Healthy adult male ICR mice (21 weeks old, weighting between 33 and 46g) were used for animal experiments. All animals were purchased from Charles River Laboratories JAPAN Inc., Kanagawa, Japan and were housed individually at controlled temperature (25°C), with a 12/12 h light/dark cycle, and had access to food and water ad libitum. After acclimatization, the mice were randomly assigned into four experimental groups (n = 6/group): Control group (PBS), the depression model group: lipopolysaccharide group (LPS), control group treated with Luteolin (PBS+L) and lipopolysaccharide group treated with Luteolin (LPS+L). All experimental animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals, and the protocol was approved by the Animal Ethics Committee of the University of Tsukuba, Japan . At day 1 and prior to depression induction by LPS, the behavioral test: Tail Suspension Test (TST) was performed to all mice to screen their initial stress status. The TST methodology used in our study was as described by Steru (Steru, Chermat et al. 1985). Briefly, the duration of the test was 6 min and the immobility time was measured on the last 4 min of the test. The mouse was considered immobile only when it is hanged passively, showing no resistance to the stress applied by the test. The experiment was recorded using a camera and scored by videos observations. Immediately after performing the TST test, LPS (850µg/kg) was injected via the intraperitoneal route (i.p.) to the LPS and LPS+L groups and PBS was injected by the same route to the PBS and PBS+L groups. Starting from Day 2 and during eight consecutive days, luteolin (10mg/kg) and PBS were orally administrated to mice once per day. Luteolin (10mg/kg) was administrated to the LPS+L and PBS+ L groups and PBS to both PBS and LPS groups. At day 9, mice were sacrified. Two mice brains were used to extract total RNA from mice hippocampus.
Extracted molecule
total RNA
Extraction protocol
The total RNA was extracted from the cells using ISOGEN kit (Nippon Gene Co. Ltd., Japan) following the manufacturer’s instructions. Total RNA was purified using chloroform and Isopropanol (Wako, Japan) and quantified and assessed for quality with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 9.4 ug total RNA
Hybridization protocol
Following fragmentation, 100 ng of cRNA were hybridized for 16 hr at 45C on GeneChip Mouse Genome Array (MG-430). GeneChips were washed and stained in the Gene Atlas Fluidics Station 400
Scan protocol
GeneChips were scanned using the GeneAtlas Imaging Station
Data processing
The raw data were normalized using Expression Console Software provided by the Affymetrix following robust multichip average (RMA) algorithm (http://www.affymetrix.com). Subsequent analysis of the gene expression data was carried out in the freely available Transcriptome Analysis Console (TAC) version 4 (Thermofisher inc.). Further analysis was conducted using an online data mining tool DAVID (Database for Annotation, Visualization and Integrated Discovery, ver. 6.8). We used 'Functional Annotation' tool of DAVID to identify the most relevant biological terms, including gene ontology (GO) terms, biological pathways. Expression console signal intensity. For calculating average value, standard deviation, fold change, and p-value of signal intensity of replicates, we used freely available Transcriptome Analysis Console software V 4.0 (CHP files ).
