过表达HDAC1通过诱导SP1去乙酰化改善类固醇诱导的小鼠骨细胞凋亡

doi:10.12122/j.issn.1673-4254.2025.01.02

摘要/Abstract

摘要：

目的探究组蛋白脱乙酰基酶1（HDAC1）通过特异性蛋白1（SP1）调控激素性股骨头坏死（SONFH）的作用机制。方法将小鼠骨样细胞MLY-O4分为对照组、曲古抑菌素（TSA）组、类固醇组、类固醇+oe-NC组、类固醇+oe-HDAC1组、oe-NC组、oe-HDAC1组。对照组细胞正常培养，不接受试剂诱导及细胞转染；TSA组细胞接受400 nmol/L TSA处理24 h；类固醇组接受1 μmol/L地塞米松诱导24 h；类固醇+oe-NC组或类固醇+oe-HDAC1组接受oe-NC或oe-HDAC1转染，通过LipoHigh 脂质体高效转染试剂行细胞转染，转染48 h后进行类固醇诱导。oe-NC组或oe-HDAC1组接受oe-NC或oe-HDAC1转染。观察各组细胞在HDAC、HDAC1蛋白、SP1蛋白、凋亡相关蛋白（裂解capase-3及Bax）、乙酰化水平、细胞增殖、细胞凋亡方面的差异，采用通过免疫共沉淀验证HDAC1与SP1的相互作用。结果类固醇组与对照组相比细胞凋亡率升高，HDAC1下调，裂解caspase-3及Bax上调，细胞增殖显著降低，差异均有统计学意义（P<0.05）。TSA组、类固醇组与对照组相比，乙酰化水平显著升高，SP1蛋白显著上调，类固醇+oc-HDAC1组与类固醇+oe-NC组相比，乙酰化水平降低，HDAC1上调，SP1、裂解caspase-3、Bax下调，细胞增殖增强，细胞凋亡降低（P<0.05）。oe-NC组及oe-HDAC1组Input样本存在HDAC1蛋白条带，表明两组样本均可表达HDAC1，两组IgG抗体上并未检测出HDAC1蛋白，而两组SP1抗体上均存在HDAC1，表明HDAC1可与SP1相互作用。结论 HDAC1通过介导去乙酰化修饰抑制SP1表达，从而抑制类固醇诱导的骨细胞凋亡，并促进骨细胞增殖。

关键词: 激素性股骨头坏死, SP1, HDAC1, 去乙酰化

Abstract:

Objective To explore the mechanism by which histone deacetylase 1 (HDAC1) regulates steroid-induced apoptosis of mouse osteocyte-like MLO-Y4 cells. Methods MLY-O4 cells were treated with 400 nmol/L trichostatin A (TSA) or 1 mmol/L dexamethasone for 24 h or transfected with a HDAC1-overexpressing vector prior to TSA or dexamethasone treatment. The changes in the expressions of HDAC1, SP1, cleaved caspase-3 and Bax, SP1 acetylation level, cell proliferation, and cell apoptosis were examined. The interaction between HDAC1 and SP1 was determined with immunoprecipitation assay and Western blotting. Results Treatment with dexamethasone significantly increased cell apoptosis, enhanced the expressions of cleaved caspase-3 and Bax, reduced HDAC1 expression, and suppressed proliferation of MLO-Y4 cells. Both TSA and dexamethasone obviously increased SP1 acetylation level and the expression of SP1 in MLO-Y4 cells. HDAC1 overexpression in the cells significantly attenuated the effect of TSA and dexamethasone, promoted cell proliferation, lowered the expressions of SP1, cleaved caspase-3 and Bax, and inhibited dexamethasone-induced cell apoptosis. Immunoprecipitation assay and Western blotting demonstrated the interaction between HDAC1 and SP1 in the cells. Conclusion HDAC1 inhibits dexamethasone-induced apoptosis and promotes proliferation of cultured mouse osteocytes by suppressing SP1 expression via promoting its deacetylation.

Key words: steroid-induced osteonecrosis femoral head, SP1, HDAC1, deacetylation

张申尧, 卢敏, 邝高艳, 许晓彤, 付俊, 曾楚然. 过表达HDAC1通过诱导SP1去乙酰化改善类固醇诱导的小鼠骨细胞凋亡[J]. 南方医科大学学报, 2025, 45(1): 10-17.

Shenyao ZHANG, Min LU, Gaoyan KUANG, Xiaotong XU, Jun FU, Churan ZENG. HDAC1 overexpression inhibits steroid-induced apoptosis of mouse osteocyte-like MLO-Y4 cells by inducing SP1 deacetylation[J]. Journal of Southern Medical University, 2025, 45(1): 10-17.

图/表 7

图1 对照组及模型组细胞凋亡比较

Fig.1 Cell apoptosis and HDAC1 expression in control and dexamethasone-treated mouse osteocyte-like MLO-Y4 cells. A: Cell apoptosis detected by flow cytometry. B: Expression levels of cleaved caspase-3, Bax and HDAC1 in the cells detected by Western blotting. **P<0.01, ***P<0.001.

表1 类固醇诱导MLO-Y4细胞转染24、48、72 h后A450 nm

Tab.1 A450 nm in MLO-Y4 cells in CCK-8 assay 24, 48 and 72 h after dexamethasone treatment (n=3, Mean±SD)

Group	Treatment time (h)			F P
Group	24	48	72	F P
Control	0.59±0.07	1.12±0.09	1.25±0.09	52.89	0.009
Steroid	0.55±0.09	0.76±0.08	0.95±0.09	27.32	0.028
t	0.501	4.299	3.308
P	0.643	0.013	0.03

图2 鉴定HDAC1过表达载体的转染效率

Fig.2 Identification of transfection efficiency of HDAC1 overexpression vector. A: qPCR for HDAC1 expression; B: western blot for HDAC1 protein. *P < 0.05, ***P<0.001.

图3 上调HDAC1抑制SONFH细胞模型的细胞凋亡

Fig.3 Up-regulation of HDAC1 inhibits apoptosis of dexamethasone-treated MLO-Y4 cells. A: Cell apoptosis detected by flow cytometry. B: Expressions of cleaved caspase-3 and Bax detected by Western blotting. *P<0.05, ***P<0.001.

表2 HDAC1过表达载体转染24、48、72 h后A450 nm (Mean±SD)

Tab.2 A450 nm at 24, 48 and 72 h after the transfection of HDAC1 overexpressing vector

Group	Treatment time (h)			F	P
Group	24	48	72	F	P
Steroid	0.56±0.07	0.79±0.09	0.96±0.11	47.72	0.016
Steroid+oe-NC	0.45±0.04	0.80±0.09	0.97±0.12	15.12	0.048
Steroid+oe-HDAC1	0.59±0.08*	1.15±0.11*	1.32±0.10*	30.63	0.031
F	2.177	8.92	7.279
P	0.195	0.016	0.025

图4 通过免疫共沉淀鉴定HDAC1与SP1之间的相互作用

Fig.4 Identification of the interaction between HDAC1 and SP1 by immunoprecipitation.

图5 HDAC1轴对蛋白乙酰化水平的调控

Fig.5 Regulation of protein acetylation levels by the HDAC1 axis. A: Cellular acetylation levels detected by immunoprecipitation assay and Western blotting. B: SP1 protein detected by Western blotting. *P<0.05, **P<0.01.

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