目的 用Aβ1-42处理高分化PC12细胞建立体外阿尔茨海默病(AD)细胞模型,研究金钗石斛生物碱(DNLA)对Aβ1-42所致PC12细胞凋亡的抑制作用及机制。方法 用UPLC-ESI-HRMS技术分析DNLA成分。将PC12细胞分为对照组、DNLA正常给药组(0.03、0.3、3 μg·mL-1)、30 μmol·L-1 Aβ1-42模型组、DNLA低浓度组(Aβ1-42+0.03 μg·mL-1 DNLA)、DNLA中浓度组(Aβ1-42+0.3 μg·mL-1 DNLA)、DNLA高浓度组(Aβ1-42+3 μg·mL-1 DNLA)和阳性对照组(Aβ1-42+10 μmol·L-1 美金刚),观察DNLA对Aβ1-42所致细胞毒性的保护作用,探讨DNLA对钙/钙调蛋白依赖性蛋白激酶激酶2磷酸化,caspase-3和Cleaved caspase-3表达的调控作用。结果 DNLA主要由石斛碱、石斛胺碱、石斛碱氮氧化物和石斛星碱等生物碱组成。DNLA不显著影响PC12细胞存活率;0.3、3 μg·mL-1 DNLA预给药显著抑制Aβ1-42诱导 PC12细胞毒性,改善损伤细胞形态,减少凋亡细胞,下调钙/钙调蛋白依赖性蛋白激酶激酶2的Ser511位点磷酸化水平和caspase-3、Cleaved caspase-3蛋白表达(P<0.05)。结论 金钗石斛生物碱抑制Aβ1-42诱导PC12细胞凋亡,其机制与负向调控钙/钙调蛋白依赖性蛋白激酶激酶2的Ser511位点磷酸化,下调caspase-3蛋白表达、减少caspase-3蛋白活化有关。
Abstract
OBJECTIVE To investigate the neuroprotective effect of Dendrobium nobile Lindl. alkaloids (DNLA) on Aβ1-42-induced PC12 cell damage and explored its underlying molecular mechanism. METHODS The stems of Dendrobium nobile Lindl. were extracted with 95% ethanol, dissolved in acidic water, extracted with petroleum ether to remove impurities, then isolated using cation exchange resin column chromatography and Sephadex LH-20 gel column chromatography to obtain DNLA; the constituents of DNLA were analyzed using UPLC-ESI-HRMS technology. PC12 cells were divided into seven groups:control group; 0.03, 0.3, 3.0 μg·mL-1 DNLA group, 30 μmol·L-1 Aβ1-42 model group, DNLA low-concentration group (Aβ1-42+0.03 μg·mL-1 DNLA); DNLA medium-concentration group (Aβ1-42+0.3 μg·mL-1 DNLA), DNLA high-concentration group (Aβ1-42+3 μg·mL-1 DNLA), and a positive control group (Aβ1-42+10 μmol·L-1 memantine). The protective effect of DNLA on Aβ1-42-induced PC12 cell cytotoxicity was investigated and the regulation effect of DNLA on CaMKK2 phosphorylation and the expression of caspase-3 and cleaved caspase-3 were explored. RESULTS The alkaloids from D. nobile were mainly dendrobine, dendramine, dendrobine N-oxide, and dendroxine. Relative to the model group, the 0.3 and 3.0μg·mL-1 DNLA groups showed significant inhibition of cell cytotoxicity, less damage to cell morphology, fewer apoptotic cells, and downregulation of CaMKK2 (Ser511) phosphorylation, caspase-3, and cleaved caspase-3 protein expression levels in Aβ1-42-treated PC12 cells(P<0.05, P<0.01). CONCLUSION DNLA inhibites Aβ1-42-induced PC12 cell apoptosis, and the mechanism may be related to negative-regulating CaMKK2 (Ser511) phosphorylation, downregulating caspase-3 protein expression, and reducing caspase-3 activation.
关键词
金钗石斛 /
生物碱 /
PC12细胞 /
钙/钙调蛋白依赖性蛋白激酶激酶2 /
caspase-3
{{custom_keyword}} /
Key words
Dendrobium nobile Lindl. /
alkaloids /
PC12 cells /
CaMKK2 /
caspase-3
{{custom_keyword}} /
中图分类号：
R965
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] REISS A B, ARAIN H A, STECKER M M, et al. Amyloid toxicity in Alzheimer′s disease[J]. Rev Neurosci, 2018, 29(6):613-627.
[2] VIOLA K L, KLEIN W L. Amyloid beta oligomers in Alzheimer′s disease pathogenesis, treatment, and diagnosis[J]. Acta Neuropathol, 2015, 129(2):183-206.
[3] SNIGDHA S, SMITH E D, PRIETO G A, et al. caspase-3 activation as a bifurcation point between plasticity and cell death[J]. Neurosci Bull, 2012, 28(1):14-24.
[4] GHAVAMI S, SHOJAEI S, YEGANEH B, et al. Autophagy and apoptosis dysfunction in neurodegenerative disorders[J]. Prog Neurobiol, 2014, 112:24-49.
[5] ZHU Y, SUN X, GONG T, et al. Antioxidant and antiapoptotic effects of 1,1′-(biphenyl-4,4′-diyl)-bis(3-(dimethylamino)-propan-1-one) on protecting PC12 cells from Abeta-induced injury[J]. Mol Pharm, 2014, 11(2):428-435.
[6] MAIRET C G, COURCHET J, PIERAUT S, et al. The CAMKK2-AMPK kinase pathway mediates the synaptotoxic effects of Abeta oligomers through Tau phosphorylation[J]. Neuron, 2013, 78(1):94-108.
[7] ZHANG C, XIE L, GUAN F, et al. 3H-1,2-dithiole-3-thione protects PC12 cells against amyloid beta 1-42 (Aβ1-42) induced apoptosis via activation of the ERK1/2 pathway[J]. Life Sci, 2018, 213:74-81.
[8] Ch.P (2020) VolⅠ(中国药典2020年版.一部)[S]. 2020:94-97.
[9] Ch.P (2020) VolⅠ(中国药典2020年版.一部)[S]. 2020:295-296.
[10] LI L S, LU Y L, NIE J, et al. Dendrobium nobile Lindl alkaloid, a novel autophagy inducer, protects against axonal degeneration induced by Abeta25-35 in hippocampus neurons in vitro[J]. CNS Neurosci Ther, 2017, 23(4):329-340.
[11] NIE J, TIAN Y, ZHANG Y, et al. Dendrobium alkaloids prevent Abeta25-35-induced neuronal and synaptic loss via promoting neurotrophic factors expression in mice[J]. Peer J, 2016, 4:2739-2755.
[12] LI F, LIU B, SHI J S, et al. Dendrobium nobile Lindl. alkaloids inhibited Tau protein hyper-phosphorylation in hippocampus[J]. Chin J Pharm Toxicol(中国药理学与毒理学杂志), 2019, 33(9):752.
[13] ZHANG X Y, ZHAO X S, HE M Q, et al. Optimization of extraction condition for total alkaloids from Dendrobium nobile[J]. Guizhou Agric Sci(贵州农业科学), 2016, 44(6):131-134.
[14] XU Y Y, WANG L Y, HUANG B, et al. Comparison of contents of polysaccharides and alkaloids in Dendrobium from different harvest time[J]. West China J Pharm Sci(华西药学杂志), 2014, 29(3):288-291.
[15] WANG Y H, AYULA B, ABE N, et al. Tandem mass spectrometry for structural identification of sesquiterpene alkaloids from the stems of Dendrobium nobile using LC-QToF[J]. Planta Med, 2016, 82(7):662-670.
[16] SONG X Y, HU J F, SUN M N, et al. IMM-H004, a novel coumarin derivative compound, protects against amyloid beta-induced neurotoxicity through a mitochondrial-dependent pathway [J]. Neuroscie, 2013, 242:28-38.
[17] LIU J, ZHU Y, CHEN S, et al. Apocynin attenuates cobalt chloride-induced pheochromocytoma cell apoptosis by inhibiting P38-MAPK/caspase-3 pathway[J]. Cell Physiol Biochem, 2018, 48(1):208-214.
[18] SANG X X, YANG Y, TANG X L, et al. Effects of oligosaccharide esters of Polygala tenuifolia on the injury of human neuroblastoma SH-SY5Y cells induced by Aβ25-35 Fragment[J]. Chin Pharm J(中国药学杂志), 2018, 53(11):876-881.
[19] WANG F J, WANG T T, XU M Y, et al. Chemical Constituents of Drynaria fortunei and their protective effects on PC12 cell[J]. Chin Pharm J(中国药学杂志), 2018, 53(16):1359-1365.
[20] WANG H, JIANG T, LI W, et al. Resveratrol attenuates oxidative damage through activating mitophagy in an in vitro model of Alzheimer′s disease[J]. Toxicol Lett, 2018, 282:100-108.
[21] NIE J, JIANG L S, ZHANG Y, et al. Dendrobium nobile Lindl. alkaloids decreases the level of intracellular β-amyloid by improving impaired autolysosomal proteolysis in APP/PS1 mice[J]. Front Pharmacol, 2018, 9:1479-1492.
[22] JIN C, DU Z, LIN L, et al. Structural characterization of mannoglucan from Dendrobium nobile Lindl and the neuritogenesis-induced effect of its acetylated derivative on PC-12 cells[J]. Polymers (Basel). 2017, 9(9):399-411.
[23] SABBIR M G. Loss of Ca2+/calmodulin dependent protein kinase kinase 2 leads to aberrant transferrin phosphorylation and trafficking:a potential biomarker for Alzheimer′s disease[J]. Front Mol Biosci, 2018, 5:99-141.
[24] MANCZAK M, REDDY P H. Abnormal interaction of oligomeric amyloid-β with phosphorylated tau:implications to synaptic dysfunction and neuronal damage[J]. J Alzheimers Dis, 2013, 36(2):285-295.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
贵州省中医药管理局中医药、民族医药科学技术研究课题资助(QZYY-2018-134);贵州医科大学药用植物功效利用国家重点实验室开放课题(FAMP201910K);贵州省科学计划项目(黔科合平台人才[2017]5101);遵义医学院博士科研启动基金项目资助(F-859)
{{custom_fund}}
PDF(3024 KB)
