Shikonin inhibits the lipopolysaccharide-induced release of HMGB1 in RAW264.7 cells via IFN and NF-κB signaling pathways

Abstract

To study the anti-inflammation effect of Shikonin (Shik) and its mechanism, murine macrophage-like RAW264.7 cells (RAW264.7 cells) were divided into control group, LPS group (0.125, 0.25 and 0.5μg/ml), LPS (0.125, 0.25 and 0.5μg/ml) plus Shik (0.5, 1 and 2μM) group, and Shik (2μM) group. After exposure for 24h, the levels of Interleukin-6 (IL-6), nitric oxide (NO) and Tumor Necrosis Factor-α (TNF-α) in supernatant were measured with ELISA, the expression of high mobility group box 1(HMGB1) in supernatant and cytoplasm was assayed using qRT-PCR, western blot and immunofluorescence assays, the expression of IFN-β in cellular and supernatant was assayed by qRT-PCR and ELISA, and the ratio of nuclear to cytoplasm for NF-κB protein expression was assayed using western blot. The results of our investigation demonstrated that Shik could reduce significantly the levels of IL-6, NO and TNF-α in RAW264.7 cells exposed to LPS (P<0.05 or P<0.01). The expression of HMGB1, IFN-β and the ratio of nuclear to cytoplasm for NF-κB protein expression in LPS plus Shik group declined significantly as compared with LPS group (P<0.05 or P<0.01). The inhibitors of IFN-β signaling molecule JAK and NF-κB could attenuate significantly the expression of HMGB1 in supernatant. It was found in the present study that Shik could have the anti-inflammatory effects in RAW264.7 cells exposed to LPS, and one of the mechanisms may be the down-regulation of HMGB expression, which was associated with the IFN-β and NF-κB signaling pathways.

Keywords: High mobility group box 1; IFN-β; Lipopolysaccharides; Macrophages; NF-κB; Shikonin.