The Research On The Effect And The Mechanism Of Piperlonguminine/dihydropiperlonguminine Inhibit The Production Of Amyloidβ And Amyloid Precursor Protein In SK-N-SH Cells

Partâ… Inhibitory Effect of Piperlonguminine/Dihydropiperlonguminine on the Production of Amyloidβand APP in SK-N-SH CellsAim: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 Aβ₄₂ and Aβ₄₀ 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,Aβ₄₂ 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,Aβ₄₂ and Aβ₄₀ 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,Aβ₄₂ and Aβ₄₀ peptide without affecting the activity ofβ-secretase andγ-secretase in SK-N-SH cells.Partâ…¡Piperlonguminine/dihydropiperlonguminine inhibit protein binding to the proximal promoter region and expression of theβ-amyloid precursor protein gene in neuroblastoma cellsAim: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 typeâ… transmembrane 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.