目的 探討穿心蓮內(nèi)酯對脂多糖誘導(dǎo)RAW264.7細(xì)胞炎癥反應(yīng)的抑制作用及其作用機(jī)制。方法 采用噻唑藍(lán)比色（MTT）法分析穿心蓮內(nèi)酯對RAW264.7細(xì)胞活力的影響。通過脂多糖處理RAW264.7細(xì)胞24 h建立細(xì)胞炎癥模型，造模前1 h用穿心蓮內(nèi)酯2.5、5、10、20 μmol/L預(yù)處理。熒光定量PCR法檢測RAW264.7細(xì)胞內(nèi)相關(guān)抗氧化應(yīng)激酶基因和iNOS水平。穿心蓮內(nèi)酯單獨(dú)處理RAW264.7細(xì)胞24 h，Western blotting法檢測Keap1/Nrf2/HO-1信號通路相關(guān)蛋白和Keap1、Nrf2、HO-1蛋白水平。免疫熒光檢測轉(zhuǎn)錄因子Nrf2在胞質(zhì)及核內(nèi)的分布情況。結(jié)果 與對照組比較，穿心蓮內(nèi)酯劑量相關(guān)性地抑制RAW264.7細(xì)胞活力，差異具有統(tǒng)計學(xué)意義（P< 0.05、0.01、0.001）。穿心蓮內(nèi)酯顯著抑制脂多糖誘導(dǎo)RAW264.7細(xì)胞的iNOS水平（P< 0.001），增加相關(guān)抗氧化酶基因HO-1、NQO1 mRNA水平。穿心蓮內(nèi)酯抑制Keap1表達(dá)，增加Nrf2和HO-1蛋白水平的表達(dá)。結(jié)論 穿心蓮內(nèi)酯可抑制脂多糖誘導(dǎo)的炎癥反應(yīng)，其作用機(jī)制可能與激活Keap1/Nrf2/HO-1信號通路從而調(diào)控抗氧化酶HO-1、NQO1 mRNA表達(dá)水平相關(guān)。

Objective To study the anti-inflammatory effect of andrographolide on inflammation suppression induced by lipopolysaccharide, and explore its mechanism. Methods Cell viability of RAW264.7 cells treated with andrographolide were analyzed by MTT assay. Cell inflammatory model was established with RAW264.7 cells treated with lipopolysaccharide for 24 h prior to incubation of 2.5, 5, 10, and 20 μmol/L andrographolide for 1 h. Anti-oxidative enzymes and iNOS were evaluated by quantitative real-time polymerase chain reaction. RAW264.7 cells were treated with andrographolide for 24 h, and Keap1, Nrf2, and HO-1 expression were detected by Western blotting. Nrf2 distribution between cytosome and nucleus were analyzed by immunocytochemistry. Results Compared with the control group, cell survival rate of RAW264.7 cells were decreased treated by andrographolide in a dose-dependent manner (P < 0.05, 0.01, and 0.001). Andrographolide could significantly inhibit iNOS levels in RAW264.7 cells induced by lipopolysaccharide (P < 0.001), and could increase the levels of related antioxidant enzymes genes HO-1, NQO1 and mRNA. Andrographolide inhibited the expression of Keap1 and increased the expression of Nrf2 and HO-1 protein levels. Conclusion Andrographolide can inhibit the inflammatory response induced by lipopolysaccharide, and its mechanism may be related to activation of Keap1/Nrf2/HO-1 signaling pathway and regulation of expression levels of antioxidant enzymes HO-1 and NQO1 mRNA.

國家自然科學(xué)基金資助項(xiàng)目（81360625）