Background and purpose: 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol (RSCL-0520) is a phenanthrene isolated from Eulophia ochreata, one of the Orchidaceae family, known by local tradition to exhibit medicinal properties. However, no anti-inflammatory activity or any molecular mechanisms involved have been reported or elucidated. Here, for the first time, we evaluate the anti-inflammatory properties of RSCL-0520 on responses induced by lipopolysaccharide (LPS) and mediated via Toll-like receptors (TLRs). Experimental approach: The in vitro anti-inflammatory activities of RSCL-0520 were investigated in LPS-stimulated monocytic cells, measuring activation of cytokine and inflammatory genes regulated by nuclear factor-kB (NF-kB). Tumour necrosis factor (TNF)-a levels in serum following LPS stimulation in mice and carrageenan-induced paw oedema in rats were used as in vivo models. Key results: Pretreatment with RSCL-0520 effectively inhibited LPS-induced, TLR4-mediated, NF-kB-activated inflammatory genes in vitro, and reduced both LPS-induced TNF-a release and carrageenan-induced paw oedema in rats. Treatment with RSCL-0520 reduced LPS-stimulated mRNA expression of TNF-a, COX-2, intercellular adhesion molecule-1, interleukin (IL)-8 and IL-1b, all regulated through NF-kB activation. RSCL-0520, however, did not interfere with any cellular processes in the absence of LPS. Conclusions and implications: RSCL-0520 blocked signals generated by TLR4 activation, as shown by down-regulation of NF-kB-regulated inflammatory cytokines. The inhibitory effect involved both MyD88-dependent and -independent signalling cascades. Our data elucidated the molecular mechanisms involved, and support the search for plant-derived TLR antagonists, as potential anti inflammatory agents.

The regulation of cytokines and pro‐inflammatory genes is an absolute essentiality to combat inflammatory diseases. The present study investigated the effects of 4‐O‐chloroacetyl‐2,3‐di‐O‐acetyl‐6‐O‐levulinoyl‐β‐d‐glucopyranosyl]‐(1‐3)‐1‐O‐(p‐methoxyphenyl)‐2‐deoxy‐2‐N‐trichloroacetyl‐4,6‐O‐benzylidene‐α‐d‐glucopyranoside (RSCL‐0409), a novel small molecule Toll‐like receptor (TLR) signalling antagonist, and its mechanism of action in human monocytic (THP‐1) cells stimulated with lipopolysaccharide (LPS). In THP‐1 and RAW264.7 cells, RSCL‐0409 suppressed LPS‐induced production of tumour necrosis factor‐α (TNF‐α) with a 50% inhibitory concentration of 10.6 μm and mRNA expression of ICAM‐1, Cox‐2 and interleukin‐8 with no evidence of cytotoxicity. RSCL‐0409 also suppressed TNF‐α production from LPS‐stimulated human peripheral blood mononuclear cells. Similar results were obtained in vivo in a murine model of LPS‐induced inflammation, where pretreatment with RSCL‐0409 resulted in significant inhibition of TNF‐α. It is also noteworthy that RSCL‐0409 suppressed the cytokine production induced by TLR2 and ‐4 ligands and not for any other TLR ligands. RSCL‐0409 significantly inhibited p65 nuclear translocation induced by LPS. In conclusion, RSCL‐0409, a novel small molecule, is the first of its kind in the category of carbohydrate‐derived TLR signalling antagonists and could definitely be a promising therapeutic agent for inflammatory diseases whose pathogenesis involves TLR2‐ or TLR4‐mediated signalling processes.

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