The Effect of p65 siRNA on the Expression of Pro-Inflammatory Factors under Conditions Mimicking Ischemia/Reperfusion in Human Umbilical Vein Endothelial Cells

ZENG Min; WU Zhiyong; ZHENG Yin; HE Yangli; FEI Yi; LIU Xiaojun

doi:10.3969/j.issn.0253-9896.2014.02.001

Abstract：Objective To investigate the effect and regulation mechanism of mimic ischemia/reperfusion (I/R) cul-ture of human umbilical vein endothelial cells (HUVECs) on levels of tumor necrosis factor(TNF)-αand intercellular adhe-sion molecule (ICAM)-1. Methods HUVECs were randomly divided into four groups:control group (normal media cell cul-ture+control siRNA transfection), mimic I/R+control siRNA transfection group (HUVECs were transfected with control siR-NA, for 48 h ,and then received mimic ischemic media culture for 30 min followed by normal media culture for 4 h), normal culture+p65 siRNA transfection group and mimic I/R+p65 siRNA transfection group. The expression levels of TNF-αand ICAM-1 mRNA and protein were determined by real-time PCR and enzyme linked immunosorbent assay (ELISA), respec-tively. Results The mRNA and supernatant protein levels of TNF-αand ICAM-1 were significantly increased in mimic I/R culture group (4.96±0.16 and 3.33±0.30)μg/L than those of other tree groups (P<0.05). The level of ICAM-1 mRNA was significantly higher in I/R+p65 siRNA transfection group (1.87±0.21)μg/L than that of control group (1.58±0.15) μg/L and normal culture+p65 siRNA transfection group [(1.69±0.21)μg/L, P<0.05]. The levels of TNF-αand ICAM-1proteins were (329.98 ± 12.18) μg/L and (654.74 ± 64.79) μg/L in mimic I/R+control siRNA transfection group, which were significantly higher than those of other three groups (P<0.05). The levels of TNF-αand ICAM-1 proteins were (129.65±22.42)μg/L and (185.76±11.27)μg/L in mimic I/R+p65 siRNA transfection group, which were significantly lower than those of contro group [(183.50±11.77)μg/L and (280.43±13.76)μg/L, P<0.05]. Conclusion The silencing of p65 through transfection of p65 siRNA in HUVECs inhibited mimic I/R-induced mRNA and protein expressions of TNF-αand ICAM-1.