4-PBA通过抑制内质网应激减轻原代海马神经元的损伤
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摘要
目的:探讨内质网应激后原代海马神经元树突棘密度及突触蛋白表达的变化,以及通过内质网应激分子伴侣4-苯基丁酸(4-phenylbutyric acid,4-PBA)抑制内质网应激对这种神经元损伤的抑制作用。方法:原代培养新生大鼠海马神经元,将表达增强型绿色荧光蛋白的质粒转染到原代培养5～7 d(DIV 5～7)的大鼠海马神经元内持续培养,DIV 20时分为对照组、衣霉素(tunicamycin,Tm)处理组和Tm+4-PBA预处理组(Tm处理前1 h给予4-PBA),采用Western blot法检测内质网应激标志蛋白Bi P和突触蛋白的表达水平,激光共聚焦显微镜下观察神经元,分析树突棘密度,采用MTT法分析细胞活力。结果:Tm处理后使Bi P蛋白水平明显升高,而4-PBA预处理使Bi P蛋白水平显著下降(P <0. 05)。Tm引起的原代海马神经元树突棘密度下降及突触蛋白的表达下降能够被4-PBA抑制。Tm引起的细胞活力下降可被4-PBA抑制。结论:Tm能够通过诱导内质网应激而引起原代海马神经元树突棘密度下降及突触蛋白表达下降,而提前给予4-PBA预处理可明显降低内质网应激反应,抑制树突棘密度下降及突触蛋白表达下降,从而减轻原代海马神经元的损伤。
AIM: To investigate the inhibitory effect of 4-phenylbutyric acid( 4-PBA),an aromatic shortchain fatty acid with the functions of endoplasmic reticulum( ER) molecule chaperon,on ER stress-induced decreases in both dendritic spine density and synaptic protein expression. METHODS: Primary hippocampal neurons were cultured from neonatal rats. The primary hippocampal neurons were transfected with enhanced green fluorescent protein plasmids at DIV( day in vitro) 5 ～ 7. At DIV 20,the neurons were divided into DMSO group,tunicamycin group and tunicamycin + 4-PBA( treated with 4-PBA 1 h before tunicamycin) group. The expression levels of ER stress marker protein Bi P and synaptic proteins were detected by Western blot. After immunofluorescence staining,the neurons were morphologically observed under confocal laser scanning microscope,and the density of dendritic spines was analyzed. At last,the cell viability was measured by MTT assay. RESULTS: Tunicamycin induced ER stress in the primary hippocampal neurons,characterized by significantly increased level of Bi P in tunicamycin group,which was reduced in tunicamycin + 4-PBA group( P <0. 05). 4-PBA inhibited tunicamycin-induced decreases in both dendritic spine density and synaptic protein expression in the primary hippocampal neurons. 4-PBA attenuated tunicamycin-induced decrease in the cell viability. CONCLUSION:4-PBA not only attenuates ER stress of primary hippocampal neurons but also inhibits the decreases in both dendritic spine density and synaptic protein expression induced by tunicamycin.
AIM: To investigate the inhibitory effect of 4-phenylbutyric acid( 4-PBA),an aromatic shortchain fatty acid with the functions of endoplasmic reticulum( ER) molecule chaperon,on ER stress-induced decreases in both dendritic spine density and synaptic protein expression. METHODS: Primary hippocampal neurons were cultured from neonatal rats. The primary hippocampal neurons were transfected with enhanced green fluorescent protein plasmids at DIV( day in vitro) 5 ～ 7. At DIV 20,the neurons were divided into DMSO group,tunicamycin group and tunicamycin + 4-PBA( treated with 4-PBA 1 h before tunicamycin) group. The expression levels of ER stress marker protein Bi P and synaptic proteins were detected by Western blot. After immunofluorescence staining,the neurons were morphologically observed under confocal laser scanning microscope,and the density of dendritic spines was analyzed. At last,the cell viability was measured by MTT assay. RESULTS: Tunicamycin induced ER stress in the primary hippocampal neurons,characterized by significantly increased level of Bi P in tunicamycin group,which was reduced in tunicamycin + 4-PBA group( P <0. 05). 4-PBA inhibited tunicamycin-induced decreases in both dendritic spine density and synaptic protein expression in the primary hippocampal neurons. 4-PBA attenuated tunicamycin-induced decrease in the cell viability. CONCLUSION:4-PBA not only attenuates ER stress of primary hippocampal neurons but also inhibits the decreases in both dendritic spine density and synaptic protein expression induced by tunicamycin.
引文
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[15] Xiang Q,Li XH,Yang B,et al. Allicin attenuates tunicamycin-induced cognitive deficits in rats via its synaptic plasticity regulatory activity[J]. Iran J Basic Med Sci,2017,20(6):676-682.
[16] Sharma D,Singh JN,Sharma SS. Effects of 4-phenyl butyric acid on high glucose-induced alterations in dorsal root ganglion neurons[J]. Neurosci Lett,2016,635:83-89.
[2] Endres K,Reinhardt S. ER-stress in Alzheimer’s disease:turning the scale?[J]. Am J Neurodegener Dis,2013,2(4):247-265.
[3] Lin L,Yang SS,Chu J,et al. Region-specific expression of tau,amyloid-βprotein precursor,and synaptic proteins at physiological condition or under endoplasmic reticulum stress in rats[J]. J Alzheimers Dis,2014,41(4):1149-1163.
[4] Liu ZC,Fu ZQ,Song J,et al. Bip enhanced the association of GSK-3βwith tau during ER stress both in vivo and in vitro[J]. J Alzheimers Dis,2012,29(4):727-740.
[5] Mimori S,Okuma Y,Kaneko M,et al. Protective effects of 4-phenylbutyrate derivatives on the neuronal cell death and endoplasmic reticulum stress[J]. Biol Pharm Bull,2012,35(1):84-90.
[6] Hoozemans JJ,Scheper W. Endoplasmic reticulum:the unfolded protein response is tangled in neurodegeneration[J]. Int J Biochem Cell Biol,2012,44(8):1295-1298.
[7] Ricobaraza A, Cuadrado-Tejedor M, Garcia-Osta A.Long-term phenylbutyrate administration prevents memory deficits in Tg2576 mice by decreasing Aβ[J]. Front Biosci,2011,3:1375-1384.
[8] Lindholm D,Wootz H,Korhonen L. ER stress and neurodegenerative diseases[J]. Cell Death Differ,2006,13(3):385-392.
[9] Tabira T,Chui DH,Kuroda S. Significance of intracellular Aβ42 accumulation in Alzheimer’s disease[J]. Front Biosci,2002,7:a44-a49.
[10] Verfaillie T,Rubio N,Garg AD,et al. PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress[J]. Cell Death Differ,2012,19(11):1880-1891.
[11] Liang J,Kulasiri D,Samarasinghe S. Ca2+dysregulation in the endoplasmic reticulum related to Alzheimer’s disease:a review on experimental progress and computational modeling[J]. Biosystems,2015,134:1-15.
[12] Devi L,Ohno M. PERK mediates e IF2αphosphorylation responsible for BACE1 elevation,CREB dysfunction and neurodegeneration in a mouse model of Alzheimer’s disease[J]. Neurobiol Aging,2014,35(10):2272-2281.
[13] Morrison JH. Which synapses are affected in aging and what is the nature of their vulnerability? A commentary on“life span and synapses:will there be a primary senile dementia?”[J]. Neurobiol Aging,2001,22(3):349-350.
[14] Selkoe DJ. Alzheimer’s disease is a synaptic failure[J].Science,2002,298(5594):789-791.
[15] Xiang Q,Li XH,Yang B,et al. Allicin attenuates tunicamycin-induced cognitive deficits in rats via its synaptic plasticity regulatory activity[J]. Iran J Basic Med Sci,2017,20(6):676-682.
[16] Sharma D,Singh JN,Sharma SS. Effects of 4-phenyl butyric acid on high glucose-induced alterations in dorsal root ganglion neurons[J]. Neurosci Lett,2016,635:83-89.