Nuclear Factor κB Decoy Oligodeoxynucleotides Prevent Endotoxin–Induced Fatal Liver Failure in A Murine Model

Abstract

Endotoxin syndrome is a systemic inflammatory response mediated by inflammatory cytokines. Nuclear factor B (NF-B) is the dominant regulator of the production of these cytokines by inflammatory cells. The aim of this study was to assess the efficacy of in vivo transfer of synthetic double-stranded oligodeoxynucleotides (ODN) with high affinity against NF-B (NF-B/decoy/ODN) as a therapeutic strategy for treating endotoxin-induced fatal liver injury. Liver injury was induced by administration of lipopolysaccharide (LPS) to Propionibacterium acnes-primed BALB/C mice. NF-B/decoy/ODN was transferred into the portal vein using a fusigenic liposome with hemagglutinating virus of Japan. NF-B/decoy/ODN was preferentially transferred to Kupffer cells, and activation of NF-B after the LPS challenge was suppressed, leading to decreased inflammatory cytokine production. As a result, the massive necrosis and hepatocyte apoptosis observed in the control mice was dramatically attenuated and the survival rate improved. In conclusion, NF-B/decoy/ ODN transfer in vivo effectively suppressed endotoxin-induced fatal liver injury in mice. (HEPATOLOGY 2003;38:335-344.)

Contribution of Toll‐like receptor/myeloid differentiation factor 88 signaling to murine liver regeneration†

“…In WT mice, an increase in the proportion of hepatocytes that were BrdU positive after PH was seen 33 ; this proprtion reached a peak at 36 hours and had subsequently decreased by 72 hours (Fig. 1B-C).…”

“…Reverse transcription-polymerase chain reaction (RT-PCR) for TNF-␣ and ␤-actin were undertaken as previously described. 33 The amplifying cycle was 25 for ␤-actin and 34 for TNF-␣.…”

“…Liver specimens were fixed in 10% buffered formalin for subsequent histological and immunohistological analyses according to methods described in previous reports. 12,33 For monitoring nuclear translocation of the NF-B p65 subunit, liver sections were incubated with goat anti-NF-B p65 Ab (Santa Cruz Biotechnology), as described previously. 33 For a positive control, we used liver specimens obtained from WT (wild-type) mice 30 minutes after intraperitoneal injection of 500 ng TNF-␣.…”

“…12,33 For monitoring nuclear translocation of the NF-B p65 subunit, liver sections were incubated with goat anti-NF-B p65 Ab (Santa Cruz Biotechnology), as described previously. 33 For a positive control, we used liver specimens obtained from WT (wild-type) mice 30 minutes after intraperitoneal injection of 500 ng TNF-␣. To assess bromodeoxyuridine (BrdU) staining, 1 hour before harvest of liver specimens animals were injected intraperitoneally with BrdU (Sigma, St. Louis, MO) (50 mg/kg of a 0.2% solution in phosphate-buffered saline).…”

“…Liver sections were incubated with mouse monoclonal anti-BrdU Ab (DAKO, Kyoto, Japan), as described previously. 33 BrdUpositive hepatocytes were counted in three low-power (80ϫ) fields on each slide; the percentage of total hepatocytes that were BrdU positive was calculated. To assess the number of hepatocyte mitoses, liver sections were stained with hematoxylin-eosin.…”