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

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+siPAI-1组小鼠分别腹腔注射300μL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+siNC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。

Objective To explore the expression and function of plasminogen activator inhibitor1(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+siNC and PF+si-PAI-1(n=12 each).Mice in PF+si-NC and PF+si-PAI-1 groups were intraperitoneally injected with 300μL 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-1group(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.