State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shanxi Key Laboratory of Stomatology, Department of Operative Dentistry and Endodontics, School of Stomatology, The Fourth Military Medical University, Xi'an, China.
Department of Operative Dentistry and Endodontics, School of Stomatology, The Guizhou Medical University, Guiyang, China.
Int Endod J. 2018 Jul;51(7):767-778. doi: 10.1111/iej.12779. Epub 2017 May 10.
To investigate the growth, migratory and adhesive effects of trichostatin A (TSA) and valproic acid (VPA), two histone deacetylase inhibitors (HDACis), on human dental pulp stem cells (hDPSCs).
To verify that TSA or VPA functions as an HDAC inhibitor, the expressions of histones H3 and H4 were examined using Western blotting analysis. hDPSC growth and metabolic activity was evaluated by MTT viability analysis at different time-points and by cell count experiments. The expression of cell cycle regulatory proteins and apoptosis-associated proteins was examined by Western blot analysis. Migration effects were investigated using wound healing and transwell migration assays. An adhesion assay was also performed in the presence and absence of HDACis. The levels of chemokines and adhesion molecules relevant to repair in hDPSCs were also assessed by qRT-PCR and Western blot analysis. The data were analysed, where appropriate, using Student's t-test or one-way anova followed by the Student-Newman-Keuls test using SPSS software.
Trichostatin A and VPA enhanced acetylation of histones H3 and H4 (P < 0.05). Significant increases (P < 0.05) in MTT levels in hDPSCs were observed after treatment with TSA (2 and 20 nmol L ) or VPA (1 and 10 mmol L ). Cell numbers were not significantly affected at the concentration of TSA (0.2-10 nmol L ) or VPA (0.01-100 mmol L ) applied compared with the control at 3, 5 or 7 days (P > 0.05). At the same time, the expression of Cdx2 and cyclin A was upregulated by 2 nmol L TSA and 1 mmol L VPA (P < 0.05). Higher TSA or VPA concentrations induced apoptosis in hDPSCs in the cell count and apoptosis experiments (P < 0.05). Moreover, TSA and VPA significantly depressed the expression of Cdx2 and cyclin A (P < 0.05), whilst it significantly improved the level of p21 (P < 0.05). TSA and VPA promoted migration and adhesion of hDPSCs (P < 0.05). The levels of chemokines and adhesion molecules were significantly upregulated after exposure of hDPSCs to 20 nmol L TSA or 1 mmol L VPA (P < 0.05).
Histone deacetylase inhibitors at specific concentrations promoted proliferation, migration and adhesion of hDPSCs, which may contribute to novel regenerative therapies for pulpal disease treatment.
研究两种组蛋白去乙酰化酶抑制剂（HDACi）曲古抑菌素 A（TSA）和丙戊酸（VPA）对人牙髓干细胞（hDPSCs）的生长、迁移和黏附作用。
通过 Western blot 分析检测组蛋白 H3 和 H4 的表达，以验证 TSA 或 VPA 是否具有 HDAC 抑制作用。采用 MTT 活力分析和细胞计数实验分别在不同时间点和细胞计数实验中评估 hDPSC 的生长和代谢活性。通过 Western blot 分析检测细胞周期调节蛋白和凋亡相关蛋白的表达。采用划痕愈合和 Transwell 迁移实验研究迁移作用。还在存在和不存在 HDACi 的情况下进行了黏附实验。通过 qRT-PCR 和 Western blot 分析评估与修复相关的趋化因子和黏附分子在 hDPSCs 中的水平。使用 SPSS 软件，适当的情况下，使用学生 t 检验或单因素方差分析，然后使用 Student-Newman-Keuls 检验对数据进行分析。
曲古抑菌素 A 和 VPA 增强了组蛋白 H3 和 H4 的乙酰化（P<0.05）。用 TSA（2 和 20 nmol/L）或 VPA（1 和 10 mmol/L）处理后，hDPSCs 的 MTT 水平显著升高（P<0.05）。与对照组相比，在 TSA（0.2-10 nmol/L）或 VPA（0.01-100 mmol/L）的浓度下，细胞数量在 3、5 或 7 天时没有明显变化（P>0.05）。同时，2 nmol/L TSA 和 1 mmol/L VPA 上调了 Cdx2 和细胞周期蛋白 A 的表达（P<0.05）。较高浓度的 TSA 或 VPA 诱导细胞计数和细胞凋亡实验中的 hDPSC 凋亡（P<0.05）。此外，TSA 和 VPA 显著下调 Cdx2 和细胞周期蛋白 A 的表达（P<0.05），同时显著上调 p21 的水平（P<0.05）。TSA 和 VPA 促进了 hDPSCs 的迁移和黏附（P<0.05）。hDPSCs 暴露于 20 nmol/L TSA 或 1 mmol/L VPA 后，趋化因子和黏附分子的水平显著上调（P<0.05）。
在特定浓度下，组蛋白去乙酰化酶抑制剂促进了 hDPSCs 的增殖、迁移和黏附，这可能有助于牙髓疾病治疗的新型再生疗法。
