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目的观察芹菜素对脂多糖(LPS)诱导小鼠巨噬细胞分泌细胞因子的影响,并探讨其分子机制。方法体外培养小鼠巨噬细胞RAW 264.7,用不同浓度的芹菜素处理后,加入LPS刺激0~16 h。Western blot检测血红素氧合酶-1(HO-1)的表达以及c-Src和NADPH氧化酶p47phox亚基的磷酸化;荧光探针H2DCFDA检测活性氧(ROS)的产生;ELISA检测TNF-α和IL-6的产生。结果芹菜素作用RAW 264.7细胞30 min即可诱导c-Src和NADPH氧化酶亚基p47phox磷酸化。采用50μmol/L c-Src抑制剂PP1预处理细胞后,p47phox磷酸化水平明显降低。同时,芹菜素能上调RAW264.7细胞内ROS的含量,而NADPH氧化酶抑制剂DPI可抑制其产生。芹菜素也能诱导RAW264.7细胞表达HO-1,而采用PP1、DPI或ROS抑制剂N-乙酰半胱氨酸(NAC)预处理细胞后,HO-1表达水平明显降低。此外,芹菜素处理能下调LPS诱导RAW264.7细胞分泌TNF-α和IL-6,而采用siRNA沉默HO-1表达后,能在一定程度上降低芹菜素对细胞因子的抑制效应。结论芹菜素可能通过激活c-Src/NADPH氧化酶/ROS通路诱导HO-1表达,从而抑制小鼠巨噬细胞过度分泌细胞因子。
Objective To observe the effect of apigenin on cytokines secreted by lipopolysaccharide (LPS) -induced murine macrophages and its molecular mechanism. Methods Mouse RAW 264.7 macrophages were cultured in vitro and treated with different concentrations of apigenin and then stimulated with LPS for 0-16 h. The expression of heme oxygenase-1 (HO-1) and the phosphorylation of c-Src and p47phox subunit of NADPH oxidase were detected by Western blot. The production of reactive oxygen species (ROS) was detected by fluorescent probe H2DCFDA. And IL-6 production. Results Apigenin induced the phosphorylation of c-Src and NADPH oxidase subunit p47phox in RAW 264.7 cells for 30 min. After pretreatment of cells with 50μmol / L c-Src inhibitor PP1, the phosphorylation level of p47phox was significantly decreased. At the same time, apigenin can upregulate ROS content in RAW264.7 cells, while NADPH oxidase inhibitor DPI can inhibit its production. Apigenin also induced HO-1 expression in RAW264.7 cells, while HO-1 expression was significantly decreased after pretreatment of cells with PP1, DPI or ROS inhibitor N-acetylcysteine (NAC). In addition, apigenin treatment could down-regulate LPS-induced TNF-α and IL-6 secretion in RAW264.7 cells. However, silencing of HO-1 expression by siRNA suppressed the inhibitory effect of apigenin on cytokines to a certain extent. Conclusions Apigenin may induce HO-1 expression through activation of c-Src / NADPH oxidase / ROS pathway, thereby inhibiting murine macrophages to over-secrete cytokines.