姜黄素对口腔鳞癌细胞中细胞骨架及Rho/ROCK信号通路的影响

摘要

目的:探讨姜黄素对口腔鳞癌细胞系HN13细胞骨架的影响及其相关的分子机制.方法:用考马斯亮蓝染色分析法检测不同浓度姜黄素(0、10、20、40lμmol/L)作用24h的细胞,观察姜黄素对鳞癌细胞骨架的影响.用免疫组织化学法半定量检测不同浓度姜黄素(0、10、20、40 μmol/L)作用24h后细胞内F-actin含量的变化.用Western印迹法检测不同浓度姜黄素(0、10、20、40 μmol/L)作用24 h后ROCK2及p-ROCK2蛋白的表达.结果:在低浓度姜黄素(10 μmol/L)作用24 h后细胞骨架结构收缩、染色变淡、稀疏、变细,排列方式发生变化,但尚存一部分应力纤维；随着姜黄素浓度的增加(20 μmol/L),细胞骨架变化更加明显,表现为细胞骨架含量明显减少,放射状应力纤维几乎完全消失;当姜黄素浓度为40 μmol/L时,细胞骨架轮廓已不清晰,染色更淡,减少更加明显,并有断裂,多数细胞变形明显.不同浓度姜黄素干预细胞24 h后,细胞F-actin含量减少,各组间差异有统计学意义(P＜0.01).Western印迹法检测结果显示不同浓度姜黄素(0、10、20、40 μmol/L)作用24h后细胞内ROCK2及p-ROCK2蛋白的表达显著下调.结论:姜黄素可诱导鳞癌细胞骨架改变,其作用机制与抑制Rho/ROCK信号通路的效应分子ROCK2及p-ROCK2活化有关.%Objective:The present article aimed to study whether the effects of curcumin are related to cytoskeleton variation in OSCC cell line HN13 and its mechanism.Methods:HN13 cells were treated and exposed with different concentrations of curcumin (0,10,20,40 μmol/L).After 24 h,Coomassie blue staining was used to measure the cystoskeleton o f HN13 cells.Immunohistochemistry was used to observe the expression of F-actin.The expression of ROCK2 and phosphated ROCK2 were detected by Western blot.Results:Qualitative morphological evaluation revealed that HN13 cell exposed to curcumin with different concentration became obvious deformation.The cytoskeletal structures shrinked,the staining became faded,stress fibers reduced.Immunohistochemistry revealed the staining of F-actin appeared somewhat weaker in HN13 cells which exposed in high concentration of curcumin.When treated for 24 h,the expression of ROCK2 and p-ROCK2 in HN13 cells was decreased.Conclusion:Curcumin may trigger alterations of the cystoskeleton of HN13 cell line by inhibiting activity of Rho/ROCK2 pathway.