蛇床子素上调miRNA-9抑制BACE-1表达治疗阿尔茨海默病
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摘要
目的探讨蛇床子素上调miRNA-9治疗AD的作用机制。方法基因芯片技术筛选蛇床子素治疗后差异表达的microRNAs;数据库和Cytoscape预测miRNA-9调控的靶基因;脂质体2000建立APP高表达的SH-SY5Y细胞模型,MTT法检测蛇床子素对细胞活力的影响;将miRNA-9抑制剂转入到APP高表达的SH-SY5Y细胞中,RT-PCR法检测各组miR-9、BACE-1 mRNA的表达情况;Western blot方法检测细胞中BACE-1蛋白的表达情况。结果数据库结果显示,蛇床子素调控的miRNA-9可以与BACE-1靶向结合。本实验成功建立APP高表达的SH-SY5Y细胞模型。MTT结果显示,50μmol·L~(-1)蛇床子素对细胞有较好的保护作用。RT-PCR结果显示,蛇床子素可以上调miR-9的表达,抑制剂组miRNA-9表达最低。与模型组相比,蛇床子素可以抑制BACE-1 mRNA和蛋白的表达,且抑制剂组的BACE-1 mRNA和蛋白表达最多。结论蛇床子素治疗阿尔茨海默病可能与上调miRNA-9,从而抑制BACE-1表达有关。
Aim To explore the mechanism of osthole regulating the expression of miR-9 to delay the occurance of Alzheimer's disease(AD). Methods The miRNA which expressed differently with osthole treatment was selected by microarray; the target gene binding to miRNA-9 was verified by databases and Cytoscape; the SH-SY5 Y cells with over expression of APP were established using Lipofectamine2000. The cell viability was determined by MTT assay. The miRNA-9 inhibitor was transfected into cells, and the expression of miRNA-9 and BACE-1 mRNA was determined by RT-PCR; the changes of BACE-1 protein were determined by Western blot. Results The results of database showed that osthole-mediated miRNA-9 was combined with BACE-1 genes. Our lab had established SH-SY5 Y cells with over expression of APP. The results of MTT assay showed that 50 μmol·L~(-1) osthole had a protective effect on cells. Osthole could increase the expression of miRNA-9, and the expression of miR-9 was lowest in inhibitor group determined by RT-PCR. Osthole decreased the expression of BACE-1 mRNA and protein compared with APP group, and the expression of BACE-1 was highest in inhibitor group. Conclusion Osthole plays a protective role in AD partly through suppressing the expression of BACE-1 by up-regulating miRNA-9.
Aim To explore the mechanism of osthole regulating the expression of miR-9 to delay the occurance of Alzheimer's disease(AD). Methods The miRNA which expressed differently with osthole treatment was selected by microarray; the target gene binding to miRNA-9 was verified by databases and Cytoscape; the SH-SY5 Y cells with over expression of APP were established using Lipofectamine2000. The cell viability was determined by MTT assay. The miRNA-9 inhibitor was transfected into cells, and the expression of miRNA-9 and BACE-1 mRNA was determined by RT-PCR; the changes of BACE-1 protein were determined by Western blot. Results The results of database showed that osthole-mediated miRNA-9 was combined with BACE-1 genes. Our lab had established SH-SY5 Y cells with over expression of APP. The results of MTT assay showed that 50 μmol·L~(-1) osthole had a protective effect on cells. Osthole could increase the expression of miRNA-9, and the expression of miR-9 was lowest in inhibitor group determined by RT-PCR. Osthole decreased the expression of BACE-1 mRNA and protein compared with APP group, and the expression of BACE-1 was highest in inhibitor group. Conclusion Osthole plays a protective role in AD partly through suppressing the expression of BACE-1 by up-regulating miRNA-9.
引文
[1] Mayeux R, Stern Y. Epidemiology of Alzheimer disease[J]. Cold Spring Harb Perspect Med, 2012,2(8): 1-18.
[2] Aron L, Yankner B A. Neurodegenerative disorders: neural synchronization in Alzheimer’s disease[J]. Nature, 2016,540(7632):207-8.
[3] Revett T J, Baker G B, Jhamandas J, et al. Glutamate system, amyloid peptides and tau protein: functional interrelationships and relevance to Alzheimer disease pathology[J]. J Psychiatry Neurosci, 2013,38(1):6-23.
[4] Swomley A M, F?rster S, Keeney J T, et al. Abeta, oxidative stress in Alzheimer disease: evidence based on proteomics studies[J]. Biochim Biophys Acta, 2014,1842(8):1248-57.
[5] 刘爽, 吴东媛, 徐建华, 等. 蛇床子素的抗焦虑作用研究[J].中国医药导报, 2012,9(29):19-21.[5] Liu S, Wu D Y, Xu J H, et al. Research of anti-anxiety effect of osthole[J]. China Med Herald, 2012, 9(29): 19-21.
[6] 孔亮, 姚璎珈, 教亚男, 等. 蛇床子素对颅脑损伤模型小鼠的神经保护作用[J].中国药房, 2015,26(22):3046-9.[6] Kong L, Yao Y J, Jiao Y N, et al. Neuroprotective effect of osthole on mice with brain injury model[J]. Chin Pharm, 2015,26(22):3046-9.
[7] 孔亮,姚璎珈,教亚男,等.蛇床子素对机械性脑损伤小鼠的抗炎抗凋亡作用研究[J].中国药理学通报, 2015,31(7):999-1004.[7] Kong L, Yao Y J, Jiao Y N, et al. Anti-inflammatory and anti-apoptotic effects of osthole on mice with mechanical brain injury[J]. Chin Pharmacol Bull, 2015,31(7):999-1004.
[8] Li S H, Gao P, Wang L T, et al. Osthole stimulated neural stem cells differentiation into neurons in an Alzheimer’s disease cell model via upregulation of microRNA-9 and rescued the functional impairment of hippocampal neurons in APP/PS1 transgenic mice[J]. Front Neurosci, 2017,11:340.
[9] Li S, Yan Y, Jiao Y, et al. Neuroprotective effect of osthole on neuron synapses in an Alzheimer's disease cell model via upregulation of microRNA-9[J]. J Mol Neurosci, 2016,60(1):71-81.
[10] 教亚男,姚璎珈,孔亮,等.蛇床子素对转染APP595/596基因的SH-SY5Y细胞的保护作用[J].中国病理生理杂志, 2015,31(11):2053-8.[10] Jiao Y N, Yao Y J, Kong L, et al. Protective effect of osthole on SH-SY5Y cells transfected with APP595/596 gene[J]. Chin J Pathophysiol, 2015,31(11):2053-8.
[11] 王雅萌,教亚男,闫宇辉,等.蛇床子素对APP/PS1双转基因AD小鼠的治疗作用[J].中国新药杂志, 2017,26(21):2573-7.[11] Wang Y M, Jiao Y N,Yan Y H, et al. Therapeutic effect of osthole on APP/PS1 double transgenic AD mice[J]. Chin J New Drugs, 2017,26(21):2573-7.
[12] Tang G, Tang X, Mendu V, et al. The art of microRNA: various strategies leading to gene silencing via an ancient pathway[J]. Biochim Biophys Acta, 2008,1779(11):655-62.
[13] McGeer P L, McGeer E G. NSAIDs and Alzheimer disease: epidemiological, animal model and clinical studies[J]. Neurobiol Aging, 2007, 28(5): 639-47.
[14] Ling Y, Morgan K, Kalsheker N. Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer’s disease[J]. Int J Biochem Cell Biol, 2003,35(11):1505-35.
[15] Kowall N W, Beal M F, Busciglio J, et al. An in vivo model for the neurodegenerative effects of β-amyloid and protection by substance P[J]. Proc Natl Acad Sci USA, 1991, 88(16): 7247-51.
[2] Aron L, Yankner B A. Neurodegenerative disorders: neural synchronization in Alzheimer’s disease[J]. Nature, 2016,540(7632):207-8.
[3] Revett T J, Baker G B, Jhamandas J, et al. Glutamate system, amyloid peptides and tau protein: functional interrelationships and relevance to Alzheimer disease pathology[J]. J Psychiatry Neurosci, 2013,38(1):6-23.
[4] Swomley A M, F?rster S, Keeney J T, et al. Abeta, oxidative stress in Alzheimer disease: evidence based on proteomics studies[J]. Biochim Biophys Acta, 2014,1842(8):1248-57.
[5] 刘爽, 吴东媛, 徐建华, 等. 蛇床子素的抗焦虑作用研究[J].中国医药导报, 2012,9(29):19-21.[5] Liu S, Wu D Y, Xu J H, et al. Research of anti-anxiety effect of osthole[J]. China Med Herald, 2012, 9(29): 19-21.
[6] 孔亮, 姚璎珈, 教亚男, 等. 蛇床子素对颅脑损伤模型小鼠的神经保护作用[J].中国药房, 2015,26(22):3046-9.[6] Kong L, Yao Y J, Jiao Y N, et al. Neuroprotective effect of osthole on mice with brain injury model[J]. Chin Pharm, 2015,26(22):3046-9.
[7] 孔亮,姚璎珈,教亚男,等.蛇床子素对机械性脑损伤小鼠的抗炎抗凋亡作用研究[J].中国药理学通报, 2015,31(7):999-1004.[7] Kong L, Yao Y J, Jiao Y N, et al. Anti-inflammatory and anti-apoptotic effects of osthole on mice with mechanical brain injury[J]. Chin Pharmacol Bull, 2015,31(7):999-1004.
[8] Li S H, Gao P, Wang L T, et al. Osthole stimulated neural stem cells differentiation into neurons in an Alzheimer’s disease cell model via upregulation of microRNA-9 and rescued the functional impairment of hippocampal neurons in APP/PS1 transgenic mice[J]. Front Neurosci, 2017,11:340.
[9] Li S, Yan Y, Jiao Y, et al. Neuroprotective effect of osthole on neuron synapses in an Alzheimer's disease cell model via upregulation of microRNA-9[J]. J Mol Neurosci, 2016,60(1):71-81.
[10] 教亚男,姚璎珈,孔亮,等.蛇床子素对转染APP595/596基因的SH-SY5Y细胞的保护作用[J].中国病理生理杂志, 2015,31(11):2053-8.[10] Jiao Y N, Yao Y J, Kong L, et al. Protective effect of osthole on SH-SY5Y cells transfected with APP595/596 gene[J]. Chin J Pathophysiol, 2015,31(11):2053-8.
[11] 王雅萌,教亚男,闫宇辉,等.蛇床子素对APP/PS1双转基因AD小鼠的治疗作用[J].中国新药杂志, 2017,26(21):2573-7.[11] Wang Y M, Jiao Y N,Yan Y H, et al. Therapeutic effect of osthole on APP/PS1 double transgenic AD mice[J]. Chin J New Drugs, 2017,26(21):2573-7.
[12] Tang G, Tang X, Mendu V, et al. The art of microRNA: various strategies leading to gene silencing via an ancient pathway[J]. Biochim Biophys Acta, 2008,1779(11):655-62.
[13] McGeer P L, McGeer E G. NSAIDs and Alzheimer disease: epidemiological, animal model and clinical studies[J]. Neurobiol Aging, 2007, 28(5): 639-47.
[14] Ling Y, Morgan K, Kalsheker N. Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer’s disease[J]. Int J Biochem Cell Biol, 2003,35(11):1505-35.
[15] Kowall N W, Beal M F, Busciglio J, et al. An in vivo model for the neurodegenerative effects of β-amyloid and protection by substance P[J]. Proc Natl Acad Sci USA, 1991, 88(16): 7247-51.