雪胆素甲通过破坏微丝结构诱导LPS活化的RAW264.7细胞凋亡

Cucurbitacin IIa induces apoptosis of LPS-stimulated RAW264.7 cells through disrupting microfilament cytoskeleton

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。

This study aimed to explore the potential anti-inflammatory activity and underlying action mechanism of cucurbitacin IIa （CuIIa） by using lipopolysaccharide （LPS）-activated RAW264.7 macrophages as a model. The effect of CuIIa on cell proliferation was measured by MTS assay. Phase contrast microscopy was used to observe the morphology changes of cells while immunofluoreseence staining was adopted to examine actin cytoskeletorr Sub-G0/G1 （apoptotie） peak was identified by flow cytometry. Immunoblotting was used to evaluate the levels of cleaved capase-3, survivin, G-actin and F-actin. Our results showed that CuIIa inhibited the proliferation of RAW264.7 cells in a time- and dose-dependent manner, but CuIIa alone could not effectively induce apoptosis. CuIIa treatment caused cell shrinkage and disappearance of pseudopodia protrusions of LPS-activated cells, and led to significant G-actin decrease and dramatic actin aggregation within cells, which indicating a disruption of microfilament structures. The hypodiploid peaks （apoptotic peaks） were increased in LPS-activated RAW264.7 cells treated with increased doses of CuIIa. Immunoblotting analysis revealed that CuIIa significantly induced the activation of caspase-3 and decreased survivin expression. All the result demonstrated that CuIIa can induces apoptosis in LPS-activated RAW264.7 cells through aggregating actin and disrupting microfilament cytoskeleton, thus exhibits its anti-inflammatory activity.