Neural Regeneration Research ›› 2020, Vol. 15 ›› Issue (12): 2278-2285.doi: 10.4103/1673-5374.285004

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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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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