The anti‐inflammatory carbazole, LCY‐2‐CHO, inhibits lipopolysaccharide‐induced inflammatory mediator expression through inhibition of the p38 mitogen‐activated protein kinase signaling pathway in macrophages

Abstract

1. The present study was undertaken to investigate the anti-inflammatory effects of a synthetic compound, LCY-2-CHO, on the expression of inducible nitric oxide synthase (iNOS), COX-2, and TNF-alpha in murine RAW264.7 macrophages. 2. Within 1-30 microm, LCY-2-CHO concentration-dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor-alpha (TNF-alpha) formation, with IC(50) values of 2.3, 1, and 0.8 microm, respectively. Accompanying inhibition of LPS-induced iNOS, cyclooxygenase-2 (COX-2), and pro-TNF-alpha proteins was observed. 3. Reverse transcription-polymerase chain reaction (RT-PCR) and promoter analyses indicated that iNOS expression was inhibited at the transcriptional level (IC(50)=2.3 microm), that inhibition of COX-2 expression only partially depended on gene transcription (IC(50)=7.6 microm), and that TNF-alpha transcription was unaffected. 4. Transcriptional assays revealed that activation of AP-1, but not NF-kappaB, was concomitantly blocked by LCY-2-CHO. Our results showed that LCY-2-CHO was capable of interfering with post-transcriptional regulation, altering the stability of COX-2 and TNF-alpha mRNAs. 5. Since the 3'-untranslated region (3' UTR) of both COX-2 and TNF-alpha mRNA contains a p38 mitogen-activated protein kinase (MAPK)-regulated element involved in mRNA stability, we assessed the effect of LCY-2-CHO on p38 MAPK. Our data clearly indicated an inhibition (IC(50)=1.7 microm) of LPS-mediated p38 MAPK activity, but not of extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activity. However, kinase assays ruled out a direct inhibition of p38 MAPK action. The selective p38 MAPK inhibitor, SB203580, inhibited the promoter activities of iNOS and COX-2 rather than that of TNF-alpha. 6. In conclusion, LCY-2-CHO downregulates inflammatory iNOS, COX-2, and TNF-alpha gene expression in macrophages through interfering with p38 MAPK and AP-1 activation.