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

Zhang Can; Zhang Qiong; Zhang Junhui; Wang Fan; Zhang Jiaping

doi:10.3760/cma.j.cn501120-20200519-00274

Volume 37 Issue 9

Sep. 2021

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Article Navigation > CHINESE JOURNAL OF BURNS AND WOUNDS > 2021 > 37(9): 853-859

Zhang C,Zhang Q,Zhang JH,et al.Effects and molecular mechanism of histone deacetylase 6 inhibitor Tubastatin A on the proliferation and movement of human skin fibroblasts[J].Chin J Burns,2021,37(9):853-859.DOI: 10.3760/cma.j.cn501120-20200519-00274.

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

doi: 10.3760/cma.j.cn501120-20200519-00274

1.
Department of Plastic Surgery, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China
2.
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
3.
Department of Endocrinology, the First Affiliated Hospital of Army Medical University (the Third Military Medical University), Chongqing 400038, China

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General Program of National Natural Science Foundation of China 81873936

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Corresponding author: Zhang Jiaping, Email: japzhang@aliyun.com
Received Date: 2020-05-19

Abstract

Abstract

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, 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. Results 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 (P<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 (P<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 (P<0.05 or P<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 (P<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 (P<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 (P<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 (P<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 (P<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 (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.
- Cell proliferation,
- Cell movement,
- Histone deacetylase 6,
- Tubastatin A,
- Human skin fibroblast,
- Extracellular signal-regulated kinase 1/2

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

doi: 10.3760/cma.j.cn501120-20200519-00274