Modulation of microglia-mediated amyloid-beta clearance with nanoformulated polyphenol through the SIRT1-LDLR pathway

Light-controlled in situ synthesis of DNA microarrays (Affymetrix GeneChip® Mouse Genome 430 2.0) were performed to determine the altered gene expression. Affymetrix algorithm and multiple analysis comparison software were used for assessing gene expression differences, and mRNAs that increased or decreased in the brains of NP(α-M)-treated mice relative to that in the brains of Blank-NP-treated mice were identified. For the assay, total RNA was extracted using Takara RNAiso Plus Kit (Cat#9109, Takara, DaLian, LiaoNing, CN) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany). Array hybridization and wash was performed using GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US) and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) followed the manufacturer’s instructions. Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US) and Command Console Software 4.0 (Affymetrix, Santa Clara, CA, US) with default settings. Raw data were normalized by MAS 5.0 algorithm, Gene Spring Software12.6.1 (Agilent technologies, Santa Clara, CA, US). To elucidate the regulatory mechanism of NP(α-M) on LDLR expression, we conducted gene microarray analysis, and interpreted the data via Ingenuity Pathway Analysis, finding that the key genes related to LDLR expression were SIRT1, sterol regulatory element binding protein-2 (SREBP2), cAMP-response element binding protein (CREB) and proprotein convertase subtilisin/kexin type 9 (PCSK9).