加味脑泰方对大鼠缺氧/复氧损伤海马神经元炎性通路SIRT1/NF-κB的影响
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摘要
目的观察加味脑泰方对大鼠缺氧/复氧损伤海马神经元炎性通路SIRT1/NF-κB的影响,初步探讨其作用机制。方法原代分离、培养及鉴定SD大鼠胎鼠海马神经元,采用缺氧24 h、复氧2 h干预构建缺氧/复氧细胞模型。将培养的原代海马神经元随机分为正常组、空白血清对照组、模型组、阳性药药物血清组和加味脑泰方药物血清组,待缺氧24h后,除正常组和模型组给予等量培养液外,其余各组分别给予相应体积分数10%药物血清或空白血清,继续复氧共孵育培养2h;采用MTT法检测海马神经元细胞活力,高内涵细胞成像分析系统检测海马神经元沉默信息调节蛋白1(SIRT1)、核因子-κB抑制蛋白(IκBα)、核因子-κB(NF-κB)蛋白表达。结果与正常组比较,模型组海马神经元细胞活力显著降低(P<0.01),神经网络及树突、树突棘断裂或减少,细胞明显受损,且神经元内SIRT1、IκBα蛋白表达显著降低(P<0.05),NF-κB蛋白表达明显增加(P<0.05);与模型组比较,阳性药药物血清组和加味脑泰方药物血清组海马神经元细胞活力明显增加(P<0.05),神经网络、树突棘受损明显改善,且神经元内SIRT1、IκBα蛋白表达明显上调(P<0.05),NF-κB蛋白表达明显下调(P<0.05)。结论加味脑泰方对缺氧/复氧损伤海马神经元内炎性相关信号通路SIRT1/NF-κB具有明显的调控作用,这可能是其保护缺氧/复氧损伤海马神经元继而抗血管性痴呆的重要机制之一。
Objective To observe the effects of modified Naotai Prescription on inflammatory pathway of SIRT1/NF-κB in hypoxia/reoxygenation injured hippocampal neurons; To explore the mechanism of action. Methods Hippocampal neurons from SD rats were primitively isolated, cultured and identified. Hypoxia/reoxygenation(H/R) cell model was established by hypoxia 24 h and reoxygenation 2 h intervention. The cultured cells were randomly divided into normal group, blank serum group, model group, positive medicine serum group and modified Naotai Prescription group. Normal group and model group were given same amount culture medium, while other groups were given relevant amount of 10% medicated serum or blank serum to continue for reoxygenation and incubate for 2 h. Hippocampal neuronal cell viability was detected by MTT assay. The expressions of SIRT1, IκBαand NF-κB in hippocampal neurons were detected by HCA. Results Compared with the normal group, hippocampal neuronal cell viability significantly decreased(P<0.01), the neural network and dendrites and dendritic spines were broken or reduced, the cells were obviously damaged, and the expressions of SIRT1 and IκBα protein in neurons significantly decreased(P<0.05), and the expression of NF-κB protein significantly increased(P<0.05) in the model group. Compared with the model group, hippocampal neuronal cell viability significantly increased(P<0.05), the neural network and dendritic spine were significantly improved, and the expressions of SIRT1 and IκBα protein in the neurons increased(P<0.05), and the expression of NF-κB protein was down-regulated(P<0.05) in positive medicine serum group and modified Naotai Prescription group. Conclusion Modified Naotai Prescription has a significant regulatory effect on the inflammatory-related signaling pathway SIRT1/NF-κB in hippocampal neurons after hypoxia/reoxygenation injury, which may be an important mechanism for protecting hypoxic/reoxygenated hippocampal neurons and then anti-vascular dementia.
Objective To observe the effects of modified Naotai Prescription on inflammatory pathway of SIRT1/NF-κB in hypoxia/reoxygenation injured hippocampal neurons; To explore the mechanism of action. Methods Hippocampal neurons from SD rats were primitively isolated, cultured and identified. Hypoxia/reoxygenation(H/R) cell model was established by hypoxia 24 h and reoxygenation 2 h intervention. The cultured cells were randomly divided into normal group, blank serum group, model group, positive medicine serum group and modified Naotai Prescription group. Normal group and model group were given same amount culture medium, while other groups were given relevant amount of 10% medicated serum or blank serum to continue for reoxygenation and incubate for 2 h. Hippocampal neuronal cell viability was detected by MTT assay. The expressions of SIRT1, IκBαand NF-κB in hippocampal neurons were detected by HCA. Results Compared with the normal group, hippocampal neuronal cell viability significantly decreased(P<0.01), the neural network and dendrites and dendritic spines were broken or reduced, the cells were obviously damaged, and the expressions of SIRT1 and IκBα protein in neurons significantly decreased(P<0.05), and the expression of NF-κB protein significantly increased(P<0.05) in the model group. Compared with the model group, hippocampal neuronal cell viability significantly increased(P<0.05), the neural network and dendritic spine were significantly improved, and the expressions of SIRT1 and IκBα protein in the neurons increased(P<0.05), and the expression of NF-κB protein was down-regulated(P<0.05) in positive medicine serum group and modified Naotai Prescription group. Conclusion Modified Naotai Prescription has a significant regulatory effect on the inflammatory-related signaling pathway SIRT1/NF-κB in hippocampal neurons after hypoxia/reoxygenation injury, which may be an important mechanism for protecting hypoxic/reoxygenated hippocampal neurons and then anti-vascular dementia.
引文
[1] BENISTY S. Current concepts in vascular dementia[J]. Geriatr Psychol Neuropsychiatr Vieil,2013,11(2):171-180.
[2] IMFELD P, BODMER M, SCHUERCH M, et al. Risk of incident stroke in patients with Alzheimer disease or vascular dementia[J].Neurology,2013,81(10):910-919.
[3] MORI E. How treatable is vascular dementia?[J]. Brain Nerve,2016,68(4):441-450.
[4] RAMADORI G, LEE C E, BOOKOUT A L, et al. Brain SIRT 1:Anatomical distribution and regulation by energy availability[J]. Jeurosci,2008,28(40):9989-9996.
[5]易亚乔,肖碧跃,刘英飞,等.加味脑泰方对血管性痴呆大鼠学习记忆和海马区NR2A及NR2B表达的影响[J].中南医学科学杂志,2014,42(1):17-20.
[6]廖君,张薇,夏兴,等.脑泰方对局灶性脑缺血大鼠脑组织核因子-κB、基质金属蛋白酶9及其抑制剂表达的影响[J].中国中医药信息杂志,2013,20(9):28-30.
[7]赵严冬,刘潘虹,李学敏,等.远志皂苷元抗H/R诱导皮层神经元凋亡的机制初探[J].中国病理生理杂志,2014,30(7):1166-1171.
[8] FARKAS E, LUITEN P G, BARI F. Permanent,bilateral common carotid artery occlusion in the rat:a model for chronic cerebral hypoperfusion-related neurodegenerative diseases[J]. Brain Res Rev,2007,54(1):162-180.
[9] MICHAN S, LI Y, CHOU M M, et al. SIRT1 is essential for normal cognitive function and synaptic plasticity[J]. The Journal of Neuroscience,2010,30(29):9695-9707.
[10] YANG H, ZHANG W, PAN H, et a1. SIRT1 activators suppress inflanrmtory responses through promotion of p65 deacctylation and inhibition of NF-κB activity[J]. PLoS One,2012,7:e46364.
[11] JUNG Y J, LEE J E, LEE A S, et al. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-κB p65 subunitand cytotoxicity in renal proximal tubule cells[J]. Biochem Biophys Res Commun,2012,419:206-210.
[12]郭清,宫洪涛.从中医五脏论血管性痴呆的病机[J].中国中医药现代远程教育,2018,16(1):71-72.
[13]曾晖,胡洁.当归小分子多糖对血管性痴呆大鼠学习与记忆功能的影响[J].中国临床药理学杂志,2015,31(11):954-956.
[14]周瑜,方锐,王国佐,等.脑泰方对SD大鼠凝血功能的影响[J].中华中医药学刊,2015,33(7):1603-1605.
[15]石咏梅,马英民,廖君,等.内质网应激PERK通路在脑泰方提取物保护局灶性脑缺血大鼠海马神经元中的作用[J].中国老年学杂志,2017,37(22):5512-5515.
[2] IMFELD P, BODMER M, SCHUERCH M, et al. Risk of incident stroke in patients with Alzheimer disease or vascular dementia[J].Neurology,2013,81(10):910-919.
[3] MORI E. How treatable is vascular dementia?[J]. Brain Nerve,2016,68(4):441-450.
[4] RAMADORI G, LEE C E, BOOKOUT A L, et al. Brain SIRT 1:Anatomical distribution and regulation by energy availability[J]. Jeurosci,2008,28(40):9989-9996.
[5]易亚乔,肖碧跃,刘英飞,等.加味脑泰方对血管性痴呆大鼠学习记忆和海马区NR2A及NR2B表达的影响[J].中南医学科学杂志,2014,42(1):17-20.
[6]廖君,张薇,夏兴,等.脑泰方对局灶性脑缺血大鼠脑组织核因子-κB、基质金属蛋白酶9及其抑制剂表达的影响[J].中国中医药信息杂志,2013,20(9):28-30.
[7]赵严冬,刘潘虹,李学敏,等.远志皂苷元抗H/R诱导皮层神经元凋亡的机制初探[J].中国病理生理杂志,2014,30(7):1166-1171.
[8] FARKAS E, LUITEN P G, BARI F. Permanent,bilateral common carotid artery occlusion in the rat:a model for chronic cerebral hypoperfusion-related neurodegenerative diseases[J]. Brain Res Rev,2007,54(1):162-180.
[9] MICHAN S, LI Y, CHOU M M, et al. SIRT1 is essential for normal cognitive function and synaptic plasticity[J]. The Journal of Neuroscience,2010,30(29):9695-9707.
[10] YANG H, ZHANG W, PAN H, et a1. SIRT1 activators suppress inflanrmtory responses through promotion of p65 deacctylation and inhibition of NF-κB activity[J]. PLoS One,2012,7:e46364.
[11] JUNG Y J, LEE J E, LEE A S, et al. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-κB p65 subunitand cytotoxicity in renal proximal tubule cells[J]. Biochem Biophys Res Commun,2012,419:206-210.
[12]郭清,宫洪涛.从中医五脏论血管性痴呆的病机[J].中国中医药现代远程教育,2018,16(1):71-72.
[13]曾晖,胡洁.当归小分子多糖对血管性痴呆大鼠学习与记忆功能的影响[J].中国临床药理学杂志,2015,31(11):954-956.
[14]周瑜,方锐,王国佐,等.脑泰方对SD大鼠凝血功能的影响[J].中华中医药学刊,2015,33(7):1603-1605.
[15]石咏梅,马英民,廖君,等.内质网应激PERK通路在脑泰方提取物保护局灶性脑缺血大鼠海马神经元中的作用[J].中国老年学杂志,2017,37(22):5512-5515.