地黄饮子调节PERK/elF2α通路抑制能量代谢障碍AD小鼠脑内β淀粉样蛋白累积的作用机制
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摘要
目的:以能量代谢障碍淀粉样前体蛋白(APP)/早老素基因1(PS1)转基因小鼠为阿尔茨海默病(Alzheimer's disease,AD)模型,探讨地黄饮子通过调控蛋白激酶R样内质网激酶(protein kinase r-like endoplasmic reticulum kinase,PERK),真核细胞起始因子2α(eukaryotic initiation factor 2α,eI F2α)通路抑制内质网应激导致的β-淀粉样蛋白(Aβ)累积的作用机制。方法:4月龄APP/PS1转基因小鼠随机分为正常组、模型组、阳性药(安理申)组、地黄饮子低、中、高剂量组。除正常组小鼠腹腔注射无菌生理盐水外,其余各组小鼠均腹腔注射100 mg·kg-13-硝基丙酸(3-nitropropionic acid,3-NP) 1次。小鼠造模后,立即给药。灌胃给药1周后,Morris水迷宫实验检测小鼠学习记忆能力,高效液相色谱法检测小鼠脑内三磷酸腺苷(ATP),二磷酸腺苷(ADP),一磷酸腺苷(AMP)含量,并计算脑能荷(energy charge,EC)。蛋白免疫印迹法(Western blot)检测小鼠脑内PERK,eI F2α磷酸化水平,β-淀粉样前体蛋白水解酶1(BACE1)表达水平。实时荧光定量聚合酶链式反应(Real-time PCR)检测BACE1 mRNA表达水平。酶联免疫吸附测定法(ELISA)检测小鼠脑内Aβ含量。结果:与正常组比较,Morris水迷宫实验结果显示,在定位巡航实验中,地黄饮子可以明显缩短模型小鼠的逃避潜伏期(P <0. 05,P <0. 01);空间探索实验中,地黄饮子可以增加模型小鼠穿越目标区域的次数(P <0. 05,P <0. 01)和在目标象限的停留时间(P <0. 01),减少相对象限的停留时间(P <0. 05,P <0. 01)。地黄饮子可以显著降低AMP水平,明显升高ATP,ADP水平,显著提高模型小鼠脑内EC水平(P <0. 05,P <0. 01),抑制PERK,eI F2α的磷酸化(P <0. 01)和BACE1的蛋白水平(P <0. 01),但对BACE1 mRNA转录没有显著影响。地黄饮子能够减少模型小鼠脑内Aβ的含量(P <0. 01)。结论:地黄饮子可以通过阻止能量代谢障碍导致的内质网应激通路PERK/eI F2α激活,抑制BACE1的翻译,从而减少能量代谢障碍APP/PS1小鼠脑内Aβ的累积。
Objective: To explore the effect and mechanism of Dihuang Yinzi on β-amyloid protein( Aβ)accumulation via protein kinase r-like endoplasmic reticulum kinase( PERK),eukaryotic initiation factor 2α( e IF2α) pathway in energy inhibited APP/PS1 transgenic Alzheimer's disease( AD) mice. Method: APP/PS1 transgenic mice at 4-month-old were randomly divided into normal control group,model group,positive drug group( Aricept),Dihuang Yinzi low-,medium-and high-dose groups. Mice were intraperitoneally injected with100 mg·kg-13-nitropropionic acid( 3-NP) except those in normal control group( intraperitoneally injected with normal saline) to establish models. Drug administration was given immediately modeling,and after 1 week of intragastric administration,the learning and memory ability of mice was tested by Morris water maze test; the contents of adenosine triphosphate( ATP),adenosine diphosphate( ADP),and adenosine monophosphate( AMP)in the brain of mice were detected by high performance liquid chromatography( HPLC); and the energy charge( EC) was calculated. Western blot was performed to detect the levels of PERK,e IF2α phosphorylation andβ-amyloid precursor protein hydrolase 1( BACE1) expression in the brain of mice. The transcriptional level of BACE1 gene was detected by Real-time fluorescence quantitative polymerase chain reaction( PCR). The content of cerebral Aβ was detected by enzyme-linked immunosorbent assay( ELISA). Result: Morris water maze test showed that as compared with the normal group,Dihuang Yinzi can significantly shorten the escape latency of model mice in navigation test( P < 0. 05, P < 0. 01). In spatial probe test,Dihuang Yinzi can increase the number passing the target area( P < 0. 05,P < 0. 01) and the residence time in target quadrant( P < 0. 01),reduce the residence time in opposite quadrant( P < 0. 05,P < 0. 01). Dihuang Yinzi can significantly decrease AMP level,increase ATP and ADP levels,and significantly improve the cerebral EC level( P < 0. 05,P < 0. 01),inhibit the phosphorylation of PERK and e IF2α( P < 0. 01),decrease the protein level of BACE1( P < 0. 01),but had no significant effect on BACE1 gene transcription. Besides,Dihuang Yinzi can reduce the cerebral Aβ content in model mice( P < 0. 01). Conclusion: Dihuang Yinzi could prevent energy metabolism disturbance-induced endoplasmic reticulum stress via inhibiting PERK/e IF2α activation,reduce cerebral Aβ accumulation,and improve cognition in 3-NP induced energy inhibited APP/PS1 mice.
Objective: To explore the effect and mechanism of Dihuang Yinzi on β-amyloid protein( Aβ)accumulation via protein kinase r-like endoplasmic reticulum kinase( PERK),eukaryotic initiation factor 2α( e IF2α) pathway in energy inhibited APP/PS1 transgenic Alzheimer's disease( AD) mice. Method: APP/PS1 transgenic mice at 4-month-old were randomly divided into normal control group,model group,positive drug group( Aricept),Dihuang Yinzi low-,medium-and high-dose groups. Mice were intraperitoneally injected with100 mg·kg-13-nitropropionic acid( 3-NP) except those in normal control group( intraperitoneally injected with normal saline) to establish models. Drug administration was given immediately modeling,and after 1 week of intragastric administration,the learning and memory ability of mice was tested by Morris water maze test; the contents of adenosine triphosphate( ATP),adenosine diphosphate( ADP),and adenosine monophosphate( AMP)in the brain of mice were detected by high performance liquid chromatography( HPLC); and the energy charge( EC) was calculated. Western blot was performed to detect the levels of PERK,e IF2α phosphorylation andβ-amyloid precursor protein hydrolase 1( BACE1) expression in the brain of mice. The transcriptional level of BACE1 gene was detected by Real-time fluorescence quantitative polymerase chain reaction( PCR). The content of cerebral Aβ was detected by enzyme-linked immunosorbent assay( ELISA). Result: Morris water maze test showed that as compared with the normal group,Dihuang Yinzi can significantly shorten the escape latency of model mice in navigation test( P < 0. 05, P < 0. 01). In spatial probe test,Dihuang Yinzi can increase the number passing the target area( P < 0. 05,P < 0. 01) and the residence time in target quadrant( P < 0. 01),reduce the residence time in opposite quadrant( P < 0. 05,P < 0. 01). Dihuang Yinzi can significantly decrease AMP level,increase ATP and ADP levels,and significantly improve the cerebral EC level( P < 0. 05,P < 0. 01),inhibit the phosphorylation of PERK and e IF2α( P < 0. 01),decrease the protein level of BACE1( P < 0. 01),but had no significant effect on BACE1 gene transcription. Besides,Dihuang Yinzi can reduce the cerebral Aβ content in model mice( P < 0. 01). Conclusion: Dihuang Yinzi could prevent energy metabolism disturbance-induced endoplasmic reticulum stress via inhibiting PERK/e IF2α activation,reduce cerebral Aβ accumulation,and improve cognition in 3-NP induced energy inhibited APP/PS1 mice.
引文
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[12]高变娥,姚伟洁,杨鑫伟,等.糖络宁对DPN大鼠和雪旺细胞内质网应激PERK通路的影响[J].中国实验方剂学杂志,2018,24(4):115-123.
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[14]王澍,张雪,马樱芮,等.补肾化痰益智方联合丁苯酞对阿尔茨海默病患者氧化应激、血流变学及认知功能的影响[J].中国实验方剂学杂志,2018,24(7):212-216.
[15]牛英才,王建明,董妙先,等.海马注射β-淀粉样蛋白对APP mRNA的影响及加减地黄饮子的干预作用[J].中国中医基础医学杂志,2007,13(12):901-903.
[16]何秀丽,孟菲,周妍妍,等.地黄饮子含药脑脊液对受损PC12细胞β-淀粉样前体蛋白mRNA表达的干预[J].中医药信息,2011,28(4):26-28.
[17]于淼,谢宁,关慧波,等.地黄饮子对AD模型大鼠脑内Aβ生成的干预机制[J].中华中医药杂志,2016,31(6):2281-2284.
[18]Collins J M,King A E,Woodhouse A,et al.The effect of focal brain injury on beta-amyloid plaque deposition,inflammation and synapses in the APP/PS1 mouse model of Alzheimer's disease[J].Exp Neurol,2015,267(3):219-229.
[19]Aso E,Sánchez-Pla A,Vegas-Lozano E,et al.Cannabis-based medicine reduces multiple pathological processes in AβPP/PS1 mice[J].J Alzheimers Dis,2015,43(3):977-991.
[20]于修芳,雷霞,曹玲,等.补阳还五汤对阿尔茨海默病小鼠海马凋亡因子及学习记忆能力的影响[J].中国实验方剂学杂志,2018,24(3):109-113.
[21]马涛,王乐,温彬宇,等.地黄饮子对β淀粉样蛋白1-42致SH-SY5Y细胞线粒体损伤及细胞凋亡的影响[J].中国中医药信息杂志,2015,22(12):59-63.
[22]Devi L,Ohno M.PERK mediates e IF2αphosphorylation responsible for BACE1 elevation,CREBdysfunction and neurodegeneration in a mouse model of Alzheimer's disease[J].Neurobiol Aging,2014,35(10):2272-2281.
[2]Bloom G S.Amyloid-βand tau:the trigger and bullet in Alzheimer disease pathogenesis[J].JAMA Neurol,2014,71(4):505-508.
[3]CHENG X,WU J,GENG M,et al.Role of synaptic activity in the regulation of amyloid beta levels in Alzheimer's disease[J].Neurobiol Aging,2014,35(6):1217-1232.
[4]Mamelak M.Energy and the Alzheimer brain[J].Neurosci Biobehav Rev,2017,doi:10.1016/j.neubiorev.2017.02.001.
[5]Velliquette R A,O'Connor T,Vassar R.Energy inhibition elevates beta-secretase levels and activity and is potentially amyloidogenic in APP transgenic mice:possible early events in Alzheimer's disease pathogenesis[J].J Neurosci,2005,25(47):10874-10883.
[6]O'Connor T,Sadleir K R,Maus E,et al.Phosphorylation of the translation initiation factor e IF2alpha increases BACE1 levels and promotes amyloidogenesis[J].Neuron,2008,60(6):988-1009.
[7]金曦.地黄饮子化裁治疗老年性痴呆43例临床观察[J].中国医药科技,2010,17(4):358-359.
[8]李金桂.地黄饮子治疗老年性痴呆症疗效观察[J].内蒙古中医药,2012,31(22):18-19.
[9]马涛,王新祥,张允岭,等.地黄饮子对阿尔茨海默病小鼠学习记忆及脑组织能量代谢的影响[J].国际中医中药杂志,2014,36(6):539-543.
[10]Biswas J,Gupta S,Verma D K,et al.Involvement of glucose related energy crisis and endoplasmic reticulum stress:insinuation of streptozotocin induced Alzheimer's like pathology[J].Cell Signal,2018,doi:10.1016/j.cellsig.2017.10.018.
[11]Park-York M,Kim Y,York D A.Cage food location alters energy balance and endoplasmic reticulum stress in the brain of mice[J].Physiol Behav,2012,106(2):158-163.
[12]高变娥,姚伟洁,杨鑫伟,等.糖络宁对DPN大鼠和雪旺细胞内质网应激PERK通路的影响[J].中国实验方剂学杂志,2018,24(4):115-123.
[13]Ridler C.Alzheimer disease:BACE1 inhibitors block new Aβplaque formation[J].Nat Rev Neurol,2018,14(3):126.
[14]王澍,张雪,马樱芮,等.补肾化痰益智方联合丁苯酞对阿尔茨海默病患者氧化应激、血流变学及认知功能的影响[J].中国实验方剂学杂志,2018,24(7):212-216.
[15]牛英才,王建明,董妙先,等.海马注射β-淀粉样蛋白对APP mRNA的影响及加减地黄饮子的干预作用[J].中国中医基础医学杂志,2007,13(12):901-903.
[16]何秀丽,孟菲,周妍妍,等.地黄饮子含药脑脊液对受损PC12细胞β-淀粉样前体蛋白mRNA表达的干预[J].中医药信息,2011,28(4):26-28.
[17]于淼,谢宁,关慧波,等.地黄饮子对AD模型大鼠脑内Aβ生成的干预机制[J].中华中医药杂志,2016,31(6):2281-2284.
[18]Collins J M,King A E,Woodhouse A,et al.The effect of focal brain injury on beta-amyloid plaque deposition,inflammation and synapses in the APP/PS1 mouse model of Alzheimer's disease[J].Exp Neurol,2015,267(3):219-229.
[19]Aso E,Sánchez-Pla A,Vegas-Lozano E,et al.Cannabis-based medicine reduces multiple pathological processes in AβPP/PS1 mice[J].J Alzheimers Dis,2015,43(3):977-991.
[20]于修芳,雷霞,曹玲,等.补阳还五汤对阿尔茨海默病小鼠海马凋亡因子及学习记忆能力的影响[J].中国实验方剂学杂志,2018,24(3):109-113.
[21]马涛,王乐,温彬宇,等.地黄饮子对β淀粉样蛋白1-42致SH-SY5Y细胞线粒体损伤及细胞凋亡的影响[J].中国中医药信息杂志,2015,22(12):59-63.
[22]Devi L,Ohno M.PERK mediates e IF2αphosphorylation responsible for BACE1 elevation,CREBdysfunction and neurodegeneration in a mouse model of Alzheimer's disease[J].Neurobiol Aging,2014,35(10):2272-2281.