摘要
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 investigate the effect and the mechanism of Apigenin on lipopolysaccharides（ LPS）-induced cytokines production in murine macrophages. Methods The murine macrophage cell line RAW 264. 7 cells were cultured in vitro,and were treated with different concentration of Apigenin followed by LPS stimulation. Expression of heme oxygenase-1（ HO-1）,phosphorylation of c-Src and p47 phox,the subunit of NADPH oxidase were detected by Western blot; The intracellular formation of reactive oxygen species（ ROS） was detected by using the fluorescent probe H2DCFDA; Secretion of TNF-αand IL-6 were detected by ELISA. Results Western blot indicated that phosphorylated c-Src and p47 phoxwere induced by apigenin after 30 min of incubation. While 50 μmol / L of PP1,an inhibitor of c-Src,could significantly inhibit p47 phoxphosphorylation. In addition,Apigenin could also induce RAW264. 7 cells accumulation of ROS,and this effect could be abrogated by the NAPDH oxidase inhibitor,DPI. HO-1 could be induced by Apigenin,and pretreatment of PP1,DPI and the ROS scavenger N-acetyl-cysteine（ NAC） could block HO-1 production. Furthermore,apigenin could inhibit LPS-induced secretion of TNF-αand IL-6,and transfection of HO-1 siRNA could antagonize this action. Conclusion Apigenin can induce HO-1 expression by c-Src / NADPH oxidase / ROS to protect against excessive cytokines secretion in murine macrophages.
出处
《中南医学科学杂志》
CAS
2015年第4期369-373,共5页
Medical Science Journal of Central South China
基金
国家自然科学基金(31370207)
