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目的分析雪胆素甲(CuIIa)对脂多糖(LPS)活化的RAW264.7巨噬细胞的影响,探讨其潜在的抗炎效应及作用机制。方法MTS法检测CuIIa对RAW264.7细胞增殖的作用,相差显微镜观察CuIIa对LPS活化的细胞形态的影响,荧光显微镜观察CuIIa对细胞骨架的影响,流式细胞仪检测亚二倍体(凋亡)峰的变化,免疫印迹检测Cleaved caspase-3、生存素以及G-actin与F-actin的变化。结果 CuIIa能明显抑制RAW264.7细胞的增殖,并呈时间和剂量依赖性,但单独CuIIa不能有效诱导细胞凋亡。CuIIa使LPS活化的伸展细胞收缩变圆,伪足突起消失,G-actin水平下降,细胞内发生严重的Actin聚集,说明微丝结构被破坏。随着CuIIa浓度的增加,其诱导LPS活化的RAW264.7细胞处于sub-G0/G1(凋亡峰)的数量明显增加。免疫印迹分析显示CuIIa使Caspase-3明显活化,生存素急剧下降。结论 CuIIa可能通过引起Actin聚集而破坏微丝细胞骨架,诱导LPS活化的RAW264.7细胞发生凋亡,从而发挥其抗炎效应。
Objective To investigate the effects of CuIIa on lipopolysaccharide (LPS) -activated RAW264.7 macrophages and to explore its potential anti-inflammatory effects and its mechanism of action. Methods The effect of CuIIa on the proliferation of RAW264.7 cells was detected by MTS method. The effect of CuIIa on the morphology of LPS-activated cells was observed by phase contrast microscopy. The effect of CuIIa on cytoskeleton was observed by fluorescence microscopy. The sub-diploid (apoptosis) Peak changes, Western blot analysis of Cleaved caspase-3, survivin and G-actin and F-actin changes. Results CuIIa significantly inhibited the proliferation of RAW264.7 cells in a time and dose-dependent manner, but CuIIa alone did not induce apoptosis effectively. CuIIa prolonged the contractility of LPS-activated stretched cells, disappeared the pseudopods, decreased the level of G-actin, and resulted in the accumulation of Actin in the cells, indicating that the microfilament structure was destroyed. With the increase of CuIIa concentration, the number of sub-G0 / G1 (apoptotic peak) of RAW264.7 cells that induced LPS activation increased significantly. Immunoblot analysis showed that CuIIa markedly activated Caspase-3, with a sharp decrease in survivin. Conclusion CuIIa may induce apoptosis of RAW264.7 cells induced by LPS, which may lead to antiinflammatory effect by inducing actin aggregation and disrupting actin cytoskeleton.