纳米氧化钴对组蛋白H3修饰的影响及其机制

Effects of Cobalt Oxide Nanoparticles on Histone H3 Modification and Its Mechanism

纳米氧化钴因其优异的性能被广泛应用于组织工程等领域,然而,广泛的应用增加了其对人类健康和环境暴露的风险。本文在详细表征纳米氧化钴的基础上,选用人永生化角质形成细胞HaCaT为模型,采用台盼蓝染色法检测纳米氧化钴对细胞生存率的影响,利用蛋白免疫印记法观察纳米氧化钴对组蛋白H3常见修饰位点的影响,通过对细胞内蓄积量、组蛋白H3修饰水平及DNA损伤的定量分析,探究了纳米氧化钴对组蛋白H3修饰的影响机制。表征结果显示,在分散介质中纳米氧化钴明显团聚,比表面积减少,二次粒径增大。生存率测定发现,实验最大剂量(1 mg·mL^(-1))作用24 h后,细胞生存率无明显变化。0.1 mg·mL^(-1)纳米氧化钴暴露1 h后,诱导了组蛋白H3第10位丝氨酸的磷酸化(p-H3S10)、第9位赖氨酸的乙酰化(Ac-H3K9)及第4位赖氨酸的三甲基化(Me3-H3K4)的上调,并持续长达24 h。同时,观察到第14位赖氨酸的乙酰化(Ac-H3K14)在纳米氧化钴暴露4 h后明显上调。第27位赖氨酸的三甲基化(Me3-H3K27)升高4 h后出现下降趋势。定量分析表明,纳米氧化钴的细胞内蓄积是其诱导组蛋白H3修饰变化的关键因素之一,且组蛋白H3修饰可能涉及DNA损伤修复途径。本研究通过体外实验证明了纳米氧化钴对组蛋白H3修饰的影响及可能的机制,为进一步评价纳米氧化钴的生物毒性及致癌风险提供了科学依据。

Cobalt(Ⅱ,Ⅲ)oxide nanoparticles(Co_(3)O_(4)-NPs)have been widely used in tissue engineering and other fields due to their excellent properties.However,the extensive application of Co_(3)O_(4)-NPs increases the risk to human health and environmental exposure.In this paper,Co_(3)O_(4)-NPs were first characterized in detail.Subsequently,the effects of Co_(3)O_(4)-NPs on cell viability were evaluated by trypan blue assay,and the effects of Co_(3)O_(4)-NPs on histone H3 modification were observed by Western blotting using human skin keratinocytes HaCaT as model cells.The influence mechanism of Co_(3)O_(4)-NPs on histone H3 modification was explored through the quantitative analysis of intracellular accumulation,histone H3 modification,and DNA damage.After dispersion treatment,Co_(3)O_(4)-NPs showed obvious agglomeration,and their specific surface area decreased whereas the secondary particle size increased.The cell viability measurement indicated that cell viability didn’t change significantly after exposed to the maximum dose(1 mg·mL^(-1))for 24 h.The phosphorylation of histone H3 at serine 10(p-H3S10),the acetylation of histone H3 at lysine 9(Ac-H3K9)and the trimethylation of histone H3 at lysine 4(Me3-H3K4)were induced to upregulate after exposure to Co_(3)O_(4)-NPs(0.1 mg·mL^(-1))for 1 h and lasted for 24 h.Meanwhile,the acetylation of histone H3 at lysine 14(Ac-H3K14)was significantly upregulated after exposure to Co_(3)O_(4)-NPs for 4 h.In addition,the trimethylation of histone H3 at lysine 27(Me3-H3K27)increased after exposure to Co_(3)O_(4)-NPs,and then decreased after 4 h.Quantitative analysis suggested that the intracellular accumulation of Co_(3)O_(4)-NPs is one of the key factors in its induction of changes in histone H3 modification,and histone H3 modification may be involved in DNA damage repair pathways.This study demonstrated the effects of Co_(3)O_(4)-NPs on histone H3 modification and its possible mechanism through in vitro experiments.It can provide a scientific basis for further evaluation of the biological toxicity and carcinogenic risk of Co_(3)O_(4)-NPs.