组蛋白脱乙酰酶6抑制剂Tubastatin A对人皮肤成纤维细胞增殖和运动的影响及其分子机制

Effects and molecular mechanism of histone deacetylase 6 inhibitor Tubastatin A on the prolifera- tion and movement of human skin fibroblasts

目的探讨组蛋白脱乙酰酶6(HDAC6)抑制剂Tubastatin A对人皮肤成纤维细胞(HSF)增殖及运动性的影响及其可能的分子机制。方法采用实验研究方法。取对数生长期HSF,按随机数字表法分为阴性对照组及1μmol/L Tubastatin A组、5μmol/L Tubastatin A组、10μmol/L Tubastatin A组。阴性对照组加入含终体积分数0.1%二甲基亚砜的DMEM培养液(以下简称完全培养液),其余3组分别加入含相应终物质的量浓度Tubastatin A的完全培养液。常规培养24 h后,采用细胞计数试剂盒8(CCK-8)法和5-乙炔基-2'-脱氧尿嘧啶核苷(EdU)染色检测细胞增殖活力;在活细胞工作站下观察细胞3 h内运动范围,计算细胞曲线运动速度;采用蛋白质印迹法检测胞外信号调节激酶1/2(ERK1/2)及磷酸化ERK1/2(p-ERK1/2)的蛋白表达量,并计算p-ERK1/2与ERK1/2比值,以此表示ERK1/2活性。CCK-8法行细胞增殖活力检测样本数为6,其余实验样本数为3。对数据行单因素方差分析及LSD检验。结果培养24 h后,CCK-8法和EdU染色显示,与阴性对照组比较,1μmol/L Tubastatin A组、5μmol/L Tubastatin A组、10μmol/L Tubastatin A组细胞增殖活力均显著下降(P<0.01)。培养24 h后,CCK-8法显示,与1μmol/L Tubastatin A组比较,10μmol/L Tubastatin A组细胞增殖活力显著下降(P<0.05);EdU染色显示,与1μmol/L Tubastatin A组比较,5μmol/L Tubastatin A组、10μmol/L Tubastatin A组细胞增殖活力显著下降(P<0.05或P<0.01)。观察3 h内,1μmol/L Tubastatin A组、5μmol/L Tubastatin A组、10μmol/L Tubastatin A组细胞运动范围较阴性对照组明显缩小。观察3 h内,阴性对照组细胞曲线运动速度为(0.780±0.028)μm/min,明显快于1μmol/L Tubastatin A组、5μmol/L Tubastatin A组、10μmol/L Tubastatin A组细胞的(0.594±0.023)、(0.469±0.028)、(0.391±0.021)μm/min(P<0.01);1μmol/L Tubastatin A组细胞曲线运动速度明显快于5μmol/L Tubastatin A组和10μmol/L Tubastatin A组(P<0.01);5μmol/L Tubastatin A组细胞曲线运动速度明显快于10μmol/L Tubastatin A组(P<0.05)。培养24 h后,与阴性对照组比较,1μmol/L Tubastatin A组、5μmol/L Tubastatin A组、10μmol/L Tubastatin A组细胞ERK1/2的活性显著下降(P<0.01);与1μmol/L Tubastatin A组比,5μmol/L Tubastatin A组和10μmol/L Tubastatin A组细胞ERK1/2的活性显著下降(P<0.01);与5μmol/L Tubastatin A组比较,10μmol/L Tubastatin A组细胞ERK1/2的活性显著下降(P<0.05)。结论HDAC6抑制剂Tubastatin A可能通过抑制ERK1/2活性,从而抑制HSF增殖及运动。

Objective To explore the effects and possible molecular mechanism of histone deacetylase 6(HDAC6)inhibitor Tubastatin A on the proliferation and movement of human skin fibroblasts(HSFs).Methods The experimental research method was used.HSFs in logarithmic growth phase were taken and divided into negative control group,1μmol/L Tubastatin A group,5μmol/L Tubastatin A group,and 10μmol/L Tubastatin A group according to the random number table.The HSFs in negative control group were added with Dulbecco′s modified eagle medium with the final volume fraction of 0.1%dimethyl sulfoxide(hereinafter referred to as the complete medium),and the other three groups were added with the complete medium with the corresponding final molarity of Tubastatin A.After 24 h of conventional culture,the cell proliferation activity was detected using cell counting kit 8(CCK-8)method and 5-ethynyl-2'-deoxyuridine(EdU)staining;the range of motion of cells within 3 h was observed under the living cell workstation,and the curve movement velocity of the cells was calculated.The protein expressions of extracellular signal-regulated kinase 1/2(ERK1/2)and phosphorylated ERK1/2(p-ERK1/2)were detected by Western blotting,and the ratio of p-ERK1/2 to ERK1/2 was calculated to represent the activity of ERK1/2.The sample number in cell proliferation activity detection with CCK-8 method was 6,while the sample numbers in other experiments were 3.Data were statistically analyzed with one-way analysis of variance and least significant difference test.Results After 24 h of culture,CCK-8 method and EdU staining showed that compared with negative control group,the cell proliferation activities in 1μmol/L Tubastatin A group,5μmol/L Tubastatin A group,and 10μmol/L Tubastatin A group were significantly decreased(P<0.01).After 24 h of culture,CCK-8 method showed that compared with 1μmol/L Tubastatin A group,the cell proliferation activity in 10μmol/L Tubastatin A group was significantly decreased(P<0.05);EdU staining showed that compared with 1μmol/L Tubastatin A group,the cell proliferation activities in 5μmol/L Tubastatin A group and 10μmol/L Tubastatin A group were significantly decreased(P<0.05 or P<0.01).Within 3 h of observation,the ranges of cell motion in 1μmol/L Tubastatin A group,5μmol/L Tubastatin A group,and 10μmol/L Tubastatin A group were obviously reduced compared with that in negative control group.Within 3 h of observation,the curve movement velocity of cells in negative control group was(0.780±0.028)μm/min,which was obviously faster than(0.594±0.023),(0.469±0.028),and(0.391±0.021)μm/min of 1μmol/L Tubastatin A group,5μmol/L Tubastatin A group,and 10μmol/L Tubastatin A group(P<0.01);the curve movement velocity of cells in 1μmol/L Tubastatin A group was obviously faster than those in 5μmol/L Tubastatin A group and 10μmol/L Tubastatin A group(P<0.01);the curve movement velocity of cells in 5μmol/L Tubastatin A group was obviously faster than that in 10μmol/L Tubastatin A group(P<0.05).After 24 h of culture,compared with negative control group,the activities of ERK1/2 of cells in 1μmol/L Tubastatin A group,5μmol/L Tubastatin A group,and 10μmol/L Tubastatin A group were decreased significantly(P<0.01);compared with 1μmol/L Tubastatin A group,the activities of ERK1/2 of cells in 5μmol/L Tubastatin A group and 10μmol/L Tubastatin A group were decreased significantly(P<0.01);compared with 5μmol/L Tubastatin A group,the activity of ERK1/2 of cells in 10μmol/L Tubastatin A group was decreased significantly(P<0.05).Conclusions HDAC6 inhibitor Tubastatin A may mediate the inhibitory effect on proliferation and movement of HSFs by inhibiting the activity of ERK1/2.