Objective To explore the role of protein kinase C (PKC)- extracellular signal-regulated kinase (ERK)1/2 signal pathway in the process of plasminogen activator inhibitor-1(PAI-1) protein and mRNA expression in cultured human umbilical vein endothelial cells(HUVECs) induced by nicotine. Methods HUVECs were cultured to examine the effect of nicotine on the expression of secreting PAI-1 in HUVECs on different experimental conditions. The expression of PAI-1 protein was measured by ELISA. PKC inhibitor staurosporine (STS)and ERK1/2 inhibitor PD98059 were used to detect PKC or ERK1/2 function on the expression of PAI-1 in HUVECs induced by nicotine. The PAI-1 mRNA expression was determined by RT-PCR. Results The expression level of PAI-1 protein in 100 μmol/L nicotine treated group [(22.6±1.1) μg/L] increased significantly compared to the control group [(14.2±2.8) μg/L; q=5.64,P<0.05]. After stimulation with 100 μmol/L nicotine for 0,4,6,8,12 and 24 h, the levels of PAI-1 protein increased over time and reached the peak at 12 h (F=32.063,P<0.05).The PAI-1 mRNA and protein expression in nicotine treated group [(1.32±0.20), (21.08 ± 0.83) μg/L] increased significantly compared to the control group [(0.73±0.10), (13.39±0.93) μg/L; q=8.43,11.97,all P<0.05].Compared with nicotine treated group , the PAI-1 mRNA and protein expression in nicotine and STS treated group [(1.07±0.10),(16.19±2.15) μg/L] decreased significantly(q=5.61,7.61, all P<0.05), but still higher than the control group (q=7.84,4.36, all P<0.05). In nicotine and PD98059 treated group, the PAI-1 mRNA and protein expression [(1.12±0.11),(17.52±1.72) μg/L] decreased significantly compared to the nicotine treated group(q= 4.68,5.54, all P<0.05), still higher than the control group (q=8.77,6.43, all P<0.05). Conclusion PKC-ERK1/2 signal pathway may play a partial role in the up-regulation of PAI-1 induced by nicotine in HUVECs.%目的 探讨蛋白激酶C(PKC)-胞外信号调节激酶(ERK)1/2信号通路在尼古丁诱导人脐静脉内皮细胞(HUVECs)表达纤维溶解酶原激活物抑制物-1(PAI-1)中的作用.方法 体外培养HUVECs,采用不同实验条件尼古丁进行干预,ELISA法测定细胞上清液中PAI-1的浓度,观察尼古丁作用的最佳浓度和时间.进一步分别用PKC的抑制剂星型胞菌素staurosporine (STS)和ERK的抑制剂PD98059干预HUVECs,观察PKC或ERK被阻断后对尼古丁诱导的HUVECs 表达PAI-1的影响,ELISA测定各组细胞上清液中PAI-1蛋白的表达水平,RT-PCR检测各组细胞PAI-1 mRNA的表达.结果 100 μmol/L尼古丁组PAI-1蛋白水平[(22.6 ± 1.1)μg/L]明显高于对照组[(14.2± 2.8)μg/L,q=5.64,P＜0.05];以100 μmol/L 尼古丁分别与HUVECs孵育0、4、6、8、12及24 h,各组PAI-1蛋白表达呈时间依赖性升高,并在12 h达到高峰(F=32.063,P＜0.05);尼古丁组PAI-1 mRNA及蛋白含量[(1.32±0.20),(21.08±0.83)μg/L]明显高于对照组[(0.73±0.10),(13.39± 0.93)μg/L,q=8.43、11.97,均P＜0.05];尼古丁+STS组PAI-1 mRNA及蛋白含量[(1.07±0.10),(16.19±2.15)μg/L]较尼古丁组降低(q=5.61、7.61,均P＜0.05),但仍高于对照组(q=7.84、4.36,均P＜0.05);尼古丁+ PD98059组PAI-1 mRNA及蛋白表达[(1.12±0.11),(17.52±1.72)μg/L]低于尼古丁组(q=4.68、5.54,均P＜0.05),仍高于对照组(q=8.77、6.43,均P＜0.05).结论 PKC-ERK1/2信号通路在尼古丁诱导的血管内皮细胞PAI-1表达上调中发挥一定作用.
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