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): 2278-2285.doi: 10.4103/1673-5374.285004

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

适当水平自噬可减轻乳化异氟烷诱导胚胎神经干细胞的凋亡

  

  • 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（81401279）；中国上海自然科学基金（18ZR1443100）；中国上海交通大学医学院转化医学合作创新中心（TM201729）； 2014年中国上海交通大学医学院国际和平妇幼保健医院青年人才基金；2019年上海交通大学医学院国际和平妇幼保健院“WUXIN”项目（2018-38）。

An appropriate level of autophagy reduces emulsifed isofurane-induced apoptosis in fetal neural stem cells

Ze-Yong Yang1, Lei Zhou2, Qiong Meng1, Hong Shi3 , Yuan-Hai Li2   

  1. 1 Department of Anesthesiology, International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Embryo Original Disease, Shanghai Municipal Key Clinical Specialty, Shanghai, China
    2 Department of Anesthesiology, First Afliated Hospital of Anhui Medical University, Hefei, Anhui Province, China 
    3 Department of Anesthesiology, Shanghai Pulmonary Hospital, Tongji University, Shanghai, China
      
  • Online:2020-12-15 Published:2020-08-05
  • Contact: Ze-Yong Yang, MD, PhD,zeyongy2018@aliyun.com; Yuan-Hai Li, MD, PhD,yuanhaili2014@sina.com.
  • Supported by:
    This work was financially supported by the National Natural Science Foundation of China, No. 81401279 (to ZYY); the Natural Science Foundation of Shanghai, China, No. 18ZR1443100 (to ZYY); the Innovation Center of Translational Medicine Collaboration, Shanghai Jiao Tong University School of Medicine of China, No. TM201729 (to ZYY); the Youth Talent Fund of International Peace Maternity and Child Health Hos- pital, Shanghai Jiao Tong University School of Medicine of China in 2014 (to ZYY); the “WUXIN” Project of International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine of China in 2019, No. 2018-38 (to ZYY).

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摘要：

自噬在细胞存活中起着至关重要的作用。但麻醉药诱导的发育性神经毒性过程中，自噬相关蛋白Atg5，LC3B和Beclin 1的功能和调控作用尚不清楚。实验旨在认识自噬对麻醉药乳化异氟烷(EI)诱导大鼠胚胎神经干细胞神经毒性的途径及机制。(1)实验外培养胚胎神经干细胞，以麻醉药EI干预诱发细胞神经毒性，分别观察采用自噬抑制剂3-MA和BAF进行预处理及采用siRNA-Atg5 转染后的干预效果；(2)使用MTT测定法确定细胞存活，流式细胞仪检测细胞凋亡，透射电子显微镜观察细胞超微结构，蛋白印迹分析定量自噬相关蛋白LC3B和Beclin 1，Atg5，P62和促凋亡蛋白caspase 3的表达：(3)麻醉药EI干预的胚胎神经干细胞增殖受到抑制，细胞凋亡率增加，细胞内溶酶体降解形成单层膜的自噬小体，细胞中自噬相关蛋白P62、LC3B、Beclin-1、Atg5和促凋亡蛋白caspase 3的表达上调，这些结果证实了EI可诱导胚胎神经干细胞出现毒性和自噬；(4)采用自噬抑制剂3-MA和BAF预处理的EI组中，细胞凋亡率升高，说明自噬被完全抑制后并不能减轻麻醉药EI诱导的胚胎神经干细胞毒性，而采用siRNA-Atg5 转染EI干预的细胞后，细胞中Atg5蛋白表达显著降低，此时细胞增殖受到抑制；(5)上述研究结果表明，通过调节Atg5的自噬途径，使自噬维持在适当水平，有助于抑制麻醉药EI诱导的胚胎神经干细胞毒性。

orcid: 0000-0003-1331-0836 (Ze-Yong Yang)
         0000-0003-4526-8775 (Yuan-Hai Li)

关键词:

细胞凋亡, Atg5, 自噬胚胎乳化异氟烷(EI), FNSC, LC3B, MTT, 神经变性神经毒性

Abstract: Autophagy plays essential roles in cell survival. However, the functions and regulation of the autophagy-related proteins Atg5, LC3B, and Beclin 1 during anesthetic-induced developmental neurotoxicity remain unclear. Tis study aimed to understand the autophagy pathways and mechanisms that afect neurotoxicity, induced by the anesthetic emulsifed isofurane, in rat fetal neural stem cells. Fetal neural stem cells were cultured, in vitro, and neurotoxicity was induced by emulsifed isofurane treatment. Te efects of pretreatment with the auto- phagy inhibitors 3-methyladenine and baflomycin and the efects of transfection with small interfering RNA against ATG5 (siRNA-Atg5) were observed. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was assessed using fow cytometry. Ultrastructural changes were analyzed through transmission electron microscopy. Te levels of the autophagy-related proteins LC3B, Beclin 1, Atg5, and P62 and the pro-apoptosis-related protein caspase-3 were analyzed using western blot assay. Te inhibition of cell proliferation and that of apoptosis rate increased afer treatment with emulsifed isofurane. Autophagoly- sosomes, monolayer membrane formation due to lysosomal degradation, were observed. Te autophagy-related proteins LC3B, Beclin 1, Atg5, and P62 and caspase-3 were upregulated. Tese results confrm that emulsifed isofurane can induce toxicity and autophagy in fetal neural stem cells. Pre-treatment with 3-methyladenine and baflomycin increased the apoptosis rate in emulsifed isofurane-treated fetal neural stem cells, which indicated that the complete inhibition of autophagy does not alleviate emulsifed isofurane-induced fetal neural stem cell toxicity. Atg5 expression was decreased significantly by siRNA-Atg5 transfection, and cell proliferation was inhibited. These results verify that the Atg5 autophagy pathway can be regulated to maintain appropriate levels of autophagy, which can inhibit the neuro- toxicity induced by emulsifed isofurane anesthetic in fetal neural stem cells.

Key words: apoptosis, Atg5, autophagy, emulsifed isofurane, fetal neural stem cells, LC3B, MTT, neurodegenerative, neurotoxicity