组蛋白去乙酰化酶SIRT1调控氧化低密度脂蛋白诱导巨噬细胞凋亡的表达

doi:10.6040/j.issn.1671-7554.0.2021.0449

摘要/Abstract

摘要： 目的观察组蛋白H3赖氨酸残基9乙酰化(H3K9Ac)在氧化低密度脂蛋白(oxLDL)诱导的巨噬细胞凋亡模型中的表达,探讨组蛋白去乙酰化酶——炎症因子沉默信息调节蛋白1(SIRT1)对组蛋白乙酰化的影响,及其通过基因表观遗传学作用,经氧化物酶体增殖物激活受体γ(PPARγ)通路调控巨噬细胞凋亡的机制。方法培养BALB/c小鼠单核巨噬细胞(RAW264.7),并加入oxLDL构建巨噬细胞模型。将细胞分为对照组(加入双蒸水)和实验组(加入50 μg/mL oxLDL),分别检测两组细胞凋亡及白细胞介素(IL-6)、SIRT1、H3K9Ac和PPARγ的蛋白表达水平。另外,将实验组细胞分别给予SIRT1兴奋剂(白藜芦醇,终浓度50 nmoL/L)和SIRT1抑制剂(尼克酰胺,终浓度50 nmoL/L),观察SIRT1过表达或抑制对oxLDL诱导巨噬细胞模型中细胞凋亡及SIRT1、H3K9Ac、PPARγ和磷酸化过氧化物酶体增殖物激活受体γ(Ser112位点)［pPPARγ(S112)］的蛋白表达水平的影响。采用Hoechst荧光凋亡染色法检测各组细胞凋亡;采用Western blotting法检测各组细胞IL-6、SIRT1、H3K9Ac、PPARγ和pPPARγ(S112)的蛋白表达。结果 (1)实验组细胞凋亡数(84.88±5.89)高于对照组(7.13±3.31)(P<0.01)。实验组IL-6蛋白相对表达水平(0.50±0.01)高于对照组(0.20±0.02)(P<0.01)。实验组SIRT1蛋白相对表达水平(0.20±0.01)低于对照组(0.30±0.02)(P<0.01)。实验组H3K9Ac蛋白相对表达水平(0.32±0.02)高于对照组(0.22±0.02)(P<0.01)。实验组PPARγ蛋白相对表达水平(0.11±0.02)低于对照组(0.20±0.03)(P<0.01)。(2)SIRT1兴奋剂组细胞凋亡数(28.63±6.44)低于实验组(84.88±5.89)(P<0.01);SIRT1抑制剂组细胞凋亡数(266.88±35.10)高于实验组(84.88±5.89)(P<0.01)。SIRT1兴奋剂组SIRT1蛋白相对表达水平(0.27±0.03)高于实验组(0.20±0.01)(P<0.01);SIRT1抑制剂组SIRT1蛋白相对表达水平(0.10±0.01)低于实验组(0.20±0.01)(P<0.01)。SIRT1兴奋剂组H3K9Ac蛋白相对表达水平(0.21±0.02)低于实验组(0.32±0.02)(P<0.01);SIRT1抑制剂组H3K9Ac蛋白相对表达水平(0.56±0.01)高于实验组(0.32±0.02)(P<0.01)。SIRT1兴奋剂组PPARγ蛋白相对表达水平(0.20±0.02)高于实验组(0.11±0.02)(P<0.01);SIRT1抑制剂组PPARγ蛋白相对表达水平(0.06±0.01)低于实验组(0.11±0.02)(P<0.01)。SIRT1兴奋剂组pPPARγ(S112)蛋白相对表达水平(0.04±0.00)低于实验组(0.12±0.02)(P<0.01);SIRT1抑制剂组pPPARγ(S112)蛋白相对表达水平(0.18±0.03)高于实验组(0.12±0.02)(P<0.01)。结论组蛋白乙酰化修饰异常的基因表观遗传学参与oxLDL暴露巨噬细胞凋亡的发生发展。在oxLDL诱导的巨噬细胞凋亡模型中,组蛋白去乙酰化酶SIRT1表达减少,使H3K9Ac呈高水平表达,而下游PPARγ呈低水平表达且PPARγ磷酸化表达增加。上调SIRT1可逆转上述因子表达,改善巨噬细胞凋亡。SIRT1对PPARγ存在正向调控作用,具有抗炎和抗凋亡作用,而这种作用不仅与组蛋白在基因转录水平调控PPARγ表达有关,还与其影响PPARγ翻译后磷酸化修饰相关。

关键词: 基因表观遗传学, 组蛋白, 组蛋白去乙酰化酶, 乙酰化修饰, 氧化物酶体增殖物激活受体γ, 凋亡

Abstract: Objective To observe the expression of histone H3 lysine residue 9 acetylation(H3K9Ac)in macrophage apoptosis model induced by oxidized low density lipoprotein(oxLDL), and to explore the controlling mechanism of histone deacetylase-inflammatory factor silencing information regulating protein1(SIRT1)on macrophage apoptosis by gene epigenetics, which was realized by peroxisome proliferator activated receptor γ(PPARγ)signaling pathway. Methods Mouse BALB/c macrophage cell line RAW264.7 was cultured with 50 μg/mL oxLDL. The cells were divided into control group(treated with double distilled water)and experimental group(treated with 50 μg/mL oxLDL). The number of apoptotic cells and protein expressions of interleukin(IL-6), SIRT1, H3K9Ac and PPARγ were detected. In addition, the experimental group was treated with SIRT1 stimulant(resveratrol, final concentration 50 nmoL/L)and SIRT1 inhibitor(nicotinamide, final concentration 50 nmoL/L). The apoptosis and protein expressions of SIRT1, H3K9Ac, PPARγ and phosphorylated peroxisome proliferator-activated receptor γ(Ser112 site)［pPPARγ(S112)］ after SIRT1 overexpression or inhibition were detected. The cell apoptosis was detected with Hoechst fluorescence apoptosis staining. The protein expressions of IL-6, SIRT1, H3K9Ac, PPARγ and pPPARγ(S112)were detected with Western blotting. Results The number of apoptotic cells in the experimental group was higher than that in the control group ［(84.88±5.89)vs(7.13±3.31), P<0.01］. The relative protein expressions of IL-6 and H3K9Ac in the experimental group were higher than those in the control group ［(0.50±0.01)vs(0.20±0.02),(0.32±0.02)vs(0.20±0.03), P<0.01］, while the relative protein expressions of SIRT1 and PPARγ in the experimental group were lower than those in the control group ［(0.20±0.01)vs(0.30±0.02),(0.11±0.02)vs(0.20±0.03), P<0.01］. The number of apoptotic cells in the SIRT1 stimulant group was lower than that in the experimental group ［(28.63±6.44)vs(84.88±5.89), P<0.01］, while the number of apoptotic cells in the SIRT1 inhibitor group was higher than that in the experimental group ［(266.88±35.10)vs(84.88±5.89), P<0.01］. In the SIRT1 stimulant group, the relative protein expressions of SIRT1 and PPARγ were higher than those in the experimental group ［(0.27±0.03)vs(0.20±0.01),(0.20±0.02)vs.(0.11±0.02), P<0.01］, while the expressions of H3K9Ac and pPPARγ(S112)were lower ［(0.21±0.02)vs(0.32±0.02),(0.04±0.00)vs(0.12±0.02), P<0.01］. In the SIRT1 inhibitor group, the relative expressions of SIRT1 and PPARγ were lower than those in the experimental group ［(0.10±0.01)vs(0.20±0.01),(0.06±0.01)vs(0.11±0.02), P<0.01］, while the expressions of H3K9Ac and pPPARγ(S112)were higher ［(0.56±0.01)vs(0.32±0.02),(0.18±0.03)vs(0.12±0.02), P<0.01］. Conclusion The gene epigenetics with abnormal histone acetylation modification is involved in the occurrence and development of macrophages apoptosis exposed to oxLDL. In oxLDL-induced macrophage apoptosis model, the expression of histone deacetylase SIRT1 decreases, resulting in a high expression of H3K9Ac, while the downstream PPARγ expressed at a low level and the expression of PPARγ phosphorylation increases. Up-regulation of SIRT1 can reverse the expression of those factors, and improve macrophage apoptosis. SIRT1 has positive regulation on PPARγ signal channel with the anti-inflammatory and anti-apoptosis effects, which are not only related to histone regulating PPARγ expression at gene transcription level, but also to the effects on PPARγ phosphorylation modification after post-translational.

Key words: Gene epigenetics, Histone, Histone deacetylation, Acetylation modification, Peroxisome proliferator activated receptor γ, Apoptosis

中图分类号:

R331.3

李卉,姜朝阳,刘岩,张曼. 组蛋白去乙酰化酶SIRT1调控氧化低密度脂蛋白诱导巨噬细胞凋亡的表达[J]. 山东大学学报 (医学版), 2022, 60(1): 6-12.

LI Hui, JIANG Chaoyang, LIU Yan, ZHANG Man. Effects and mechanism of histone deacetylase SIRT1 controlled macrophage apoptosis induced by oxidized low density lipoprotein[J]. Journal of Shandong University (Health Sciences), 2022, 60(1): 6-12.

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