目的 研究对映-贝壳杉烷二萜wangzaozin A诱导A549细胞骨架重组与迁移抑制的机制。方法 采用MTT、显微观察、免疫荧光染色、Western blotting、划痕实验等方法考察wangzaozin A对A549细胞毒性、细胞形态、细胞骨架、蛋白表达和细胞迁移的影响。结果 Wangzaozin A（0.2～0.8 μmol/L）处理A549细胞24、48、72 h后，细胞形态显著改变，细胞伪足增多并拉伸延长；细胞核扁化呈肾形改变；微管和角蛋白纤维网络的排布方式显著改变，表明细胞骨架经历了持续的重组过程。Wangzaozin A可显著上调细胞外信号调节蛋白激酶（extracellular regulated protein kinase，ERK）、微管相关蛋白4（microtubule-associated protein 4，MAP4）和角蛋白8（keratin 8，K8）的磷酸化水平（P<0.05、0.01），ERK抑制剂U0126可显著抑制wangzaozin A诱导的ERK、MAP4和K8的磷酸化水平上调（P<0.05、0.01）。Wangzaozin A可显著抑制A549细胞的迁移，呈剂量和时间相关性。结论 Wangzaozin A可以通过激活ERK信号通路，上调MAP4和K8磷酸化水平，增加微管和角蛋白纤维的动态性，干扰细胞微管动力学过程，从而抑制A549细胞迁移。

Objective To investigate the mechanism of cytoskeletal recombination and migration inhibition induced by wangzaozin A, ent-kaurane diterpenoid, in A549 cells. Methods The effects of wangzaozin A on cytotoxicity, cell morphology, cytoskeleton and protein expression as well as cell migration were detected in A549 cells by using MTT, microscope observation, Western blotting, immunofluorescence assay and scratch assay. Results Wangzaozin A induced significant changes in cell morphology at 24, 48 and 72 h, including increased pseudopods, stretched pseudopods and flattened nucleus in A549 cells. Moreover, microtubules and keratin fibers networks in A549 cells also showed obvious rearrangement, which indicated the cytoskeleton had gone through a continuous recombination process. Further, wangzaozin A significantly increased the phosphorylation of extracellular regulated protein kinase (ERK), microtubule-associated protein 4 (MAP4), keratin 8 (K8) (P<0.05, 0.01), while wangzaozin A-induced phosphorylation of MAP4 and K8 were suppressed in A549 cells treated with ERK inhibitors U0126 (P<0.05, 0.01); Wangzaozin A inhibited the migration of A549 cells with a correlation between concentration and time. Conclusion Wangzaozin A can upregulate the phosphorylation of MAP4 and K8 by activating ERK signaling pathway, which can significantly increase the dynamics of MTs and KFs, disturb the dynamic balance of the cytoskeleton, and inhibit the migration of A549 cells.

R285.5

国家自然科学基金资助项目（31660101）；国家自然科学基金资助项目（30960464）