title: [Effect of retroviral vector mediating RNA interference on the release of tumor necrosis factor-alpha in lipopolysaccharide-stimulating macrophage].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Respiratory Medicine, Affiliated Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China.
authors.forename: Chun-ting
authors.initials: CT
authors.lastname: Wu
authors.forename: Guang-fa
authors.initials: GF
authors.lastname: Zhu
authors.forename: Jia-hui
authors.initials: JH
authors.lastname: Zhao
authors.forename: Xiang-feng
authors.initials: XF
authors.lastname: Zhang
authors.forename: Li-yan
authors.initials: LY
authors.lastname: Jin
authors.forename: Shu-feng
authors.initials: SF
authors.lastname: Yan
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D008070:Lipopolysaccharides / Q000009:adverse effects; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D051996:Transcription Factor RelA / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha