毕拨明宁碱/二氢毕拨明宁碱抑制SK-N-SH细胞Aβ产生及作用机制研究
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摘要
第一部分毕拨明宁碱/二氢毕拨明宁碱通过抑制SK-N-SH细胞APP的表达抑制Aβ的产生
目的:阿尔茨海默病(Alzheimer's disease,AD)是一种逐渐进展的神经系统退行性疾病,其特征为逐渐加重的痴呆和认知功能的障碍。AD最为显著的组织学特征为大脑皮质细胞外出现由β类淀粉多肽(amyloidβ,Aβ)积聚构成的老年斑(senile plaques,SP)。β淀粉样前体蛋白(amyloid precursor protein,APP)是一种Ⅰ型跨膜糖蛋白,APP经β分泌酶和γ分泌酶水解能够产生Aβ。β分泌酶和γ分泌酶是APP水解产生Aβ的关键酶,能够调控Aβ的产生。以往研究发现中药海风藤提取成分毕拨明宁碱/二氢毕拨明宁碱能有效抑制人神经母细胞瘤细胞(SK-N-SH细胞)的APP的蛋白表达水平,但是该药是否能够最终影响Aβ的水平以及β分泌酶和γ分泌酶的活性,需要进一步实验证实。本实验着重研究中药海风藤提取物毕拨明宁碱/二氢毕拨明宁碱能否抑制SK-N-SH细胞的Aβ产生和药物对β分泌酶和γ分泌酶的影响,以及药物可能的作用机制。
方法:用生药学的萃取和层析方法从中药海风藤中提取了毕拨明宁碱/二氢毕拨明宁碱(1:0.8)成分,然后用浓度3.13μg/mL,6.25μg/mL和12.50μg/mL的毕拨明宁碱/二氢毕拨明宁碱处理SK-N-SH细胞22小时。之后,分别采用MTT法检测细胞活性和乳酸脱氢酶(lactate dehydrogenase,LDH)释放法检测药物对细胞毒性的影响。采用Western Blot法检测SK-N-SH细胞Aβ_(42)和Aβ_(40)多肽的表达,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测SK-N-SH细胞Aβ_(42)和Aβ_(40)的产生的浓度。另外,采用Western Blot法检测APP、Notch胞内区多肽(Notch1)和β-位APP切割酶(BACE-1)蛋白的表达。同时,采用免疫细胞化学染色法检测SK-N-SH细胞Aβ_(42)的表达。另外,采用荧光分析法检测药物对SK-N-SH细胞β-分泌酶和γ-分泌酶活性的影响。
结果:实验结果显示,实验浓度的毕拨明宁碱/二氢毕拨明宁碱(3.13μg/mL,6.25μg/mL和12.50μg/mL)和0.1%DMSO对SK-N-SH细胞的活性无明显影响,无明显的细胞毒性(P＞0.05)。实验浓度的毕拨明宁碱/二氢毕拨明宁碱(3.13μg/mL,6.25μg/mL和12.50μg/mL)能有效抑制SK-N-SH细胞APP蛋白的表达和减少Aβ_(42)和Aβ_(40)肽的产生(P＜0.05),这种作用与药物溶剂0.1%DMSO无关。免疫细胞化学染色检测发现经毕拨明宁碱/二氢毕拨明宁碱处理的SK-N-SH细胞Aβ_(42)表达显著减少(P＜0.05)。但是对SK-N-SH细胞的BACE-1蛋白的表达和Notch1蛋白的表达无明显影响(P＞0.05)。实验浓度的毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞的β-分泌酶和γ-分泌酶的活性无明显影响(P＞0.05)。
结论:实验结果提示浓度为3.13μg/mL,6.25μg/L和12.50μg/mL的毕拨明宁碱/二氢毕拨明宁碱复合物能有效的通过抑制APP蛋白的表达,减少SK-N-SH细胞Aβ_(42)和Aβ_(40)多肽的产生。各浓度的毕拨明宁碱/二氢毕拨明宁碱不能影响SK-N-SH细胞的β-分泌酶和γ-分泌酶的表达和活性。
第二部分毕拨明宁碱/二氢毕拨明宁碱抑制SK-N-SH细胞β类淀粉前体蛋白基因启动子主要启动调控区蛋白结合活性及机制研究
目的:Alzheimer病(Alzheimer's disease,AD)即阿尔茨海默病,又称老年性痴呆,是一种逐渐进展的神经变性疾病。AD最为显著的组织学特征为大脑皮质细胞外出现由β类淀粉多肽(amyloidβ,Aβ)构成的老年斑。β淀粉样前体蛋白(amyloid precursor protein,APP)经β分泌酶和γ分泌酶水解能够产生Aβ。APP基因定位于人类21号染色体2区1带,APP基因的表达主要由其启动子调控,还受到激活蛋白-1(activator protein-1,AP-1)和白介素-1(interleukin-1,IL-1)的调控。在APP的启动子内,起主要作用的是APP基因的启动调控区(proximal promoterregion,PPR),位于其翻译起始位点上游-46至-1碱基之间。在APP的PPR区内有7个碱基也同时为AP-1的结合位点(TGA CTC G),能够接受AP-1的调节。
夏文等采用RT-PCR法和Western Blot法证实了从海风藤中提取的毕拨明宁碱/二氢毕拨明宁碱组成成分能够有效的选择性地抑制SK-N-SH细胞APP基因mRNA和蛋白的表达。为了研究毕拨明宁碱和二氢毕拨明宁碱抑制APP基因表达的可能的机制,本课题通过凝胶电泳阻滞实验(Electrophoretic mobility shift assay,EMSA)研究药物毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞APP启动子PPR区的蛋白结合活性的影响,同时观察药物对AP-1探针与核蛋白结合能力的影响。另外,在本研究中,还检测了药物对IL-1β,c-Jun,c-Fos和APP蛋白表达的影响,以探讨药物作用的可能的机制。
方法:用生药学方法从中药海风藤的水提物浸膏中依次用石油醚、乙酸乙酯、正丁醇萃取,加硅胶(100-200目)层析,收集洗脱流份,提取毕拨明宁碱/二氢毕拨明宁碱(1:0.8)组成成分。用浓度为3.13μg/mL,6.25μg/mL和12.50μg/mL的毕拨明宁碱/二氢毕拨明宁碱(1:0.8)处理SK-N-SH细胞22小时,同时用正常对照和0.1%DMSO药物溶剂对照。流式细胞仪检测药物毕拨明宁碱/二氢毕拨明宁碱对细胞周期的影响,细胞活力分析仪Beckman coulter Vi-CELL进行活力细胞计数。实时定量PCR法检测毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞APP基因mRNA的表达的影响。Western Blot法检测药物对SK-N-SH细胞IL-1β,c-Jun,c-Fos和APP蛋白表达的影响。EMSA检测APP基因启动子主要启动区(proximalpromoter region,PPR)以及激活蛋白-1(activator protein-1,AP-1)探针与SK-N-SH细胞核蛋白结合活性,研究药物对核蛋白结合活性的影响。
结果:经流式细胞仪和细胞活力分析仪检测,实验浓度的毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞的细胞周期和细胞活力无明显影响(P＞0.05)。经实时定量PCR和Western Blot检测,不同浓度的毕拨明宁碱/二氢毕拨明宁碱(3.13μg/mL,6.25μg/mL和12.50μg/mL)均能显著减少SK-N-SH细胞APP基因mRNA的表达水平和蛋白表达的水平,其抑制作用随剂量增加而增加(P＜0.05)。然而,毕拨明宁碱/二氢毕拨明宁碱不能影响SK-N-SH细胞IL-1β,c-Jun和c-Fos蛋白表达的水平(P＞0.05)。EMSA结果显示,毕拨明宁碱/二氢毕拨明宁碱能显著抑制SK-N-SH细胞APP基因PPR区和AP-1探针与核蛋白的结合活性(P＜0.05)。
结论:结果提示实验浓度的毕拨明宁碱/二氢毕拨明宁碱(3.13μg/mL,6.25μg/mL和12.50μg/mL)能有效负向调控SK-N-SH细胞AP-1探针和APP基因PPR区与核蛋白的结合活性。该药物有可能通过抑制APP启动子的蛋白结合活性来减少APP基因mRNA的表达和翻译,并且减少APP蛋白的表达。
PartⅠInhibitory Effect of Piperlonguminine/Dihydropiperlonguminine on the Production of Amyloidβand APP in SK-N-SH Cells
Aim:Alzheimer's disease(AD) is a progressive neurodegenerative disease characterized by progressive dementia and deterioration of cognitive function.The histopathological hallmarks of AD are the presence of extracellular deposits of amyloidβ(Aβ) peptide in senile plaques of cerebral cortex and the intracellular aggregation of tau protein in neurofibrillary tangles.Aβis a toxic 40-42 residue peptide derived from proteolysis of the amyloid precursor protein(APP).Certain mutations of APP gene may cause excessive cleavage of the protein byβ-secretase andγ-secretase,leading to the excessive aggregation of neurotoxic Aβpeptide.
Futokadsura stem is the petiole of piper plant Kadsura.Experimental studies showed that Futokadsura Stem is an efficient anti-inflammatory and anti-platelet drug. In previous experiments,Futokadsura stem showed selective inhibition on the expression of APP.Subsequently,we demonstrated that such inhibition on APP gene expression in human neuroblastoma cells(SK-N-SH) was mediated through the piperlonguminine/dihydropiperlonguminine components extracted from Futokadsura stem.Although piperlonguminine/dihydropiperlonguminine could efficiently inhibit the expression of APP at the protein level,whether they also affect the activities of Aβand Aβ-generating enzymes remains unclear.To answer this question,we investigated the effects of piperlonguminine/dihydropiperlonguminine on the production and peoteolytic processing of Aβ.
Methods:Piperlonguminine/dihydropiperlonguminine components(1:0.8) were extracted from Futokadsura stem,and then used to treat SK-N-SH cells at three different concentrations:3.13μg/mL,6.25μg/mL and 12.50μg/mL.Cell viability was assayed with the 3-[4,5-dimenthylthiazol-2-yl]-2,5- dimethyltetrazolium bromide (MTT) method and the lactate dehydrogenase(LDH) release method.Subsequently, the productions of Aβ_(42) and Aβ_(40) were measured by Western Blot analysis and enzyme linked imrnunosorbent assay(ELISA).On the other hand,the expressions of amyloid precursor protein(APP),Notchl(Notch intracellular domain) andβ-site amyloid precursor protein cleavage enzyme(BACE-1) were also examined by Western Blot assay.The activities ofβ-secretase andγ-secretase were detected at the same time.Furthermore,Aβ_(42) level was detected by immunocytochemistry staining.
Results:The treatment of piperlonguminine/dihydropiperlonguminine did not influence the cell viability of SK-N-SH cells under the experimental concentrations(P＞0.05).We demonstrated that the treatment of piperlonguminine/dihydropiperlonguminine could significantly decrease the levels of APP,Aβ_(42) and Aβ_(40) peptide in SK-N-SH cells(P＜0.05),despite the fact that the activities ofβ-secretase andγ-secretase were not affected significantly(P＞0.05).
Conclusion:These data suggested that piperlonguminine/dihydropiperlonguminine components could significantly decrease the level of APP,Aβ_(42) and Aβ_(40) peptide without affecting the activity ofβ-secretase andγ-secretase in SK-N-SH cells.
PartⅡPiperlonguminine/dihydropiperlonguminine inhibit protein binding to the proximal promoter region and expression of theβ-amyloid precursor protein gene in neuroblastoma cells
Aim:Alzheimer disease(AD) is a progressive neurodegenerative disease first described by Alois Alzheimer in 1906 that causes progressive dementia and deterioration of cognitive function.The pathological hallmarks of AD are extracellular deposits of amyloidβ(Aβ) peptide in senile plaques in the cerebral cortex and intracellular aggregation of tau protein in neurofibrillary tangles.Aβis a toxic 40-42 residue peptide derived from proteolysis of the amyloid precursor protein(APP).APP is an expressed typeⅠtransmembrane glycoprotein,and the APP gene is located on chromosome 21 q21.
The expression of APP is mainly regulated through its promoter,especially the proximal promoter region(PPR;-46/-1 in the human sequence;+1 is the transcription start site).The PPR plays an important role in APP expression,and it acts as a drug target for the treatment of AD.The promoter of APP can be upregulated by many factors,such as nerve growth factor,basic fibroblast growth factor,phorbol ester, retinoic acid and interleukin-1,which could increase the expression of APP and lead to Aβdeposition.
There is a region located between -485 and -305 upstream from the transcription start site of the APP promoter that is necessary for interleukin-1(IL-1) mediated transcription of the gene and contains an activator protein-1(AP-1) binding site.IL-1 could upregulate APP expression through a pathway mediated by protein kinase C utilizing the upstream AP-1 binding site of the APP promoter.
Piperlonguminine/dihydropiperlonguminine is a novel alkaloid derived from the Chinese folk medicine "Futokadsura stem".In previous experiments,we found that Futokadsura stem could selectively inhibit expression of APP.Subsequently it has been demonstrated that it is the piperlonguminine and dihydropiperlonguminine components of futokadsura stem that selectively inhibit the expression of the APP gene at both the mRNA and protein levels in SK-N-SH cells.
In order to investigate the mechanism by which piperlonguminine/dihydropiperlonguminine decreases the expression of APP,we explored the DNA binding activity of AP-1 to the PPR of APP in SK-N-SH cells treated with piperlonguminine/dihydropiperlonguminine.The protein levels of IL-1, cJun,cFos and APP were measured simultaneously.Furthermore,the transcription of APP was detected by Real-time PCR analysis.
Methods:Piperlonguminine/dihydropiperlonguminine components(1:0.8) were extracted from Futokadsura stem.Cell viability was assayed with the 3-[4,5-dimenthylthiazol-2-yl]-2,5- dimethyltetrazolium bromide(MTT) method,flow cytomertry and vi-cell count method.Human neuroblastoma(SK-N-SH) cells were treated with piperlonguminine/dihydropiperlonguminine components(1:0.8) at 3.13μg/mL,6.25μg/mE or 12.50μg/mL for 22 h.The expression of APP gene was measured by Real-time PCR,the expressions of IL-1β,c-Jun,c-Fos and APP were detected by western blot assay,the binding activity of the proximal promoter region (PPR) of APP and the consensus binding sequence for activator protein-1(AP-1) by nuclear proteins were measured with Electrophoretic mobility shift assays(EMSA).
Results:The treatment of piperlonguminine/dihydropiperlonguminine did not influence the cell viability and cell cycle of SK-N-SH cells under the experimental concentrations(3.13μg/mE,6.25μg/mL or 12.50μg/mL)(P＞0.05). Piperlonguminine/dihydropiperlonguminine at 3.13μg/mL,6.25μg/mL or 12.50μg/mL significantly decreased the expression of APP mRNA and protein,as measured by Real-time PCR and western blot(P＜0.05).However,the expression of IL-1β, c-Jun and c-Fos were not affected by piperlonguminine/dihydropiperlonguminine(P＞0.05).Electrophoretic mobility shift assays showed that binding of the PPR of APP and AP-1 by nuclear proteins were reduced in nuclear extracts from SK-N-SH cells treated with piperlonguminine/dihydropiperlonguminine at 3.13μg/mL,6.25μg/mL or 12.50μg/mL(P＜0.05).
Conclusion:The data suggest that piperlonguminine/dihydropiperlonguminine can negatively regulate the binding of AP-1 to the PPR of the APP gene in SK-N-SH cells,and it could decrease transcription of the gene and production of APP protein by affecting the promoter of the APP gene.
目的:阿尔茨海默病(Alzheimer's disease,AD)是一种逐渐进展的神经系统退行性疾病,其特征为逐渐加重的痴呆和认知功能的障碍。AD最为显著的组织学特征为大脑皮质细胞外出现由β类淀粉多肽(amyloidβ,Aβ)积聚构成的老年斑(senile plaques,SP)。β淀粉样前体蛋白(amyloid precursor protein,APP)是一种Ⅰ型跨膜糖蛋白,APP经β分泌酶和γ分泌酶水解能够产生Aβ。β分泌酶和γ分泌酶是APP水解产生Aβ的关键酶,能够调控Aβ的产生。以往研究发现中药海风藤提取成分毕拨明宁碱/二氢毕拨明宁碱能有效抑制人神经母细胞瘤细胞(SK-N-SH细胞)的APP的蛋白表达水平,但是该药是否能够最终影响Aβ的水平以及β分泌酶和γ分泌酶的活性,需要进一步实验证实。本实验着重研究中药海风藤提取物毕拨明宁碱/二氢毕拨明宁碱能否抑制SK-N-SH细胞的Aβ产生和药物对β分泌酶和γ分泌酶的影响,以及药物可能的作用机制。
方法:用生药学的萃取和层析方法从中药海风藤中提取了毕拨明宁碱/二氢毕拨明宁碱(1:0.8)成分,然后用浓度3.13μg/mL,6.25μg/mL和12.50μg/mL的毕拨明宁碱/二氢毕拨明宁碱处理SK-N-SH细胞22小时。之后,分别采用MTT法检测细胞活性和乳酸脱氢酶(lactate dehydrogenase,LDH)释放法检测药物对细胞毒性的影响。采用Western Blot法检测SK-N-SH细胞Aβ_(42)和Aβ_(40)多肽的表达,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测SK-N-SH细胞Aβ_(42)和Aβ_(40)的产生的浓度。另外,采用Western Blot法检测APP、Notch胞内区多肽(Notch1)和β-位APP切割酶(BACE-1)蛋白的表达。同时,采用免疫细胞化学染色法检测SK-N-SH细胞Aβ_(42)的表达。另外,采用荧光分析法检测药物对SK-N-SH细胞β-分泌酶和γ-分泌酶活性的影响。
结果:实验结果显示,实验浓度的毕拨明宁碱/二氢毕拨明宁碱(3.13μg/mL,6.25μg/mL和12.50μg/mL)和0.1%DMSO对SK-N-SH细胞的活性无明显影响,无明显的细胞毒性(P＞0.05)。实验浓度的毕拨明宁碱/二氢毕拨明宁碱(3.13μg/mL,6.25μg/mL和12.50μg/mL)能有效抑制SK-N-SH细胞APP蛋白的表达和减少Aβ_(42)和Aβ_(40)肽的产生(P＜0.05),这种作用与药物溶剂0.1%DMSO无关。免疫细胞化学染色检测发现经毕拨明宁碱/二氢毕拨明宁碱处理的SK-N-SH细胞Aβ_(42)表达显著减少(P＜0.05)。但是对SK-N-SH细胞的BACE-1蛋白的表达和Notch1蛋白的表达无明显影响(P＞0.05)。实验浓度的毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞的β-分泌酶和γ-分泌酶的活性无明显影响(P＞0.05)。
结论:实验结果提示浓度为3.13μg/mL,6.25μg/L和12.50μg/mL的毕拨明宁碱/二氢毕拨明宁碱复合物能有效的通过抑制APP蛋白的表达,减少SK-N-SH细胞Aβ_(42)和Aβ_(40)多肽的产生。各浓度的毕拨明宁碱/二氢毕拨明宁碱不能影响SK-N-SH细胞的β-分泌酶和γ-分泌酶的表达和活性。
第二部分毕拨明宁碱/二氢毕拨明宁碱抑制SK-N-SH细胞β类淀粉前体蛋白基因启动子主要启动调控区蛋白结合活性及机制研究
目的:Alzheimer病(Alzheimer's disease,AD)即阿尔茨海默病,又称老年性痴呆,是一种逐渐进展的神经变性疾病。AD最为显著的组织学特征为大脑皮质细胞外出现由β类淀粉多肽(amyloidβ,Aβ)构成的老年斑。β淀粉样前体蛋白(amyloid precursor protein,APP)经β分泌酶和γ分泌酶水解能够产生Aβ。APP基因定位于人类21号染色体2区1带,APP基因的表达主要由其启动子调控,还受到激活蛋白-1(activator protein-1,AP-1)和白介素-1(interleukin-1,IL-1)的调控。在APP的启动子内,起主要作用的是APP基因的启动调控区(proximal promoterregion,PPR),位于其翻译起始位点上游-46至-1碱基之间。在APP的PPR区内有7个碱基也同时为AP-1的结合位点(TGA CTC G),能够接受AP-1的调节。
夏文等采用RT-PCR法和Western Blot法证实了从海风藤中提取的毕拨明宁碱/二氢毕拨明宁碱组成成分能够有效的选择性地抑制SK-N-SH细胞APP基因mRNA和蛋白的表达。为了研究毕拨明宁碱和二氢毕拨明宁碱抑制APP基因表达的可能的机制,本课题通过凝胶电泳阻滞实验(Electrophoretic mobility shift assay,EMSA)研究药物毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞APP启动子PPR区的蛋白结合活性的影响,同时观察药物对AP-1探针与核蛋白结合能力的影响。另外,在本研究中,还检测了药物对IL-1β,c-Jun,c-Fos和APP蛋白表达的影响,以探讨药物作用的可能的机制。
方法:用生药学方法从中药海风藤的水提物浸膏中依次用石油醚、乙酸乙酯、正丁醇萃取,加硅胶(100-200目)层析,收集洗脱流份,提取毕拨明宁碱/二氢毕拨明宁碱(1:0.8)组成成分。用浓度为3.13μg/mL,6.25μg/mL和12.50μg/mL的毕拨明宁碱/二氢毕拨明宁碱(1:0.8)处理SK-N-SH细胞22小时,同时用正常对照和0.1%DMSO药物溶剂对照。流式细胞仪检测药物毕拨明宁碱/二氢毕拨明宁碱对细胞周期的影响,细胞活力分析仪Beckman coulter Vi-CELL进行活力细胞计数。实时定量PCR法检测毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞APP基因mRNA的表达的影响。Western Blot法检测药物对SK-N-SH细胞IL-1β,c-Jun,c-Fos和APP蛋白表达的影响。EMSA检测APP基因启动子主要启动区(proximalpromoter region,PPR)以及激活蛋白-1(activator protein-1,AP-1)探针与SK-N-SH细胞核蛋白结合活性,研究药物对核蛋白结合活性的影响。
结果:经流式细胞仪和细胞活力分析仪检测,实验浓度的毕拨明宁碱/二氢毕拨明宁碱对SK-N-SH细胞的细胞周期和细胞活力无明显影响(P＞0.05)。经实时定量PCR和Western Blot检测,不同浓度的毕拨明宁碱/二氢毕拨明宁碱(3.13μg/mL,6.25μg/mL和12.50μg/mL)均能显著减少SK-N-SH细胞APP基因mRNA的表达水平和蛋白表达的水平,其抑制作用随剂量增加而增加(P＜0.05)。然而,毕拨明宁碱/二氢毕拨明宁碱不能影响SK-N-SH细胞IL-1β,c-Jun和c-Fos蛋白表达的水平(P＞0.05)。EMSA结果显示,毕拨明宁碱/二氢毕拨明宁碱能显著抑制SK-N-SH细胞APP基因PPR区和AP-1探针与核蛋白的结合活性(P＜0.05)。
结论:结果提示实验浓度的毕拨明宁碱/二氢毕拨明宁碱(3.13μg/mL,6.25μg/mL和12.50μg/mL)能有效负向调控SK-N-SH细胞AP-1探针和APP基因PPR区与核蛋白的结合活性。该药物有可能通过抑制APP启动子的蛋白结合活性来减少APP基因mRNA的表达和翻译,并且减少APP蛋白的表达。
PartⅠInhibitory Effect of Piperlonguminine/Dihydropiperlonguminine on the Production of Amyloidβand APP in SK-N-SH Cells
Aim:Alzheimer's disease(AD) is a progressive neurodegenerative disease characterized by progressive dementia and deterioration of cognitive function.The histopathological hallmarks of AD are the presence of extracellular deposits of amyloidβ(Aβ) peptide in senile plaques of cerebral cortex and the intracellular aggregation of tau protein in neurofibrillary tangles.Aβis a toxic 40-42 residue peptide derived from proteolysis of the amyloid precursor protein(APP).Certain mutations of APP gene may cause excessive cleavage of the protein byβ-secretase andγ-secretase,leading to the excessive aggregation of neurotoxic Aβpeptide.
Futokadsura stem is the petiole of piper plant Kadsura.Experimental studies showed that Futokadsura Stem is an efficient anti-inflammatory and anti-platelet drug. In previous experiments,Futokadsura stem showed selective inhibition on the expression of APP.Subsequently,we demonstrated that such inhibition on APP gene expression in human neuroblastoma cells(SK-N-SH) was mediated through the piperlonguminine/dihydropiperlonguminine components extracted from Futokadsura stem.Although piperlonguminine/dihydropiperlonguminine could efficiently inhibit the expression of APP at the protein level,whether they also affect the activities of Aβand Aβ-generating enzymes remains unclear.To answer this question,we investigated the effects of piperlonguminine/dihydropiperlonguminine on the production and peoteolytic processing of Aβ.
Methods:Piperlonguminine/dihydropiperlonguminine components(1:0.8) were extracted from Futokadsura stem,and then used to treat SK-N-SH cells at three different concentrations:3.13μg/mL,6.25μg/mL and 12.50μg/mL.Cell viability was assayed with the 3-[4,5-dimenthylthiazol-2-yl]-2,5- dimethyltetrazolium bromide (MTT) method and the lactate dehydrogenase(LDH) release method.Subsequently, the productions of Aβ_(42) and Aβ_(40) were measured by Western Blot analysis and enzyme linked imrnunosorbent assay(ELISA).On the other hand,the expressions of amyloid precursor protein(APP),Notchl(Notch intracellular domain) andβ-site amyloid precursor protein cleavage enzyme(BACE-1) were also examined by Western Blot assay.The activities ofβ-secretase andγ-secretase were detected at the same time.Furthermore,Aβ_(42) level was detected by immunocytochemistry staining.
Results:The treatment of piperlonguminine/dihydropiperlonguminine did not influence the cell viability of SK-N-SH cells under the experimental concentrations(P＞0.05).We demonstrated that the treatment of piperlonguminine/dihydropiperlonguminine could significantly decrease the levels of APP,Aβ_(42) and Aβ_(40) peptide in SK-N-SH cells(P＜0.05),despite the fact that the activities ofβ-secretase andγ-secretase were not affected significantly(P＞0.05).
Conclusion:These data suggested that piperlonguminine/dihydropiperlonguminine components could significantly decrease the level of APP,Aβ_(42) and Aβ_(40) peptide without affecting the activity ofβ-secretase andγ-secretase in SK-N-SH cells.
PartⅡPiperlonguminine/dihydropiperlonguminine inhibit protein binding to the proximal promoter region and expression of theβ-amyloid precursor protein gene in neuroblastoma cells
Aim:Alzheimer disease(AD) is a progressive neurodegenerative disease first described by Alois Alzheimer in 1906 that causes progressive dementia and deterioration of cognitive function.The pathological hallmarks of AD are extracellular deposits of amyloidβ(Aβ) peptide in senile plaques in the cerebral cortex and intracellular aggregation of tau protein in neurofibrillary tangles.Aβis a toxic 40-42 residue peptide derived from proteolysis of the amyloid precursor protein(APP).APP is an expressed typeⅠtransmembrane glycoprotein,and the APP gene is located on chromosome 21 q21.
The expression of APP is mainly regulated through its promoter,especially the proximal promoter region(PPR;-46/-1 in the human sequence;+1 is the transcription start site).The PPR plays an important role in APP expression,and it acts as a drug target for the treatment of AD.The promoter of APP can be upregulated by many factors,such as nerve growth factor,basic fibroblast growth factor,phorbol ester, retinoic acid and interleukin-1,which could increase the expression of APP and lead to Aβdeposition.
There is a region located between -485 and -305 upstream from the transcription start site of the APP promoter that is necessary for interleukin-1(IL-1) mediated transcription of the gene and contains an activator protein-1(AP-1) binding site.IL-1 could upregulate APP expression through a pathway mediated by protein kinase C utilizing the upstream AP-1 binding site of the APP promoter.
Piperlonguminine/dihydropiperlonguminine is a novel alkaloid derived from the Chinese folk medicine "Futokadsura stem".In previous experiments,we found that Futokadsura stem could selectively inhibit expression of APP.Subsequently it has been demonstrated that it is the piperlonguminine and dihydropiperlonguminine components of futokadsura stem that selectively inhibit the expression of the APP gene at both the mRNA and protein levels in SK-N-SH cells.
In order to investigate the mechanism by which piperlonguminine/dihydropiperlonguminine decreases the expression of APP,we explored the DNA binding activity of AP-1 to the PPR of APP in SK-N-SH cells treated with piperlonguminine/dihydropiperlonguminine.The protein levels of IL-1, cJun,cFos and APP were measured simultaneously.Furthermore,the transcription of APP was detected by Real-time PCR analysis.
Methods:Piperlonguminine/dihydropiperlonguminine components(1:0.8) were extracted from Futokadsura stem.Cell viability was assayed with the 3-[4,5-dimenthylthiazol-2-yl]-2,5- dimethyltetrazolium bromide(MTT) method,flow cytomertry and vi-cell count method.Human neuroblastoma(SK-N-SH) cells were treated with piperlonguminine/dihydropiperlonguminine components(1:0.8) at 3.13μg/mL,6.25μg/mE or 12.50μg/mL for 22 h.The expression of APP gene was measured by Real-time PCR,the expressions of IL-1β,c-Jun,c-Fos and APP were detected by western blot assay,the binding activity of the proximal promoter region (PPR) of APP and the consensus binding sequence for activator protein-1(AP-1) by nuclear proteins were measured with Electrophoretic mobility shift assays(EMSA).
Results:The treatment of piperlonguminine/dihydropiperlonguminine did not influence the cell viability and cell cycle of SK-N-SH cells under the experimental concentrations(3.13μg/mE,6.25μg/mL or 12.50μg/mL)(P＞0.05). Piperlonguminine/dihydropiperlonguminine at 3.13μg/mL,6.25μg/mL or 12.50μg/mL significantly decreased the expression of APP mRNA and protein,as measured by Real-time PCR and western blot(P＜0.05).However,the expression of IL-1β, c-Jun and c-Fos were not affected by piperlonguminine/dihydropiperlonguminine(P＞0.05).Electrophoretic mobility shift assays showed that binding of the PPR of APP and AP-1 by nuclear proteins were reduced in nuclear extracts from SK-N-SH cells treated with piperlonguminine/dihydropiperlonguminine at 3.13μg/mL,6.25μg/mL or 12.50μg/mL(P＜0.05).
Conclusion:The data suggest that piperlonguminine/dihydropiperlonguminine can negatively regulate the binding of AP-1 to the PPR of the APP gene in SK-N-SH cells,and it could decrease transcription of the gene and production of APP protein by affecting the promoter of the APP gene.
引文
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1 Goedert M, Spillantini MG A century of Alzheimer's disease. Science 2006; 314: 777-81.
2 Irvine GB, El-Agnaf OM, Shankar GM, Walsh DM. Protein aggregation in the brain: the molecular basis for Alzheimer's and Parkinson's diseases. Mol Med 2008; 14: 451-64.
3 Pietrzik C, Behl C. Concepts for the treatment of Alzheimer's disease: molecular mechanisms and clinical application. Int J Exp Pathol 2005; 86:173-85.
4 Hardy, J. The relationship between amyloid and tau. J Mol Neurosci 2003; 20: 203-6.
5 LaFerla FM, Oddo S. Alzheimer's disease: Abeta, tau and synaptic dysfunction. Trends Mol Med 2005; 11: 170-6.
6 Findeis MA. The role of amyloid beta peptide 42 in Alzheimer's disease. Pharmacol Ther 2007; 116:266-86.
7 Mattson MP. Pathways towards and away from Alzheimer's disease. Nature 2004; 430: 631-9.
8 Lahiri DK, Ge YW, Maloney B. Characterization of the APP proximal promoter and 5'-untranslated regions: identification of cell type-specific domains and implications in APP gene expression and Alzheimer's disease. FASEB J 2005; 19: 653-5.
9 Matsui T, Ingelsson M, Fukumoto H, Ramasamy K, Kowa H, Frosch MP, et al. Expression of APP pathway mRNAs and proteins in Alzheimer's disease. Brain Res 2007; 1161:116-23.
10 Lahiri DK, Nail C. Promoter activity of the gene encoding the beta-amyloid precursor protein is up-regulated by growth factors, phorbol ester, retinoic acid and interleukin-1. Brain Res Mol Brain Res 1995; 32:233-40.
11 Goldgaber D, Harris HW, Hla T, Maciag T, Donnelly RJ, Jacobsen JS, et al. Interleukin 1 regulates synthesis of amyloid beta-protein precursor mRNA in human endothelial cells, Proc Natl Acad Sci USA 1989; 86: 7606-10.
12 Han EJ, Hu HT, He XQ, Deng XM, Lu Y, Zhou HT. Selective inhibition of haifengteng in gene expression of β-amyloid precursor protein. Chinese J Clin Rehabil 2004; 8: 2592-3.
13 Han E, Rajiv J. Inhibition of P-amyloid precursor protein gene expression by haifengteng. Zhongguo Zhong Yao Za Zhi 1998; 23: 691-3.
14 Xia W, Zeng JP, Chen LB, Jiang AL, Xiang L, Xu J, et al. Inhibition of beta-amyloid precursor protein gene in SK-N-SH cells by piperlonguminine/dihydropiperlonguminine components separated from Chinese herbal medicine Futokadsura stem. Chin J of Physiol 2007; 50:157-63.
15 Deng ZH, Wen JF, Li JH, Xiao DS, Zhou JH. Activator protein-1 involved in growth inhibition by RASSF1A gene in the human gastric carcinoma cell line SGC7901. World J Gastroenterol 2008; 14:1437-43.
16 Iwashita M, Saito M, Yamguchi Y, Takagaki R, Nakahata N. Inhibitory Effect of Ethanol Extract of Piper longum L. on Rabbit Platelet Aggregation through Antagonizing Thromboxane A2 Receptor. Biol Pharm Bull 2007; 30: 1221-5.
17 Salbaum JM, Weidemann A, Lemaire HG, Masters CL, Beyreuther K. The promoter of Alzheimer's disease amyloid A4 precursor gene. EMBO J 1988; 7: 2807-13.
18 Koh JY, Yang LL, Cotman CW. Beta-amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage. Brain Res 1990; 533:315-20.
19 Lansbury PT, Lashuel HA. A century-old debate on protein aggregation and neurodegeneration enters the clinic. Nature 2006; 443: 774-9.
20 Roberson ED, Mucke L. 100 years and counting: prospects for defeating Alzheimer's disease. Science 2006; 314: 781-4.
21 Peng Y, Lee DY, Jiang L, Ma Z, Schachter SC, Lemere CA. Huperzine A regulates amyloid precursor protein processing via protein kinase C and mitogen-activated protein kinase pathways in neuroblastoma SK-N-SH cells over-expressing wild type human amyloid precursor protein 695. Neuroscience 2007; 150: 386-95.
2 LaFerla FM, Oddo S. Alzheimer's disease: Abeta, tau and synaptic dysfunction. Trends Mol Med. 2005; 11(4): 170-176.
3 Pietrzik C, Behl C. Concepts for the treatment of Alzheimer's disease: molecular mechanisms and clinical application.Int J Exp Pathol. 2005; 86(3): 173-185.
4 Hardy J. The relationship between amyloid and tau. J Mol Neurosci. 2003; 20(2): 203-206.
5 Matsui T, Ingelsson M, Fukumoto H, Ramasamy K, Kowa H, Frosch MP, Irizarry MC, Hyman BT. Expression of APP pathway mRNAs and proteins in Alzheimer's disease. Brain Res. 2007; 1161: 116-123.
6 Nowotny P, Bertelsen S, Hinrichs AL, Kauwe JS, Mayo K, Jacquart S, Morris JC, Goate A. Association Studies between Common Variants in Prolyl isomerase Pin1 and the Risk for Late-Onset Alzheimer's Disease. Neurosci Lett. 2007; 419(1): 15-17.
7 Findeis MA. The role of amyloid beta peptide 42 in Alzheimer's disease. Pharmacol Ther. 2007; 116(2): 266-286.
8 Mattson MP. Pathways towards and away from Alzheimer's disease. Nature. 2004; 430(7000): 631-639.
9 Masters CL, Multhaup G, Simms G, Pottgiesser J, Martins RN, Beyreuther K. Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels. EMBO J. 1985; 4(11): 2757-2763.
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14 Leissring MA,Murphy MP,Mead TR,Akbari Y,Sugarman MC,Jannatipour M,Anliker B,M(u|¨)ller U,Saflig P,De Strooper B,Wolfe MS,Golde TE,LaFerla FM.A physiologic signaling role for the gamma-secretase-derived intracellular fragment of APP.Proc Natl Acad Sci USA.2002;99(7):4697-4702.
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18 Xia W,Zeng JP,Chen LB,Jiang AL,Xiang L,Xu J,Cui X,Han EJ.Inhibition of β-amyloid precursor protein gene in SK-N-SH cells by piperlonguminine/dihydropiperlongurninine components separated from Chinese herbal medicine Futokadsura stem.Chin J Physiol.2007;50(4):157-163.
19 韩恩吉,许军,Rajiv Joseph.海风藤抑制淀粉样蛋白诱导神经细胞胞浆钙离子升高的研究.山东医科大学学报.1998;36(3):239-241.
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25 Weggen S,Eriksen JL,Das P,Sagi SA,Wang R,Pietrzik CU,Findlay KA,Smith TE,Murphy MP,Bulter T,Kang DE,Marquez-Sterling N,Golde TE,and Koo EH.A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooygenase activity.Nature.2001;414(6860):212-216.
26 Weggen S,Rogers M,Eriksen J.NSAIDs:small molecules for prevention of Alzheimer's disease or precursors for future drug development? Trends Pharmacol Sci.2007;28(10):536-543.
27 宋敬丽,衰林,刘艳菊,李赫宇,谭文界.海风藤化学成分和药理作用的研究进展.湖北中医学院学报.2007;9(3):70-72.
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31 Koh,JY,Yang LL,Cotman CW.Beta-amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage.Brain Res.1990;533(2):315-320.
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35 Goldgaber D, Harris HW, Hla T, Maciag T, Donnelly RJ, Jacobsen JS, Vitek MP, Gajdusek DC. Interleukin 1 regulates synthesis of amyloid β-protein precursor mRNA in human endothelial cells. Proc Natl Acad Sci USA. 1989; 86(19): 7606-7610.
36 Schliebs R, Heidel K, Apelt J, Gniezdzinska M, Kirazov L, Szutowicz A. Interaction of interleukin-1β with muscarinic acetylcholine receptor-mediated signaling cascade in cholinergically differentiated SH-SY5Y cells. Brain Res. 2006; 1122(1): 78-85.
37 Roberson ED, Mucke L.100 years and counting: prospects for defeating Alzheimer's disease. Science. 2006; 314(5800): 781-784.
38 Licastro F, Porcellini E, Caruso C, Lio D, Corder EH. Genetic risk profiles for Alzheimer's disease: Integration of APOE genotype and variants that up-regulate inflammation. Neurobiol Aging. 2007; 28(11): 1637-1643.
39 Zhu X, Smith MA, Honda K, Aliev G, Moreira PI, Nunomura A, Casadesus G, Harris PL, Siedlak SL, Perry G.Vascular oxidative stress in Alzheimer disease. J Neurol Sci. 2007; 257(1-2): 240-246.
40 Patterson C, Feightner JW, Garcia A, Hsiung GYR, MacKnight C, Sadovnick AD. Diagnosis and treatment of dementia: 1. Risk assessment and primary prevention of Alzheimer disease. CMAJ. 2008; 178(5): 548-556.
1 Goedert M,Spillantini MG.A century of Alzheimer's disease.Science.2006;314(5800):777-781.
2 Irvine GB,El-Agnaf OM,Shankar GM,Walsh DM.Protein aggregation in the brain:the molecular basis for Alzheimer's and Parkinson's diseases.Mol Med.2008;14(7-8):451-464.
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6 Findeis MA.The role of amyloid beta peptide 42 in Alzheimer's disease.Pharmacol Ther.2007;116(2):266-286.
7 Mattson MP.Pathways towards and away from Alzheimer's disease.Nature. 2004;430(7000):631-639.
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15 Xia W,Zeng JP,Chen LB,Jiang AL,Xiang L,Xu J,Cui X,Han EJ.Inhibition of β-amyloid precursor protein gene in SK-N-SH cells by piperlonguminine/dihydropiperlonguminine components separated from Chinese herbal medicine Futokadsura stem.Chin J Physiol.2007;50(4):157-163.
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7 Mattson MP. Pathways towards and away from Alzheimer's disease. Nature 2004; 430: 631-9.
8 Lahiri DK, Ge YW, Maloney B. Characterization of the APP proximal promoter and 5'-untranslated regions: identification of cell type-specific domains and implications in APP gene expression and Alzheimer's disease. FASEB J 2005; 19: 653-5.
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20 Roberson ED, Mucke L. 100 years and counting: prospects for defeating Alzheimer's disease. Science 2006; 314: 781-4.
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