目的 基于脂多糖誘導(dǎo)小鼠單核巨噬細(xì)胞（RAW 264.7）構(gòu)建的體外細(xì)胞炎癥模型，探討牡蒿Artemisia japonica Thunb.水提物的抗炎作用及其機(jī)制。方法 通過(guò)CCK-8法檢測(cè)牡蒿水提物對(duì)RAW 264.7細(xì)胞活力的影響以篩選合適的牡蒿水提物給藥質(zhì)量濃度。采用脂多糖200 ng/mL誘導(dǎo)RAW 264.7細(xì)胞建立體外炎癥細(xì)胞模型。將細(xì)胞分為對(duì)照組、模型組和牡蒿水提物（240、480、960μg/mL）組，用Griess試劑法檢測(cè)RAW 264.7細(xì)胞一氧化氮（NO）釋放水平，ELISA法檢測(cè)細(xì)胞白細(xì)胞介素（IL）-6、IL-1β含量。采用Western blotting法檢測(cè)誘導(dǎo)型一氧化氮合成酶（iNOS）、環(huán)氧合酶-2(COX-2)以及Toll樣受體4(TLR4)/核因子-κB(NF-κB)信號(hào)通路中TLR4、核因子κB抑制蛋白α(IκBα)、p-IκBα、p65蛋白（p65）、p-p65蛋白相對(duì)表達(dá)量。結(jié)果 牡蒿水提物質(zhì)量濃度為0～960μg/mL時(shí)對(duì)RAW 264.7細(xì)胞活力沒有顯著影響。與模型組相比，牡蒿水提物480、960μg/mL組能顯著抑制IL-6的釋放以及COX-2的蛋白相對(duì)表達(dá)量（P<0.01）；各質(zhì)量濃度牡蒿水提物均能顯著抑制NO、IL-1β的釋放，iNOS、p-IκBα、p65、p-p65蛋白的表達(dá)以及IκBα蛋白的降解（P<0.01）。結(jié)論 牡蒿水提物能抑制脂多糖誘導(dǎo)的RAW 264.7細(xì)胞炎性因子的分泌，其作用機(jī)制與抑制炎性蛋白iNOS和COX-2的表達(dá)及TLR4/NF-κB信號(hào)通路的激活有關(guān)。

Objective To explore the effects and mechanisms of A. japonica water extract on lipopolysaccharide-induced RAW 264.7macrophages model. Methods CCK-8 colorimetric method was used to select the appropriate concentration of A. japonica on the activity of RAW 264.7 macrophages. RAW 264.7 cells were induced by lipopolysaccharide 200 ng/mL to establish in vitro inflammatory cell model. Cells were divided into control group, model group, and A. japonica（240, 480, and 960 μg/mL） groups.Griess assay was used to assess NO concentration, IL-1β, and IL-6 content was measured by ELISA. The expression of iNOS, COX-2, and TLR4/NF-κB pathway-related proteins（TLR4, IκBα, p-IκBα, p65, p-P65） were detected by Western blotting. Results A.japonica had no significant effect on the viability of RAW 264.7 cells at the concentrations of 0 — 960 μg/mL. Compared with model group, A. japonica water extract 480 and 960 μg/mL groups significantly inhibitory effects on the release of IL-6 and protein expression of COX-2（P＜0.01）. Each dose group of A. japonica water extract displayed favorable inhibitory decreased effects on release of NO and IL-1β, expression of iNOS, p-IκBα, p65, and p-p65 proteins and degradation of IκBα. Conclusion A. japonica water extract could inhibit lipopolysaccharide-induced secretion of inflammatory factors in RAW 264.7 cells, the mechanism of which is related to inhibiting the expression of inflammatory proteins iNOS and COX-2 and inhibiting the activation of TLR4/NF-κB signaling pathway.

R285.5

國(guó)家自然科學(xué)基金資助項(xiàng)目(32260925); 貴州省科技計(jì)劃項(xiàng)目(黔科合基礎(chǔ)-ZK[2022]一般507); 貴州中醫(yī)藥大學(xué)大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計(jì)劃項(xiàng)目[貴中醫(yī)大創(chuàng)合字(2021)84號(hào)