过表达Homer1a对神经元机械性损伤模型的保护作用及可能机制
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摘要
目的:探讨过表达Homer1a对神经元机械性损伤模型细胞凋亡和AMP活化蛋白激酶(AMPK)表达的影响。方法:分离培养大鼠大脑皮层神经元,随机分组为对照组、模型组、空载组和Homer1a过表达(Exp-Homer1a)组。构建神经元机械性损伤模型,转染Homer1a过表达载体。采用MTT法检测各组细胞活力,qPCR检测各组细胞Homer1a的mRNA表达水平,使用乳酸脱氢酶(LDH)测试盒测定各组细胞上清液LDH活性,流式细胞术检测各组细胞凋亡水平,Western blot检测各组Hormer1a、cleaved caspase-3、Bax、Bcl-2、p-AMPKα和AMPKα蛋白的表达。结果:与对照组比较,机械性损伤神经元的活力显著降低,上清液LDH活性和神经元凋亡率显著增加(P<0.05),且Homer1a mRNA和蛋白表达水平显著升高(P<0.05);与模型组比较,Exp-Homer1a组的上清液LDH活性和神经元凋亡率显著降低,神经元中cleaved caspase-3和Bax的表达水平显著降低(P<0.05),Bcl-2和p-AMPKα的表达水平显著升高(P<0.05)。结论:过表达Homer1a可能通过促进激活AMPKα磷酸化及抑制神经元凋亡来提高机械性损伤神经元的存活率。
AIM: To investigate the effects of Homer1 a over-expression on the apoptosis and AMP-activated protein kinase(AMPK) protein expression in mechanically injured neurons. METHODS: The rat cortical neurons were isolated and cultured in vitro, and then ramdomly divided into control group, model group, empty vector group, and Exp-Homer1 a group. Neuron models with mechanical injury were constructed and infected with the Homer1 a over-expression vector. The mRNA expression of Homer1 a was detected by qPCR. The cell viability in each group was detected by MTT assay. The activity of lactate dehydrogenase(LDH) in the supernatant of each group was measured by LDH test kit. The apoptosis level was analyzed by flow cytometry. The protein levels of Hormer1 a, cleaved caspase-3, Bax, Bcl-2, p-AMPKα and AMPKα were determined by Western blot. RESULTS: Compared with control group, the viability of mechanically injured neurons was significantly decreased, the LDH activity in the supernatant and neuronal apoptotic rate were significantly increased(P<0.05), and Homer1 a expression at mRNA and protein levels was significantly increased(P<0.05). Compared with model group, the LDH activity in the supernatant and neuronal apoptotic rate in Exp-Homer1 a group were significantly decreased, the protein levels of cleaved caspase-3 and Bax were significantly decreased(P<0.05), and the protein levels of Bcl-2 and p-AMPKα were significantly increased(P<0.05). CONCLUSION: Over-expression of Homer1 a may increase the viability of mechanically injured neurons and inhibit their apoptosis by promoting the activation of AMPKα phosphorylation.
AIM: To investigate the effects of Homer1 a over-expression on the apoptosis and AMP-activated protein kinase(AMPK) protein expression in mechanically injured neurons. METHODS: The rat cortical neurons were isolated and cultured in vitro, and then ramdomly divided into control group, model group, empty vector group, and Exp-Homer1 a group. Neuron models with mechanical injury were constructed and infected with the Homer1 a over-expression vector. The mRNA expression of Homer1 a was detected by qPCR. The cell viability in each group was detected by MTT assay. The activity of lactate dehydrogenase(LDH) in the supernatant of each group was measured by LDH test kit. The apoptosis level was analyzed by flow cytometry. The protein levels of Hormer1 a, cleaved caspase-3, Bax, Bcl-2, p-AMPKα and AMPKα were determined by Western blot. RESULTS: Compared with control group, the viability of mechanically injured neurons was significantly decreased, the LDH activity in the supernatant and neuronal apoptotic rate were significantly increased(P<0.05), and Homer1 a expression at mRNA and protein levels was significantly increased(P<0.05). Compared with model group, the LDH activity in the supernatant and neuronal apoptotic rate in Exp-Homer1 a group were significantly decreased, the protein levels of cleaved caspase-3 and Bax were significantly decreased(P<0.05), and the protein levels of Bcl-2 and p-AMPKα were significantly increased(P<0.05). CONCLUSION: Over-expression of Homer1 a may increase the viability of mechanically injured neurons and inhibit their apoptosis by promoting the activation of AMPKα phosphorylation.
引文
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[2] Li Z,Ding K,Wang H,et al.Traumatic brain injury-induced neuronal apoptosis is reduced through modulation of PI3K and autophagy pathways in mouse by FTY720[J].Cell Mol Neurobiol,2016,36(1):131-142.
[3] Yin J,Zhang H,Chen H,et al.Hypertonic saline alle-viates brain edema after traumatic brain injury via downregulation of aquaporin 4 in rats[J].Med Sci Monit,2018,24:1863-1870.
[4] Wang Q,Chikina MD,Pincas H,et al.Homer1 alternative splicing is regulated by gonadotropin-releasing hormone and modulates gonadotropin gene expression[J].Cell Mol Neurobiol,2014,34(10):1747-1756.
[5] Shan W,Nagai T,Tanaka M,et al.Neuronal PAS domain protein 4 (Npas4) controls neuronal homeostasis in pentylenetetrazole-induced epilepsy through the induction of Homer1a[J].J Neurochem,2018,145(1):19-33.
[6] Leber SL,Llenos IC,Miller CL,et al.Homer1a protein expression in schizophrenia,bipolar disorder,and major depression[J].J Neural Transm (Vienna),2017,124(10):1261-1273.
[7] Guo W,Molinaro G,Collins KA,et al.Selective disruption of metabotropic glutamate receptor 5-Homer interactions mimics phenotypes of fragile x syndrome in mice[J].J Neurosci,2016,36(7):2131-2147.
[8] Park SY,Lee HR,Lee WS,et al.Cilostazol modulates autophagic degradation of β-amyloid peptide via SIRT1-coupled LKB1/AMPKα signaling in neuronal cells[J].PLoS One,2016,11(8):e0160620.
[9] Xu N,Zhang Y,Doycheva DM,et al.Adiponectin atte-nuates neuronal apoptosis induced by hypoxia-ischemia via the activation of AdipoR1/APPL1/LKB1/AMPK pathway in neonatal rats[J].Neuropharmacology,2018,133:415-428.
[10] Majd S,Majd Z,Koblar S,et al.Beta estradiol and norepinephrine treatment of differentiated SH-SY5Y cells enhances tau phosphorylation at (Ser396) and (Ser262) via AMPK but not mTOR signaling pathway[J].Mol Cell Neurosci,2018,88:201-211.
[11] 苏鑫洪,叶玉勤,杨永祥,等.小鼠皮层神经元机械损伤后miR-124的动态变化及意义[J].中华神经外科疾病研究杂志,2017,16(3):229-233.
[12] Xue CJ,Zhao YN,Li JM,et al.Impacts of SB203580 on the spatial memory and the expression of phosphorylated p38 MAPK and Homer1a in the hippocampus in rats with diffuse brain injury[J].Int J Clin Exp Pathol,2016,9(3):3781-3786.
[13] 黄从刚,段发亮,吴京雷,等.颅脑损伤患者Homer1a蛋白表达及其与神经元损伤、凋亡的关系研究[J].中华神经医学杂志,2017,16(6):595-598.
[14] Ango F,Prézeau L,Muller T,et al.Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein homer[J].Nature,2001,411(6840):962-965.
[15] 李建民,赵雅宁,马荣丽,等.重型弥漫性颅脑损伤大鼠海马区Homer1a表达与神经细胞丢失的关系[J].医学研究生学报,2014,27(8):797-801.
[16] Meunier JC,Mollereau C,Toll L,et al.Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor[J].Nature,1995,377(6549):532-535.
[17] 岳康异,杨甜,李新,等.Preso调控nNOS在神经元机械性损伤中的作用[J].中华神经外科疾病研究杂志,2016,15(6):510-513.
[18] Suchadolskiene O,Pranskunas A,Baliutyte G,et al.Microcirculatory,mitochondrial,and histological changes following cerebral ischemia in swine[J].BMC Neurosci,2014,15:2.
[19] 李园园,王明山,时飞,等.电针预处理对小鼠脑缺血再灌注时海马神经元AMPK活性的影响[J].中华麻醉学杂志,2015,35(1):44-47.