Zhang C, Zhang Q, Zhang J H, Wang F, Zhang J P
Department of Plastic Surgery, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China.
State Key Laboratory of Trauma, Burns, and Combined Injury, Institute of Burn Research, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China.
Zhonghua Shao Shang Za Zhi. 2021 Sep 20;37(9):853-859. doi: 10.3760/cma.j.cn501120-20200519-00274.
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). The experimental research method was used. HSFs in logarithmic growth phase were taken and divided into negative control group, 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/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. After 24 h of culture, CCK-8 method and EdU staining showed that compared with negative control group, the cell proliferation activities in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were significantly decreased (<0.01). After 24 h of culture, CCK-8 method showed that compared with 1 μmol/L Tubastatin A group, the cell proliferation activity in 10 μmol/L Tubastatin A group was significantly decreased (<0.05); EdU staining showed that compared with 1 μmol/L Tubastatin A group, the cell proliferation activities in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group were significantly decreased (<0.05 or <0.01). Within 3 h of observation, the ranges of cell motion in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/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 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group (<0.01); the curve movement velocity of cells in 1 μmol/L Tubastatin A group was obviously faster than those in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group (<0.01); the curve movement velocity of cells in 5 μmol/L Tubastatin A group was obviously faster than that in 10 μmol/L Tubastatin A group (<0.05). After 24 h of culture, compared with negative control group, the activities of ERK1/2 of cells in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were decreased significantly (<0.01); compared with 1 μmol/L Tubastatin A group, the activities of ERK1/2 of cells in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group were decreased significantly (<0.01); compared with 5 μmol/L Tubastatin A group, the activity of ERK1/2 of cells in 10 μmol/L Tubastatin A group was decreased significantly (<0.05). HDAC6 inhibitor Tubastatin A may mediate the inhibitory effect on proliferation and movement of HSFs by inhibiting the activity of ERK1/2.
探讨组蛋白去乙酰化酶6（HDAC6）抑制剂Tubastatin A对人皮肤成纤维细胞（HSFs）增殖和迁移的影响及其可能的分子机制。采用实验研究方法。取对数生长期的HSFs，根据随机数字表分为阴性对照组、1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组。阴性对照组的HSFs加入终体积分数为0.1%二甲基亚砜的杜氏改良 Eagle培养基（以下简称完全培养基），其他三组加入含相应终摩尔浓度Tubastatin A的完全培养基。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'-脱氧尿苷（EdU）染色检测细胞增殖活性；在活细胞工作站下观察细胞3 h内的运动范围，并计算细胞的曲线运动速度。采用蛋白质免疫印迹法检测细胞外信号调节激酶1/2（ERK1/2）和磷酸化ERK1/2（p-ERK1/2）的蛋白表达，并计算p-ERK1/2与ERK1/2的比值以代表ERK1/2的活性。CCK-8法检测细胞增殖活性的样本数为6，其他实验的样本数为3。数据采用单因素方差分析和最小显著差法进行统计学分析。培养24 h后，CCK-8法和EdU染色显示，与阴性对照组相比，1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组的细胞增殖活性均显著降低（<0.01）。培养24 h后，CCK-8法显示，与1 μmol/L Tubastatin A组相比，10 μmol/L Tubastatin A组的细胞增殖活性显著降低（<0.05）；EdU染色显示，与1 μmol/L Tubastatin A组相比，5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组的细胞增殖活性均显著降低（<0.05或<0.01）。观察3 h内，1 μmol/L Tubastatin A组、� μmol/L Tubastatin A组和円 μmol/L Tubastatin A组的细胞运动范围与阴性对照组相比明显减小。观察3 h内，阴性对照组细胞的曲线运动速度为（0.780±0.028）μm/min，明显快于1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组的（0.594±0.023）、（0.469±0.028）和（0.391±0.021）μm/min（<0.01）；1 μmol/L Tubastatin A组细胞的曲线运动速度明显快于5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组（<0.01）；5 μmol/L Tubastatin A组细胞的曲线运动速度明显快于10 μmol/L Tubastatin A组（<0.05）。培养24 h后，与阴性对照组相比，1 μmol/L Tubastatin A组、5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组细胞的ERK1/2活性均显著降低（<0.01）；与1 μmol/L Tubastatin A组相比，5 μmol/L Tubastatin A组和10 μmol/L Tubastatin A组细胞的ERK1/2活性均显著降低（<0.01）；与5 μmol/L Tubastatin A组相比，10 μmol/L Tubastatin A组细胞的ERK1/2活性显著降低（<0.05）。HDAC6抑制剂Tubastatin A可能通过抑制ERK1/2的活性介导对HSFs增殖和迁移的抑制作用。
