Shikonins, Phytocompounds from Lithospermum erythrorhizon, Inhibit the Transcriptional Activation of Human Tumor Necrosis Factor Promoter in Vivo*

Tumor necrosis factor (TNF) contributes to the pathogenesis of both acute and chronic inflammatory diseases and has been a target for the development of new anti-inflammatory drugs. Shikonins, the naphthoquinone pigments present in the root tissues of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), have been reported to exert anti-inflammatory effects both in vitro and in vivo. In this study, we evaluated the effects of shikonin and its derivatives on the transcriptional activation of human TNFpromoter in a gene gun-transfected mouse skin system by using a luciferase reporter gene assay. The crude plant extract of L. erythrorhizon as well as derived individual compounds shikonin, isobutyryl shikonin, acetyl shikonin, dimethylacryl shikonin and isovaleryl shikonin showed significant dose-dependent inhibition of TNFpromoter activation. Among the tested compounds, shikonin and isobutyryl shikonin exhibited the highest inhibition of TNFpromoter activation and also showed significant suppression of transgenic human TNFmRNA expression and protein production. We demonstrated that shikonin-inhibitory response was retained in the core TNFpromoter region containing the TATA box and a 48-bp downstream sequence relative to the transcription start site. Further our results indicated that shikonin suppressed the basal transcription and activator-regulated transcription of TNFby inhibiting the binding of transcription factor IID protein complex (TATA box-binding protein) to TATA box. These in vivo results suggest that shikonins inhibit the transcriptional activation of the human TNFpromoter through interference with the basal transcription machinery. Thus, shikonins may have clinical potential as anti-inflammatory therapeutics.