Reagents
Nissl Staining Solution, Antifade Mounting Medium with DAPI and RIPA Lysis Buffer were purchased from Beyotime Biotechnology (Shanghai, China). Melatonin and EX527 were obtained from MedChemExpress, LLC (Princeton, NJ, USA). Scopolamine Hydrobromide, MTT and tunicamycin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Nanjing Jiancheng Bioengineering Institute (Nanjing, China) provided the following kits: ChAT, Ach, MDA, SOD, GSH-Px, CAT. The kit of AChE was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). RNAiso Plus, TB Green Premix Ex Taq, Prime Script RT Master Mix were obtained from Takara Bio Inc. (Ostu, Shiga, Japan). Lipofectamine 3000 Transfection Reagent and anti-p-PERK antibody were purchased from Invitrogen Corporation (Carlsbad, CA, USA). The Sirt1 RNAi was synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Cell Signaling Technology, Inc (Danvers, MA, USA) offered the following antibodies: anti-SIRT1, anti-Bip, anti-PDI, anti-PERK, anti-IRE1α, anti-ACTB, Alexa Fluor® 488 Conjugated-anti-rabbit IgG, HRP-linked Goat anti-rabbit IgG, HRP-linked Horse anti-Mouse IgG. Anti-ATF6, anti-XBP1, anti-ChAT, anti-AChE antibodies were purchased from Abcam (Cambridge, MA). Anti-MT1A and anti-MT1B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-IRE1αand anti-GAPDH antibodies were purchased from Novus Biologicals (Littletone, Colorado, USA) and Beijing Biosynthesis Biotechnology Co., LTD. (Beijing, China) respectively.
Animal experiments
Male C57BL/6J mice were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). All mice were housed on standard laboratory conditions (indoor temperature 21 ± 2 ℃, relative humidity 55 ± 5 %, 12 h light/dark cycle) and allowed ad libitum access to water and food. 8-week-old mice (weighing 25-30 g) were randomly divided to four groups (n = 10 per group): Con (control); SCOP (scopolamine 4 mg/kg); Mel-L (scopolamine 4 mg/kg + melatonin 10 mg/kg); Mel-H (scopolamine 4 mg/kg + melatonin 20 mg/kg). Mice were administered intragastrically with melatonin once a day for four weeks. Intraperitoneal injection of SCOP was half hour before animal behavior test. In addition, the control group was given normal saline. These treatments were performed at 8:00-11:00 AM of the day.
Morris water maze test
The SCOP was injected 30 min before the behavioral tasks while melatonin was administrated as usual. Each mouse was trained from four water entry points one day for three consecutive days. The escape latency was recorded for each trail for five consecutive days. On the sixth day, the spatial probe test was executed. Each mouse was allowed to swim 60 s without the platform. The time spent in platform quadrant, the numbers of crossing where the platform was before and the average speed of mice swimming were measured.
Open field test
The open field test was conducted as reported by Yu Xi et al (Xi et al., 2019). with minor modification. The numbers and the distance of each mouse entered to the central area (the middle four squares) and the total distance they traveled were recorded respectively.
Novel object recognition test
On the first day, two identical objects were placed on an open arena (30 cm × 30 cm) where each mouse was allowed to move freely for 5 min. The next day, one of objects was replaced with a new one (the other one was still day one) and the mice were allowed to explore again. Exploration times of novel and familiar objects were recorded in order to calculate the discrimination index.
18F-FDG PET imaging
The mice were intraperitoneal injected with 18F-fluordeoxyglucose (18F-FDG), and then scanned by Focus 220 microPET scanner (Siemens Medical Solutions USA, Inc., Knoxville, TN, USA). The dynamic scans were conducted for 0.5 h. PET images were reconstructed by using the microPET-CT manager (Siemens Medical Solutions USA, Inc.).
Nissl staining
The Nissl staining was conducted according to those we reported earlier (S.-J. Zhang et al., 2020). Brain paraffin sections were successively treated with xylene, alcohol and distilled water. Then the sections were stained for 10 min, cleared with distilled water. Images were acquired by microscope (Leica Microsystems, Wetzlar, Germany).
Measurement the activities of Ach, AChE, ChAT and Oxidative stress biomarkers
The homogenate of mice brains was centrifuged 12,000 × g at 4 ℃ for 15 min, the supernatant was used to detected the levels of acetyl choline (Ach), acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and oxidative stress biomarkers. Measured the absorbance by Universal Microplate Spectrophotometer (Bio-Rad, Hercules, CA, USA).
Western blotting analysis
RIPA with phosphatase inhibitor and protease inhibitor was used to lyse mice brains or HT22 cells. The following primary antibodies were used: Metallothionein 1A (MT1A), Metallothionein 1B (MT1B), AChE, ChAT, brain-derived neurotrophic factor (BDNF), postsynaptic density protein-95 (PSD95), SIRT1, Bip, protein disulfide isomerase (PDI), PERK, phosphorylate PERK (p-PERK), IRE1α, phosphorylate IRE1α (p-IRE1α), activating transcription factor 6 (ATF6), X-box binding protein 1 (XBP1).
Immunofluorescence
The tissue sections or cell slides were fixed with 4 % paraformaldehyde for 30 min, permeabilized with 0.5 % TritonX-100 for 15 min, washed with PBS for 3 × 3 min. Then they were blocked with 10 % bovine serum albumin (BSA), dropped the primary antibodies (SIRT1 1:400, p-IRE1α 1:400, XBP1 1:400) at 4 ℃ overnight. The Alexa Fluor® 488 Conjugated-anti-rabbit (1:1000) was used as secondary antibody and sealed with DAPI. The samples were photographed by fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
Cell culture and treatment
HT22 cells were incubated in the dulbecco's modified eagle medium (DMEM) with 10 % fetal bovine serum (FBS) and 1 % Penicillin-Streptomycin (P/S) at 37 °C in air containing 5 % CO2. The media was changed every day, and the cells were successively used when they reached 80 % fusion. The cells were incubated in serum-free medium with melatonin for 24 h in 96-well plate, then treated SCOP (Muhammad et al., 2019) or TM (H. Kim, Baek, Chang, Yang, & Lee, 2020) for another 24 h.
RNAi transfection
HT22 cells were transfected with Sirt1 RNAi (50 nM) or control RNAi (50 nM) by using Lipofectamine 3000 transfection reagent. Transfection occurred 24 h before the addition of other treatments.
Statistical analysis
The escape latency data were analyzed by using multi-factor analysis of variance of SPSS 16.0. The other data were subjected to One-way ANOVA with Bonferroni test by using GraphPad Prism 5.0. All data were presented as the mean ± SEM and P < 0.05 was regarded as statistically significance.