重组人源galectin-1通过调节自噬水平保护神经元
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摘要
目的制备重组人源半乳糖凝集素1(rhgalectin-1),并研究其对受损神经元自噬水平的促进作用。方法利用PCR技术构建pEGX-4T-1-galectin-1原核生物蛋白表达质粒,构建工程菌。收集rhgalectin-1蛋白,并进行纯化及纯度鉴定。用细胞毒性物质鱼藤酮(rotenone)处理神经母细胞瘤SK-N-SH细胞系,检测在有无rhgalectin-1预处理的情况下自噬标志物LC3Ⅱ以及p62的水平,分析不同组别细胞的自噬水平。结果成功制备了具有生物学活性的rhgalectin-1,发现rhgalectin-1可降低鱼藤酮对SK-N-SH细胞的毒性作用。鱼藤酮处理SK-N-SH细胞后,其自噬水平有所降低。表现为LC3Ⅱ水平明显降低以及p62水平明显增加(P<0.05)。而rhgalectin-1预处理组细胞自噬水平明显增加,LC3Ⅱ水平明显升高,p62水平明显降低(P<0.05)。当用自噬阻断剂bafilomycin阻断自噬体降解后,可观察到rhgalectin-1预处理并给予鱼藤酮刺激组较单独鱼藤酮刺激组LC3Ⅱ水平明显增加(P<0.05),此现象说明了rhgalectin-1处理的确增强了自噬水平而不是抑制自噬体的降解。结论 rhgalectin-1可以增强神经元的自噬水平,这一特性可能在神经元的毒性抵抗以及损伤后修复过程中发挥重要作用。
Objective To prepare recombinant human galectin-1(rhgalectin-1) and study its role in promoting autophagy of damaged neurons. Methods Plasmid of pEGX-4 T-1-galectin-1 was constructed by PCR and then transformed into E.coli BL21(DE3). SK-N-SH cells treated with rotenone and pretreated with or without rhgalectin-1. LC3 Ⅱ and p62 levels were tested by Western blot.Results galectin-1 reduces the toxic effects of rotenone on SK-N-SH cells. After treatment of SK-N-SH cells with rotenone, the decrease of autophagy level was shown by a decrease in LC3 Ⅱ level and an increase in p62 level. In the galectin-1 pretreatment group, the autophagy level significantly increased, the level of LC3 Ⅱ increased and the level of p62 decreased. When the autophagy blocker bafilomycin was used, the level of LC3 Ⅱ in the galectin-1 pretreatment group increased, indicating that galectin-1 promoted the autophagy level of injured SK-N-SH neurons. Conclusions rhgalectin-1 can promote the autophagy response of injured neurons,which may play an important role in the mechanism of toxicity resistance of neurons.
Objective To prepare recombinant human galectin-1(rhgalectin-1) and study its role in promoting autophagy of damaged neurons. Methods Plasmid of pEGX-4 T-1-galectin-1 was constructed by PCR and then transformed into E.coli BL21(DE3). SK-N-SH cells treated with rotenone and pretreated with or without rhgalectin-1. LC3 Ⅱ and p62 levels were tested by Western blot.Results galectin-1 reduces the toxic effects of rotenone on SK-N-SH cells. After treatment of SK-N-SH cells with rotenone, the decrease of autophagy level was shown by a decrease in LC3 Ⅱ level and an increase in p62 level. In the galectin-1 pretreatment group, the autophagy level significantly increased, the level of LC3 Ⅱ increased and the level of p62 decreased. When the autophagy blocker bafilomycin was used, the level of LC3 Ⅱ in the galectin-1 pretreatment group increased, indicating that galectin-1 promoted the autophagy level of injured SK-N-SH neurons. Conclusions rhgalectin-1 can promote the autophagy response of injured neurons,which may play an important role in the mechanism of toxicity resistance of neurons.
引文
[1] Teixeira FG, Carvalho MM, Panchalingam KM, et al. Impact of the secretome of human mesenchymal stem cells on brain structure and animal behavior in a rat model of parkinson’s disease [J]. Stem Cells Translational Med, 2017, 6: 634-646.
[2] Oh SH, Kim HN, Park HJ, et al. Mesenchymal stem cells inhibit transmission of alpha-synuclein by modulating clathrin-mediated endocytosis in a Parkinsonian model [J]. Cell Reports,2016, 14: 835-849.
[3] Echigo Y, Sugiki H, Koizumi Y, et al. Activation of RAW264.7 macrophages by oxidized galectin-1 [J]. Immunol Lett, 2010, 131: 19-23.
[4] Qu WS, Wang YH, Wang JP, et al. Galectin-1 enhances astrocytic BDNF production and improves functional outcome in rats following ischemia[J]. Neurochem Res, 2010, 35:1716-1724.
[5] Nonaka M, Fukuda M. Galectin-1 for neuroprotection? [J]. Immunity, 2012, 37: 187-189.
[6] Starossom SC, Mascanfroni ID, Imitola J, et al. Galectin-1 deactivates classically activated microglia and protects from inflammation-induced neurodegeneration [J]. Immunity, 2012, 37: 249-263.
[7] Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes [J]. Apoptosis, 2008, 13: 1-9.
[8] Kochergin IA, Zakharova MN. The role of autophagy in neurodegenerative diseases [J]. Neurochem J, 2016, 10: 7-18.
[9] Sundblad V, Morosi LG, Geffner JR, et al. Galectin-1: a Jack-of-All-Trades in the resolution of acute and chronic inflammation [J]. J Immunol, 2017, 199: 3721-3730.
[10] Menzies FM, Fleming A, Caricasole A, et al, Autophagy and neuro-degeneration: pathogenic mechanisms and therapeutic opportunities [J]. Neuron, 2017, 93: 1015-1034.
[11] Rubinsztein DC, Codogno P, Levine B. Autophagy modulation as a potential therapeutic target for diverse diseases [J]. Nat Rev Drug Discov, 2012, 11: 709-784.
[12] Bento CF, Renna M, Ghislat G, et al. Mammalian autophagy: how does it work?[J]. Annu Rev Biochem, 2016, 85, 685-713.
[13] Ge L, Melville D, Zhang M, et al. The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis [J]. Elife, 2013, 2: 89-99.
[14] Liu WJ, Ye L, Huang WF,et al. p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation [J]. Cell Mol Biol Lett, 2016, 21: 115-117.
[2] Oh SH, Kim HN, Park HJ, et al. Mesenchymal stem cells inhibit transmission of alpha-synuclein by modulating clathrin-mediated endocytosis in a Parkinsonian model [J]. Cell Reports,2016, 14: 835-849.
[3] Echigo Y, Sugiki H, Koizumi Y, et al. Activation of RAW264.7 macrophages by oxidized galectin-1 [J]. Immunol Lett, 2010, 131: 19-23.
[4] Qu WS, Wang YH, Wang JP, et al. Galectin-1 enhances astrocytic BDNF production and improves functional outcome in rats following ischemia[J]. Neurochem Res, 2010, 35:1716-1724.
[5] Nonaka M, Fukuda M. Galectin-1 for neuroprotection? [J]. Immunity, 2012, 37: 187-189.
[6] Starossom SC, Mascanfroni ID, Imitola J, et al. Galectin-1 deactivates classically activated microglia and protects from inflammation-induced neurodegeneration [J]. Immunity, 2012, 37: 249-263.
[7] Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes [J]. Apoptosis, 2008, 13: 1-9.
[8] Kochergin IA, Zakharova MN. The role of autophagy in neurodegenerative diseases [J]. Neurochem J, 2016, 10: 7-18.
[9] Sundblad V, Morosi LG, Geffner JR, et al. Galectin-1: a Jack-of-All-Trades in the resolution of acute and chronic inflammation [J]. J Immunol, 2017, 199: 3721-3730.
[10] Menzies FM, Fleming A, Caricasole A, et al, Autophagy and neuro-degeneration: pathogenic mechanisms and therapeutic opportunities [J]. Neuron, 2017, 93: 1015-1034.
[11] Rubinsztein DC, Codogno P, Levine B. Autophagy modulation as a potential therapeutic target for diverse diseases [J]. Nat Rev Drug Discov, 2012, 11: 709-784.
[12] Bento CF, Renna M, Ghislat G, et al. Mammalian autophagy: how does it work?[J]. Annu Rev Biochem, 2016, 85, 685-713.
[13] Ge L, Melville D, Zhang M, et al. The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis [J]. Elife, 2013, 2: 89-99.
[14] Liu WJ, Ye L, Huang WF,et al. p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation [J]. Cell Mol Biol Lett, 2016, 21: 115-117.