小胶质细胞组蛋白去乙酰化酶3在低压低氧诱导的氧化应激中的作用

doi:10.3969/j.issn.1006-7795.2022.01.016

首都医科大学学报 ›› 2022, Vol. 43 ›› Issue (1): 91-98.doi: 10.3969/j.issn.1006-7795.2022.01.016

小胶质细胞组蛋白去乙酰化酶3在低压低氧诱导的氧化应激中的作用

李珺^1,2, 李硕硕², 彭志鑫³, 廖亚金⁴, 程金波^4*, 袁增强^1,2*

1.首都医科大学脑重大疾病研究院,北京 100069;
2.军事医学研究院军事认知与脑科学研究所,北京 100850;
3.南华大学衡阳医学院,湖南衡阳 421001;
4.中央民族大学生命与环境科学学院转化神经科学中心,北京 100081

收稿日期:2021-02-09 出版日期:2022-02-21 发布日期:2022-01-27
基金资助:
国家自然科学基金(81930029,81630026)。

Role of microglial histone deacetylases 3 in hypobaric hypoxia-induced oxidative stress

Li Jun^1,2, Li Shuoshuo², Peng Zhixin³, Liao Yajin⁴, Cheng Jinbo^4*, Yuan Zengqiang^1,2*

1. Beijing Institute for Brain Disorders, Capital Medical University, Beijing 100069, China;
2. The Brain Science Center, Beijing Institute of Basic Medical Sciences, Beijing 100850, China;
3. Institute of Neuroscience, Hengyang Medical College,University of South China, Hengyang 421001, Hunan Province, China;
4. Center on Translational Neuroscience, College of Life and Environmental Science, Minzu University of China,Beijing 100081, China

Received:2021-02-09 Online:2022-02-21 Published:2022-01-27
Contact: * E-mail:zyuan620@yahoo.com;zqyuan@bmi.ac.cn
Supported by:
National Natural Science Foundation of China(81930029,81630026).

摘要/Abstract

摘要： 目的探究低压低氧对脑内氧化应激的影响及小胶质细胞HDAC3在其中的作用。方法将野生型小鼠置于模拟海拔高度6 000米的低压舱中连续饲养7 d,随后取小鼠海马组织并提取RNA,实时定量聚合酶链反应(real-time polymerase chain reaction,real-time PCR)检测氧化应激标志物诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和超氧化物歧化酶(superoxidase dismutase,SOD)的mRNA,以及缺氧诱导因子-1α(hypoxia inducible factors-1α, HIF-1α)和组蛋白去乙酰化酶3(histone deacetylases 3,HDAC3)的mRNA。利用低氧培养箱[0.1%(体积分数)O₂]对小胶质细胞系BV2细胞进行低氧处理,蛋白质免疫印迹和real-time PCR法检测iNOS、SOD、HDAC3和HIF-1α的蛋白及mRNA水平,以及利用活性氧(reactive oxygen species, ROS)检测试剂盒和流式细胞术检测氧化应激水平。然后敲低或抑制小胶质细胞系HDAC3表达或活性,并进行低氧处理,检测上述相关因子及ROS的变化。最后,利用小胶质细胞特异性敲除HDAC3的小鼠进行低压低氧[0.1%(体积分数)O₂]处理,检测干预小胶质细胞HDAC3表达之后iNOS的表达水平。结果体内实验结果显示低压低氧处理显著增加小鼠脑内iNOS和SOD的mRNA;体外实验结果显示低氧处理导致BV2细胞iNOS、HDAC3和HIF-1α的蛋白水平升高,且iNOS和SOD的mRNA也增加。流式分析结果显示低氧处理导致BV2细胞ROS水平上升。敲低或抑制HDAC3则可以显著抑制低氧诱导的ROS产生和iNOS上调。特异性敲除小胶质细胞HDAC3可以显著抑制低压低氧处理导致的小鼠脑组织中iNOS上调。结论低压低氧可引起脑组织及小胶质细胞氧化应激水平升高,抑制小胶质细胞HDAC3可减轻低压低氧诱导的氧化应激。

关键词: 低氧, 缺氧, 氧化应激, 小胶质细胞, 组蛋白去乙酰化酶3

Abstract: Objective To investigate the effect of hypobaric hypoxiaon the brain tissue oxidative stress level and whether microglial HDAC3 is involved in this process. Methods First, wild type mice were treated in a hypobaric chamber at a simulated altitude of 6 000 meters for 7 days.The mRNA levels of hypoxia inducible factors-1α (HIF-1α), inducible nitric oxide synthase(iNOS), superoxidase dismutase(SOD) and histone deacetylases 3 (HDAC3) in the hippocampus were analyzed by real-time quantitative polymerase chain reaction (real-time PCR). Then, BV2 cells were treated in atmosphere with 0.1% O₂ in a hypoxia incubator and the mRNA and protein levels of iNOS, SOD, HDAC3 and HIF-1α were determined by Western blotting and real-time PCR.The levels of reactive oxygen species (ROS) were analyzed by flow cytometry.Furthermore, HDAC3-silenced and wild type BV2 cells were cultured in 0.1% O₂ atmosphere and the levels of ROS, iNOS and SOD were analyzed as above. Last, microglial HDAC3 conditional knockout mice and wild type mice were treated in hypoxia atmosphere, and the mRNA level of iNOS were analyzed by real-time PCR. Results The mRNA levels of iNOS, SOD and HDAC3 were upregulated in the hippocampus from mice underwent hypobaric hypoxia.The in vitro experiments indicated that the protein levels of iNOS, HDAC3 and HIF-1α were increased in BV2 cells treated 0.1% O₂, and the mRNA levels of iNOS and SOD were increased as well. By flow cytometry, we found the level of ROS was increased when BV2 cells were cultured in hypoxia atmosphere. Inhibition of HDAC3 significantly reduced the production of hypoxia-induced ROS, as well as decreased expression of SOD and iNOS. In addition, knockout of HDAC3 in microglia inhibited the hypoxia-induced upregulation iNOS. Conclusion The hypoxia treatment resulted in increased oxidative stress in the hippocampus and microglia, and inhibition of microglial HDAC3 ameliorated hypoxia-induced oxidative stress.

Key words: hypoxia, anoxia, oxidative stress, microglia, histone deacetylases 3

中图分类号:

李珺, 李硕硕, 彭志鑫, 廖亚金, 程金波, 袁增强. 小胶质细胞组蛋白去乙酰化酶3在低压低氧诱导的氧化应激中的作用[J]. 首都医科大学学报, 2022, 43(1): 91-98.

Li Jun, Li Shuoshuo, Peng Zhixin, Liao Yajin, Cheng Jinbo, Yuan Zengqiang. Role of microglial histone deacetylases 3 in hypobaric hypoxia-induced oxidative stress[J]. Journal of Capital Medical University, 2022, 43(1): 91-98.

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小胶质细胞组蛋白去乙酰化酶3在低压低氧诱导的氧化应激中的作用

Role of microglial histone deacetylases 3 in hypobaric hypoxia-induced oxidative stress

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