Methylene Blue Attenuates iNOS Induction Through Suppression of Transcriptional Factor Binding Amid iNOS mRNA Transcription.
Inducible nitric oxide synthase (iNOS) critically contributes to the development of endotoxin-mediated inflammation. It can be induced by cytokines or endotoxins via distinct signaling pathways. Lipopolysaccharide (LPS) triggers iNOS expression through activation of the inhibitor of κB-α (IκB-α)-nuclear factor κB (NF-κB) cascade, whereas interferon-γ (IFN-γ) acts primarily through Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1). Methylene blue (MB), an agent used clinically to treat numerous ailments, has been shown to reduce NO accumulation through suppression of iNOS activity. But it remains unclear whether MB affects iNOS induction. This knowledge gap is addressed in the present study using cultured cells and endotoxemic mice. With mouse macrophages, MB treatment prevented the LPS- and/or IFN-γ-stimulated iNOS protein expression. Real-time PCR experiments showed that iNOS mRNA transcription was robustly blocked by MB treatment. The inhibitory effect of MB on iNOS expression was confirmed in vivo in endotoxemic mice. Further analysis showed that MB had no significant effect on IκB-α degradation and NF-κB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-κB or STAT1 was also not affected by MB treatment. But MB treatment markedly reduced the binding of NF-κB and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that MB reduced NF-κB and STAT1 bindings to iNOS promoter inside the cell. These studies show that MB attenuates transcriptional factor binding amid iNOS mRNA transcription, providing further insight into the molecular mechanism of MB in disease therapy.
Department of Pharmacology, School of Pharmacy, Nantong University, #19 Qixiu Road, Nantong, Jiangsu Province, 226001, China.
Chao
C
Huang
Department of Pharmacology, School of Pharmacy, Nantong University, #19 Qixiu Road, Nantong, Jiangsu Province, 226001, China.
Lijuan
L
Tong
Department of Pharmacology, School of Pharmacy, Nantong University, #19 Qixiu Road, Nantong, Jiangsu Province, 226001, China.
Xu
X
Lu
Department of Pharmacology, School of Pharmacy, Nantong University, #19 Qixiu Road, Nantong, Jiangsu Province, 226001, China.
Jia
J
Wang
Department of Pharmacology, School of Pharmacy, Nantong University, #19 Qixiu Road, Nantong, Jiangsu Province, 226001, China.
Wenjuan
W
Yao
Department of Pharmacology, School of Pharmacy, Nantong University, #19 Qixiu Road, Nantong, Jiangsu Province, 226001, China.
Bo
B
Jiang
Department of Pharmacology, School of Pharmacy, Nantong University, #19 Qixiu Road, Nantong, Jiangsu Province, 226001, China.
Wei
W
Zhang
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D019446:Endotoxemia / Q000139:chemically induced / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D008751:Methylene Blue / Q000008:administration & dosage* / Q000494:pharmacology; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism*
D008070:Lipopolysaccharides; D014157:Transcription Factors; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D008751:Methylene Blue
INDUCIBLE NITRIC OXIDE SYNTHASE; INHIBITOR OF κB-α; LIPOPOLYSACCHARIDE; METHYLENE BLUE; NUCLEAR FACTOR κB; SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1