The Regulation Effects Of Conophylline On ADAM10 And Related Researches

Background:ADAM10 plays an important role in APP metabolism.It regulates APP as soluble and non-toxic product—sAPP?,which reduces the production of toxic A? and has important physiological function in the brain.In the previous study,we have found that the pancreatic ?-cell differentiation drug Conophylline can significantly enhance the luciferase activity of ADAM10 gene promoter by high-throughput drug scanning.Objective:To detect the effect and mechanism of Conophylline on ADAM10 and AD at vitro level.Methods:Using qPCR?luciferase assay to detect the transcriptional effect of Conophylline on ADAM10 at vitro level.Western Blot was used to detect the effect on APP metabolism,Tau expression.Analyzing the effect of CNP on transcrition factors belong to ADAM10 with Si-RNA.Results:(1)Conophylline could effectively promote the expression of ADAM10 mRNA or protein level in SH-SY5 Y,HT22 and HEK293 cell lines.(2)Conophylline could down-regulate the expression of BACE1 and CTFs in HEK-APP cell line by Immunoblotting.(3)The expression of autophagy-related molecules LC3-II can be enhanced by Conophylline,suggesting that Conophylline might be involved in autophagy process,and the down-regulation of CTFs could ascribe to this.(4)Conophylline reduced the protein level of total Tau and pSer396 Tau,Ser262 Tau and the related kinase CDK5.(5)Conophylline could activate the fluorescence activity of different fragments of the core promoter region of ADAM10 gene(-508--300,-467--300,-444--300),among which-508--467 region reacted most obviously.(6)Si-RNA technics was used to inhibite the transcription factor Sp1,USF1 and CAAT-BOX binding element NF-YB among ADAM10 promoter-508-33 region,respectively.The results revealed that the CAAT-BOX regulatory elements played a great role in the regulation of Conophylline on ADAM10.(7)PI3K inhibitor Wortmannin reduced the effect of Conophylline on ADAM10,indicating that PI3 K signaling pathway might be involved in the process of Conophylline.Conclusions:In summary,this study revealed the protective effects of a novel small molecule Chinese herbal compound--Conophylline,on Alzheimer's disease and its possible mechanism.This research could have provided new experimental evidence for the later vivo study.