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Title: [Regulation of hypoxia inducible factor-1α on permeability of vascular endothelial cells and the mechanism].
Author: Hu DL, Yu YX, Liang R, Zhou SY, Duan SL, Jiang ZY, Meng CY, Jiang W, Wang H, Sun YX, Fang LS.
Journal: Zhonghua Shao Shang Za Zhi; 2019 Mar 20; 35(3):209-217. PubMed ID: 30897868.
Abstract:
Objective: To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α) on permeability of rat vascular endothelial cells and the mechanism. Methods: Twelve male Sprague-Dawley rats aged 35 to 38 days were collected and vascular endothelial cells were separated and cultured. The morphology of cells was observed after 4 days of culture, and the following experiments were performed on the 2nd or 3rd passage of cells. (1) Rat vascular endothelial cells were collected and divided into blank control group, negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were transfected with GV248 empty plasmid, recombinant plasmid respectively containing HIF-1α interference sequence 1, interference sequence 2, and interference sequence 3 with liposome 2000. Cells in blank control group were only transfected with liposome 2000. After transfection of 24 h, expression levels of HIF-1α mRNA and protein of cells in each group were respectively detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction and Western blotting (the same detecting methods below) . The sequence with the highest interference efficiency was selected. (2) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α low expression group, with 3 wells in each group. Cells in blank control group were only transfected with liposome 2000, and cells in negative control group and HIF-1α low expression group were respectively transfected with GV248 empty plasmid and low expression HIF-1α recombinant plasmid selected in experiment (1) with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α in each group were detected. (3) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α high expression group, with 3 wells in each group. Cells in blank control group were transfected with liposome 2000, and cells in negative control group and HIF-1α high expression group were respectively transfected with GV230 empty plasmid and HIF-1α high expression recombinant plasmid with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α of cells in each group were detected. (4) After transfection of 24 h, cells of three groups in experiment (1) and three groups in experiment (2) were collected, and mRNA and protein expressions of myosin light chain kinase (MLCK), phosphorylated myosin light chain (p-MLC), and zonula occludens 1 (ZO-1) of cells were detected. Data were processed with one-way analysis of variance and t test. Results: After 4 days of culture, the cells were spindle-shaped, and rat vascular endothelial cells were successfully cultured. (1) The interference efficiencies of HIF-1α of cells in HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were 47.66%, 45.79%, and 62.62%, respectively, and the interference sequence 3 group had the highest interference efficiency. After transfection of 24 h, the mRNA and protein expression levels of HIF-1α of cells in interference sequence 3 group were significantly lower than those in blank control group (t=18.404, 9.140, P<0.01) and negative control group (t=15.099, 7.096, P<0.01). (2) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=21.140, 5.440, P<0.01) and negative control group (t= 14.310, 5.210, P<0.01). (3) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=19.160, 7.710, P<0.01) and negative control group (t= 19.890, 7.500, P<0.01). (4) After transfection of 24 h, the mRNA expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.709, 4.011, P<0.05 or P<0.01) and negative control group (t=2.373, 3.744, P<0.05 or P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=4.285, 5.050, P<0.01). The mRNA expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=9.118, 11.313, P<0.01) and negative control group (t=9.073, 11.280, P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α high expression group was significantly lower than that in blank control group and negative control group (t=2.889, 2.640, P<0.05). (5) After transfection of 24 h, the protein expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.652, 3.983, P<0.05 or P<0.01) and negative control group (t=2.792, 4.065, P<0.05 or P<0.01). The protein expression of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=3.881, 3.570, P<0.01). The protein expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were 1.18±0.24 and 0.68±0.22, which were significantly higher than 0.41±0.21 and 0.35±0.14 in blank control group (t=5.011, 3.982, P<0.05 or P<0.01) and 0.43±0.20 and 0.36±0.12 in negative control group (t= 4.880, 3.862, P<0.05 or P<0.01). The protein expression level of ZO-1 of cells in HIF-1α high expression group was 0.08±0.06, which was significantly lower than 0.20±0.09 in blank control group and 0.19±0.09 in negative control group (t=4.178, 3.830, P<0.05 or P<0.01). Conclusions: HIF-1α up-regulates expressions of MLCK and p-MLC and down-regulates expression of ZO-1, thereby increasing the permeability of rat vascular endothelial cells. 目的： 探讨缺氧诱导因子1α(HIF-1α)对大鼠血管内皮细胞通透性的调控作用及相关机制。 方法： 取12只雄性SD大鼠，35～38 d龄，分离主动脉培养血管内皮细胞，4 d后观察细胞形态，并取第2或3代细胞进行以下实验。(1)取大鼠血管内皮细胞，采用随机数字表法(分组方法下同)分为空白对照组、阴性对照组、HIF-1α干扰序列1组、HIF-1α干扰序列2组和HIF-1α干扰序列3组，每组3孔。阴性对照组和HIF-1α干扰序列1组、HIF-1α干扰序列2组和HIF-1α干扰序列3组细胞经脂质体2000分别转染GV248空质粒和含HIF-1α干扰序列1、干扰序列2、干扰序列3的GV248重组质粒，空白对照组细胞仅加入脂质体2000。转染24 h后，采用实时荧光定量反转录PCR法和蛋白质印迹法(检测方法下同)分别检测各组细胞HIF-1α的mRNA和蛋白的表达，选取干扰效率最高的序列。(2)另取大鼠血管内皮细胞，分为空白对照组、阴性对照组和HIF-1α低表达组，每组3孔。空白对照组细胞仅加入脂质体2000，阴性对照组和HIF-1α低表达组细胞经脂质体2000分别转染GV248空质粒及实验(1)中筛选的低表达HIF-1α重组质粒。培养14 d，检测各组细胞HIF-1α的mRNA和蛋白表达水平。(3)另取大鼠血管内皮细胞，分为空白对照组、阴性对照组、HIF-1α高表达组，每组3孔。空白对照组细胞仅加入脂质体2000，阴性对照组、HIF-1α高表达组细胞经脂质体2000分别转染GV230空质粒和HIF-1α高表达重组质粒。培养14 d，检测各组细胞HIF-1α的mRNA和蛋白表达水平。(4)取实验(1)中3组、实验(2)中3组细胞，检测肌球蛋白轻链激酶(MLCK)、磷酸化肌球蛋白轻链(p-MLC)和带状闭合蛋白1(ZO-1)的mRNA和蛋白表达水平。对数据行单因素方差分析、t检验。 结果： 培养4 d，细胞呈梭形，成功培养出大鼠血管内皮细胞。(1)HIF-1α干扰序列1组、HIF-1α干扰序列2组和HIF-1α干扰序列3组细胞HIF-1α的干扰效率分别为47.66%、45.79%、62.62%，干扰序列3组细胞干扰效率最高。转染24 h后，HIF-1α干扰序列3组细胞HIF-1α的mRNA和蛋白表达水平明显低于空白对照组(t＝18.404、9.140，P<0.01)及阴性对照组(t＝15.099、7.096，P<0.01)。(2)培养14 d后，HIF-1α低表达组细胞HIF-1α的mRNA和蛋白表达水平明显低于空白对照组(t＝21.140、5.440，P<0.01)和阴性对照组(t＝14.310、5.210，P<0.01)。(3)培养14 d后，HIF-1α高表达组细胞HIF-1α的mRNA和蛋白表达水平明显高于空白对照组(t＝19.160，7.710，P<0.01)及阴性对照组(t＝19.890、7.500，P<0.01)。(4)转染24 h后，HIF-1α低表达组细胞MLCK、p-MLC的mRNA表达水平显著低于空白对照组(t＝2.709、4.011，P<0.05或P<0.01)和阴性对照组(t＝2.373、3.744，P<0.05或P<0.01)，HIF-1α低表达组细胞ZO-1的mRNA表达水平显著高于空白对照组和阴性对照组(t＝4.285、5.050，P<0.01)。HIF-1α高表达组细胞MLCK、p-MLC的mRNA表达水平显著高于空白对照组(t＝9.118、11.313，P<0.01)和阴性对照组(t＝9.073、11.280，P<0.01)，HIF-1α高表达组细胞ZO-1的mRNA表达水平显著低于空白对照组和阴性对照组(t＝2.889、2.640，P<0.05)。(5)转染24 h后，HIF-1α低表达组细胞MLCK、p-MLC的蛋白表达水平显著低于空白对照组(t＝2.652、3.983，P<0.05或P<0.01)和阴性对照组(t＝2.792、4.065，P<0.05或P<0.01)，HIF-1α低表达组细胞ZO-1的蛋白表达显著高于空白对照组和阴性对照组(t＝3.881、3.570，P<0.01)。HIF-1α高表达组细胞MLCK、p-MLC的蛋白表达水平为1.18±0.24、0.68±0.22，显著高于空白对照组的0.41±0.21、0.35±0.14(t＝5.011、3.982，P<0.05或P<0.01)和阴性对照组的0.43±0.20、0.36±0.12(t＝4.880、3.862，P<0.05或P<0.01)。HIF-1α高表达组细胞ZO-1的蛋白表达水平为0.08±0.06，显著低于空白对照组的0.20±0.09和阴性对照组的0.19±0.09(t＝4.178、3.830，P<0.05或P<0.01)。 结论： HIF-1α通过上调MLCK、p-MLC，下调ZO-1的表达，从而增加大鼠血管内皮细胞通透性。.

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