57/BL6小鼠连续28 d腹腔注射3 mL含4.25%葡萄糖的腹膜透析液来建立腹膜纤维化（peritoneal fibrosis，PF）小鼠模型。将小鼠按随机数字表法分为3组（n=12）：对照组、PF+si-NC组和PF+si-PAI-1组。PF+si-NC组和PF+si-PAI-1组小鼠分别腹腔注射300 μL阴性对照siRNA（si-NC）或靶向PAI-1的siRNA（si-PAI-1）。通过蛋白质印迹法检测PAI-1、MMP-2、VEGF、磷酸化血管内皮细胞生长因子受体2（phosphorylated vascular endothelial growth factor 2，pVEGFR2）和磷酸化细胞外调节蛋白激酶（phosphorylated extracellular regulated protein kinases，pErk）的蛋白表达。通过免疫组织化学染色检测巨噬细胞表面标志物（macrophage surface markers，CD68）、VEGF和血小板-内皮细胞黏附分子（platelet endothelial cell adhesion molecule-1，PECAM-1/CD31）的阳性表达。结果与透析1个月时相比，透析7个月后患者透析液中PAI-1、MMP-2和VEGF的水平均显著升高（P<0.05）。与低转运组相比，高转运组患者透析液中PAI-1、MMP-2和VEGF的水平均显著升高（P<0.05）。PAI-1、MMP-2和VEGF联合诊断高转运的曲线下面积（area under curve，AUC）、敏感性和特异性依次为0.909、82.61和92.45。与PE+si-NC组比较，PE+si-PAI-1组的间质细胞外基质（extracellular matrix，ECM）沉积和炎症细胞浸润明显减轻。与PE+si-NC组比较，PE+si-PAI-1组腹膜组织的CD68、VEGF和CD31阳性率降低（P<0.05）。与PE+si-NC组比较，PE+si-PAI-1组腹膜组织的PAI-1、MMP-2、VEGF、pVEGFR2和pErk的蛋白表达水平降低（P<0.05）。结论本研究显示PAI-1、MMP-2和VEGF的联合诊断对腹膜溶质转运速率具有较高的诊断价值。下调PAI-1可能通过抑制MMP-2/VEGF/细胞外调节蛋白激酶1/2信号通路来抑制血管生成，从而抑制腹膜纤维化。

Abstract: Objective To explore the expression and function of plasminogen activator inhibitor 1 （PAI-1） in peritoneal dialysis （PD） patients.Methods A total of 100 PD patients were selected as research objects.After 7 months of dialysis，they were divided into two groups of high transport and low transport according to the results of peritoneal balance test.At Month 1/7 post-dialysis，the dialysate levels of PAI-1，matrix metalloproteinase-2 （MMP-2） and vascular endothelial growth factor （VEGF） were detected by enzyme-linked immunosorbent assay （ELISA）.C57/BL6 mice were intraperitoneally injected with 3 mL peritoneal dialysate containing 4.25% glucose for 28 consecutive days for establishing a model of peritoneal fibrosis （PF）.The mice were randomly divided into three groups of control，PF+si-NC and PF+si-PAI-1 （n=12 each）.Mice in PF+si-NC and PF+si-PAI-1 groups were intraperitoneally injected with 300 μL negative control siRNA （si-NC） or siRNA targeting PAI-1 （si-PAI-1）.The protein expressions of PAI-1，MMP-2，VEGF，pVEGFR2 and pErk were detected by Western blot.The positive expressions of CD68，VEGF and CD31 were detected by immunohistochemical staining.Results As compared with 1-month dialysis，the dialysate levels of PAI-1，MMP-2 and VEGF became significantly elevated after 7-month dialysis （P<0.05）.As compared with low transport group，the dialysate levels of PAI-1，MMP-2 and VEGF of high transport group were significantly higher （P<0.05）.The area under curve （AUC），sensitivity and specificity of PAI-1，MMP-2 and VEGF in the combined diagnosis of high transport were 0.909，82.61 and 92.45.As compared with PE+si-NC group，extracellular matrix （ECM） deposition and inflammatory cell infiltration declined markedly in PE+si-PAI-1 group.As compared with PE+si-NC group，the positive rates of CD68，VEGF and CD31 were lower in peritoneal tissue of PE+si-PAI-1 group （P<0.05）.As compared with PE+si-NC group，the protein expression levels of PAI-1，MMP-2，VEGF，pVEGFR2 and pErk declined in peritoneal tissues of PE+si-PAI-1 group （P<0.05）.Conclusion The combined diagnosis of PAI-1，MMP-2 and VEGF offers a high diagnostic value for peritoneal solute transport rate.A down-regulation of PAI-1 may arrest angiogenesis by interfering with MMP-2/VEGF/Erk1/2 signaling pathway，thereby suppressing peritoneal fibrosis.

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