State Key Lab of Diagnostic and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, 1st Affiliated Hospital of Medical School, Zhejiang University, Hangzhou 310003, China.
Department of Infectious Diseases, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province 310016, China.
Int Immunopharmacol. 2014 Mar;19(1):81-7. doi: 10.1016/j.intimp.2014.01.003. Epub 2014 Jan 19.
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.
为了研究紫草素（Shik）的抗炎作用及其机制，将鼠源巨噬细胞样 RAW264.7 细胞（RAW264.7 细胞）分为对照组、LPS 组（0.125、0.25 和 0.5μg/ml）、LPS（0.125、0.25 和 0.5μg/ml）加 Shik（0.5、1 和 2μM）组和 Shik（2μM）组。暴露 24h 后，采用 ELISA 法测定上清液中白细胞介素 6（IL-6）、一氧化氮（NO）和肿瘤坏死因子-α（TNF-α）的水平，qRT-PCR、western blot 和免疫荧光法测定上清液和细胞质中高迁移率族蛋白 1（HMGB1）的表达，qRT-PCR 和 ELISA 法测定细胞和上清液中 IFN-β的表达，western blot 法测定 NF-κB 蛋白表达的核质比。研究结果表明，Shik 可显著降低 LPS 诱导的 RAW264.7 细胞中 IL-6、NO 和 TNF-α的水平（P<0.05 或 P<0.01）。与 LPS 组相比，LPS 加 Shik 组中 HMGB1、IFN-β和 NF-κB 蛋白表达的核质比显著降低（P<0.05 或 P<0.01）。IFN-β信号分子 JAK 和 NF-κB 的抑制剂可显著减弱上清液中 HMGB1 的表达。本研究发现，Shik 对 LPS 诱导的 RAW264.7 细胞具有抗炎作用，其机制之一可能是下调 HMGB 表达，与 IFN-β 和 NF-κB 信号通路有关。
