中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (12): 2362-2368.doi: 10.4103/1673-5374.285003

• 原著:脑损伤修复保护与再生 • 上一篇    

DNA甲基转移酶保护低氧预适应小鼠海马神经元的作用机制

  

  • 出版日期:2020-12-15 发布日期:2020-08-05
  • 基金资助:
    国家自然科学基金(81460283，81660307，31860307)；中国内蒙古自治区科学基金(2018LH08078，2018LH03029); 内蒙古自治区高校青年科技人才(NJYT-18-B26)；中国包头医学院科学研究基金(BYJJ-YF 201717，BYJJ-YF 201606)；国家重点研究发展计划号(2017YFC1308405)

Neuroprotective mechanisms of DNA methyltransferase in a mouse hippocampal neuronal cell line after hypoxic preconditioning

Na Liu 1, 2, 3, Xiao-Lu Zhang 1, 2, 3, Shu-Yuan Jiang 1, 2 , Jing-Hua Shi 1, 2 , Jun-He Cui 1, 2 , Xiao-Lei Liu 1, 2 , Li-Hong Han 2 , Ke-Rui Gong 4 , Shao-Chun Yan 1, 2 , Wei Xie 1, 2, 3, Chun-Yang Zhang 5 , Guo Shao 1, 2, 3, 5   

  1. 1 Inner Mongolia Key Laboratory of Hypoxic Translational Medicine, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
    2 Biomedicine Research Center, Basic Medical College and Baotou Medical College of Neuroscience Institute, Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
    3 Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China
    4 Department of Oral and Maxillofacial Surgery, University of California San Francsico, San Francisco, CA, USA
    5 Department of Neurosurgery, the First Affiliated Hospital of Baotou Medical College, Baotou, Inner Mongolia Autonomous Region, China
  • Online:2020-12-15 Published:2020-08-05
  • Contact: Guo Shao, MD,shao_guo_china@163.com; Wei Xie, PhD, xiewei@tmu.edu.cn.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, Nos. 81460283 (to GS), 81660307 (to GS), 31860307 (to WX); the Science Foundation of Inner Mongolia Autonomous Region of China, Nos. 2018LH08078 (to GS), 2018LH03029 (to JHS); the Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region of China, No. NJYT-18-B26 (to WX); the Scientific Research Foundation of Baotou Medical College of China, Nos. BYJJ-YF 201717 (to SCY), BYJJ-YF 201606 (to WX); the National Key Research and Development Program of China, No. 2017YFC1308405 (to GS).

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摘要：

课题组以往研究表明，低氧预适应能够提升小鼠的低氧耐受能力，并且伴有脑组织中DNA甲基转移酶(DNMTs)表达和活性降低的趋势。但没有揭示DNMTs在低氧预适应的多种神经保护作用中的机制。实验旨在通过培养小鼠海马神经元HT22细胞系，建立低氧预适应体外模型，更全面的揭示DNMTs在低氧预适应此内源性神经保护作用中的机制。(1)实验将不进行低氧暴露的HT22细胞设为对照组，将低氧暴露1次的HT22细胞设为低氧组，将重复低氧暴露4次的HT22细胞设为低氧预适应组；(2)采用MTS法测定低氧预适应对HT22细胞低氧耐受能力的影响发现，与低氧组相比，低氧预适应组的细胞存活率明显提升；(3)Western blot和流式细胞术检测低氧预适应对HT22细胞周期变化和细胞凋亡的影响发现，与低氧组相比，低氧预适应组中caspase-3和spectrin蛋白表达、细胞周期S期比例和早期细胞凋亡率均明显减少；(4)使用ELISA、实时荧光定量PCR和Western blot方法检测DNMTs的表达和活性，以明确DNMTs在低氧预适应中的变化情况发现，与对照组相比，低氧预适应下调了HT22细胞中DNMT3A和DNMT3B的mRNA和蛋白表达，并降低了总DNMTs和DNMT3B的活性；(5)这些数据说明，低氧预适应对海马神经元具有多种神经保护作用，表现在抗缺氧、维持HT22细胞活力和抑制细胞凋亡方面，其神经保护机制可能与DNMT 3A和DNMT 3B的表达被抑制有关。

orcid:

0000-0003-3766-5076 (Guo Shao)
0000-0001-6744-4827 (Wei Xie)

Abstract: Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice, accompanied by the downregulation of DNA methyltrans- ferases (DNMTs) in the brain. However, the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood. This study aimed to establish an in vitro model of hypoxic preconditioning, using a cultured mouse hippocampal neuronal cell line (HT22 cells), to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning. HT22 cells were divided into a control group, which received no exposure to hypoxia, a hypoxia group, which was exposed to hypoxia once, and a hypoxic preconditioning group, which was exposed to four cycles of hypoxia. To test the ability of hypoxic preadaptation to induce hypoxic tolerance, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-car- boxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group. The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by west- ern blot assay and flow cytometry. Compared with the hypoxia group, the expression levels of caspase-3 and spectrin, which are markers of early apoptosis and S-phase arrest, respectively, noticeably reduced in the hypoxic preconditioning group. Finally, enzyme-linked im- munosorbent assay, real-time polymerase chain reaction, and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning. The results showed that compared with the control group, hypoxic preconditioning down- regulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B. In conclusion, hypoxic preconditioning may exert anti-hypoxic neuroprotective effects, maintaining HT22 cell viability and inhibiting cell apoptosis. These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

Key words: caspase-3, cells, growth, injury, plasticity, recovery, regeneration, repair