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Specific inhibition of nitric oxide production in macrophages by phosphorothioate antisense oligonucleotides
H Arima1, T Sakamoto, Y Aramaki
1School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
Abstract:
The effects of antisense oligonucleotides (ODNs) on nitric oxide (NO) production induced by lipopolysaccharide (LPS) were investigated using thioglycollate-induced mouse peritoneal macrophages. Antisense phosphorothioate ODNs (S-oligo) corresponding to a sequence in the neighborhood of the AUG initiation codon of a mouse inducible nitric oxide synthase (iNOS) mRNA, which has a G-quartet motif in its antisense sequence, inhibited NO induction in a dose-dependent manner. Antisense phosphodiester ODNs (D-oligo), 5'- and 3'-terminal phosphorothioate-modified antisense ODNs and control scramble and missense S-oligos had no such effect. In addition, control nonsense and two mismatched S-oligos, which include G-quartet motif in their sequences, inhibited NO induction to approximately 50% of those in the control. Antisense S-oligo showed the inhibitory effect on NO production by exposure of macrophages to various concentrations of LPS. Western blot analysis using anti-mouse inducible nitric oxide synthase (iNOS) antibody revealed that antisense S-oligo specifically removed an immunoreactive band at 130 kDa. In addition, the results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effect originated from a specific reduction of the targeted iNOS mRNA by hybridization with the antisense S-oligo. Furthermore, no ODNs affected beta-actin mRNA and tumor necrosis factor alpha (TNF-alpha) expression in macrophages stimulated by LPS. These findings demonstrated that antisense S-oligo inhibited NO production derived from iNOS expression in macrophages by an antisense mechanism, including the aptameric effect partially mediated by the G-quartet motif.
Insights
Antisense oligonucleotides targeting inducible nitric oxide synthase (iNOS) mRNA effectively reduced nitric oxide (NO) production in macrophages. This inhibition, mediated by an antisense mechanism involving G-quartet motifs, offers a potential therapeutic strategy.
Area of Science:
- Molecular Biology
- Immunology
- Pharmacology
Background:
- Nitric oxide (NO) plays a crucial role in inflammatory responses.
- Inducible nitric oxide synthase (iNOS) is a key enzyme in NO production during inflammation.
- Antisense oligonucleotides (ODNs) are being explored for their therapeutic potential in modulating gene expression.
Purpose of the Study:
- To investigate the effect of antisense oligonucleotides (ODNs) on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse macrophages.
- To determine the specific mechanism by which antisense ODNs inhibit iNOS expression and NO production.
Main Methods:
- Thioglycollate-induced mouse peritoneal macrophages were treated with various antisense phosphorothioate ODNs (S-oligos) targeting iNOS mRNA.
- NO production was measured, and iNOS protein levels were assessed by Western blot.
- iNOS mRNA levels were analyzed using reverse transcription-polymerase chain reaction (RT-PCR).
Main Results:
- Antisense S-oligos targeting iNOS mRNA significantly inhibited NO production in a dose-dependent manner.
- Western blot and RT-PCR confirmed that antisense S-oligo specifically reduced iNOS protein and mRNA levels.
- Control ODNs, including mismatched and nonsense sequences with G-quartet motifs, showed partial inhibition, suggesting a dual mechanism.
Conclusions:
- Antisense S-oligo effectively inhibits NO production in macrophages by targeting iNOS mRNA through an antisense mechanism.
- The G-quartet motif in the antisense sequence may contribute to the inhibitory effect through an aptameric mechanism.
- These findings highlight the potential of antisense ODNs as therapeutic agents for inflammatory conditions characterized by excessive NO production.