Effect of retroviral vector mediating RNA interference on the release of tumor necrosis factor-alpha in lipopolysaccharide-stimulating macrophage

Abstract

To explore the effect of small interfering RNA (siRNA) targeting NF-kappaB signal pathway on the expression level of tumor necrosis factor alpha (TNF-alpha) released by lipopolysaccharides (LPS)-stimulating-macrophages. Human monocytic THP-1 cell was induced by phorbol myristate acetate (PMA) and transformed into macrophage. Two groups of macrophage were infected by siRNA retroviral expression vector specific to NF-kappaB functional subunit P65 (siRNA group) and Scramble control vector (Scramble control group) constructed by molecular cloning technology. Lipopolysaccharide (50 microg/ml) was used to treat the macrophages continuously. RT-PCR was performed to detect the expression level of NF-kappaB P65 mRNA and TNF-alpha mRNA at different time-points of LPS stimulation. Western blotting was used to analyze the protein level of NF-kappaB P65. Enzyme-linked immunosorbent assay was applied to analyze the expression level of TNF-alpha released by LPS-stimulated macrophages. At Hours 12 and 24 after LPS stimulation, the expression level of NF-kappaB P65 mRNA in siRNA group (0.97 +/- 0.02, 0.89 +/- 0.01) was significantly less than that in Scramble control group (1.01 +/- 0.03, 0.97 +/- 0.01, both P < 0.05). At Hours 24 and 72 after LPS stimulation, the expression level of NF-kappaB P65 protein in siRNA group (0.95 +/- 0.04, 0.94 +/- 0.01) was obviously less than that in Scramble control group (1.07 +/- 0.06, 1.03 +/- 0.05, both P < 0.05). At Hours 4, 8, 12 and 24 after LPS stimulation, TNF-alpha mRNA released by siRNA group macrophages was far less than that by Scramble control group macrophages (0.92 +/- 0.02 vs 0.98 +/- 0.01, 0.86 +/- 0.02 vs 1.00 +/- 0.01, 0.79 +/- 0.03 vs 1.01 +/- 0.01, 0.78 +/- 0.03 vs 1.02 +/- 0.01, all P < 0.05). At Hours 2, 4, 8, 24, 36, 48, 54 and 72 after LPS stimulation, the TNF-alpha content in culture medium supernatant in siRNA group macrophage was less than that in scramble control group (P < 0.05). NF-kappaB P65 siRNA inhibits the functional activity of NF-kappaB signal pathway in PMA-induced macrophage. Then it blocks the activation of macrophage and the excessive release of TNF-alpha due to endotoxin stimulation. The RNA interference technology may be applied to prevent and treat excessive inflammatory reaction in acute lung injury.