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.
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