5-氮杂-2'-脱氧胞苷通过启动 TSC1/mTOR 通路增加小鼠神经元细胞缺氧耐受依赖性自噬。
5-Aza-2'-deoxycytidine increases hypoxia tolerance-dependent autophagy in mouse neuronal cells by initiating the TSC1/mTOR pathway.
机构信息
Department of Neurobiology and Center of Stroke, Beijing Institute for Brain Disorders, School of Basic Medical Science, Capital Medical University, Beijing, China; Inner Mongolia Key laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, China; Beijing key laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China.
Inner Mongolia Key laboratory of Hypoxic Translational Medicine, Baotou Medical College, Inner Mongolia, China; Beijing key laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China.
出版信息
Biomed Pharmacother. 2019 Oct;118:109219. doi: 10.1016/j.biopha.2019.109219. Epub 2019 Jul 17.
BACKGROUND
Our previous study found that 5-Aza-2'-deoxycytidine (5-Aza-CdR) can repress the expression and activity of protein serine/threonine phosphatase-1γ (PP1γ) in mouse hippocampus. It is well known that PP1γ regulates cell metabolism, which is related to hypoxia/ischaemia tolerance. It has been reported that it can also induce autophagy in cancer cells. Autophagy is important for maintaining cellular homeostasis associated with metabolism. In this study, we examined whether 5-Aza-CdR increases hypoxia tolerance-dependent autophagy by initiating the TSC1/mTOR/autophagy signalling pathway in neuronal cells.
METHODS
5-Aza-CdR was either administered to mice via intracerebroventricular injection (i.c.v) or added to cultured hippocampal-derived neuronal cell line (HT22 cell) in the medium for cell culture. The hypoxia tolerance of mice was measured by hypoxia tolerance time and Perl's iron stain. The mRNA and protein expression levels of tuberous sclerosis complex 1 (TSC1), mammalian target of rapamycin (mTOR) and autophagy marker light chain 3 (LC3) were measured by real-time PCR and western blot. The p-mTOR and p-p70S6k proteins were used as markers for mTOR activity. In addition, the role of autophagy was determined by correlating its intensity with hypoxia tolerance in a time-dependent manner. At the same time, the involvement of the TSC1/mTOR pathway in autophagy was also examined through transfection with TSC1 (hamartin) plasmid.
RESULTS
5-Aza-CdR was revealed to increase hypoxia tolerance and induce autophagy, accompanied by an increase in mRNA and protein expression levels of TSC1, reduction in p-mTOR (Ser2448) and p-p70S6k (Thr389) protein levels, and an increase in the ratio of LC3-II/LC3-I in both mouse hippocampus and hippocampal-derived neuronal cell line (HT22). The fluorescence intensity of hamartin was enhanced in the hippocampus of mice exposed to 5-Aza-CdR. Moreover, HT22 cells that over-expressed TSC1 showed more autophagy.
CONCLUSIONS
5-Aza-CdR can increase hypoxia tolerance by inducing autophagy by initiating the TSC1/mTOR pathway.
背景
我们之前的研究发现，5-氮杂-2′-脱氧胞苷（5-Aza-CdR）可以抑制小鼠海马体中蛋白丝氨酸/苏氨酸磷酸酶-1γ（PP1γ）的表达和活性。众所周知，PP1γ 调节细胞代谢，与缺氧/缺血耐受有关。据报道，它还可以诱导癌细胞自噬。自噬对于维持与代谢相关的细胞内稳态很重要。在这项研究中，我们通过启动 TSC1/mTOR/自噬信号通路，研究了 5-Aza-CdR 是否通过增加神经元细胞中的自噬来提高缺氧耐受。
方法
通过脑室内注射（i.c.v）将 5-Aza-CdR 给予小鼠，或在细胞培养液中添加培养的海马源性神经元细胞系（HT22 细胞）。通过缺氧耐受时间和 Perl 铁染色测量小鼠的缺氧耐受能力。通过实时 PCR 和 Western blot 测量结节性硬化复合物 1（TSC1）、雷帕霉素靶蛋白（mTOR）和自噬标志物微管相关蛋白轻链 3（LC3）的 mRNA 和蛋白表达水平。p-mTOR 和 p-p70S6k 蛋白被用作 mTOR 活性的标志物。此外，通过与时间相关的方式将其强度与缺氧耐受相关联，确定自噬的作用。同时，还通过转染 TSC1（错构瘤蛋白）质粒研究 TSC1/mTOR 通路在自噬中的作用。
结果
5-Aza-CdR 增加了缺氧耐受能力并诱导了自噬，同时增加了 TSC1 的 mRNA 和蛋白表达水平，降低了 p-mTOR（Ser2448）和 p-p70S6k（Thr389）蛋白水平，以及 LC3-II/LC3-I 的比值在小鼠海马体和海马源性神经元细胞系（HT22）中均升高。在暴露于 5-Aza-CdR 的小鼠海马体中，错构瘤蛋白的荧光强度增强。此外，过表达 TSC1 的 HT22 细胞表现出更多的自噬。
结论
5-Aza-CdR 可以通过启动 TSC1/mTOR 通路诱导自噬，从而增加缺氧耐受能力。
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