Animals and house
The five-week-old db/db mice (BKS- Leprem2Cd479/Gpt, strain number: T002407) were purchased from the GemPharmatech Co., Ltd (Nanjing, China). Mice were given 1 week to habituate to the facility and allowed ad libitum access to normal chow and water. For circadian disruption, one group (n=16) was kept on 6:18 light/dark cycle (6:18 LD cycle). The lights were switched at 7 am and off at 1 pm, which were regarded as zeitgeber time (ZT) 0 and ZT6 respectively. The control group (n=16) remained in a 12:12 light/dark cycle (12:12 LD cycle), with lights on at 7 am (ZT0) and off at 7 pm (ZT12). All animal experiments were approved by Animal Care and Use Committee of Tongji Hospital in accordance to the Public Health Service Policy on Human Care and Use of Laboratory animals.
At the age of 14 weeks, behavioral tasks were performed in db/db mice to assess their cognitive ability. Then they were euthanized every six hours in one day (ZT0, ZT6, ZT12 and ZT18) to evaluate the effect of light on diurnal rhythmicity. Their brains were removed and sagittally bisected. The right hemisphere was fixed in 4% paraformaldehyde for immunohistochemical analysis; the hippocampus was separated from the left hemisphere and frozen at −80 °C for later western blotting.
Open field test
For all behavior tests, we adapted the protocols from Volmar et al . The open field test was performed to test the locomotor activity of db/db mice. Briefly, the open field arena was made up of a standard clear plastic box (45 ×45 × 45 cm) placed in a quiet, well-lit room. Animals were individually put in the center of the box for 10 min. The tracks of mice were recorded using a computerized Ethovision detection system (Noldus, Netherlands). Total distance as well as average speed each mouse traveled during that time were automatically recorded. Arena was clean by 70% ethanol between different tests.
Novel object recognition test
A novel object recognition test aims to assess hippocampal associated contextual learning in rodents. This test was performed in the same box where the open field test was conducted. First, each mouse was allowed to explore two identical objects in the arena for 5 min and then it was sent back to home cage. After 30 min interval, this mouse was exposed to two different objects (one was the previous object and another was a novel object with different shape and color) in the arena. The frequency and time the mouse explored the novel object were used to evaluate the use of learning and recognition memory. Frequency related memory index was defined as [(frequency of novel object investigation)/(total frequency of investigation of both objects)*100] and time related index was calculated as [(novel object investigation time)/(total investigation time of both objects)*100]  .
Barnes maze test
A Barnes maze test was used as a test for spatial learning and memory assessment. Simplified Barnes maze contained “Acquisition” and “ Probe” trials.
Barnes maze Acquisition trial. We used a dark grey PVC circular platform (100 cm in diameter, elevated 90 cm above the floor) with 20 holes (5 cm in diameter) as the Barnes maze. An escape box was hidden under one of the holes. The aversive cues (the bright light and buzzer sound) were set up in the surroundings to stimulate mice to escape in 5 min. When the mice were exposed to these conditions, they were allowed to escape or were gently guided to the box. From day 1 to day 3, we trained animals for twice with an inter-trial interval of 30 min. On day 4, the mice had a rest for 24 h. Then, on the final day, we conducted the probe trial.
Barnes maze Probe trial. In Probe trial, the escape box was removed from holes. Target zone was the goal hole where the escape box was previously located in the acquisition test. The errors the mouse made before finding the target zone was used as an evaluation of spatial memory retention.
RNA isolation and RT-qPCR
Hippocampus samples were collected for RNA isolation at the different ZTs. Total RNA was extracted by TRIzol reagent according to the manufacturer’s instructions (Takara Bio, Japan). cDNA was synthesized using the Hifair II Reverse Transcription System following the manufacturer’s instructions (Yeasen Biotech Co. Ltd, Shanghai, China). Real-time reverse transciptase–PCR was conducted using the QuantStudioTM 1 system (Thermo Fisher Scientific biosystem) and SYBR Green qPCR Master Mix (Yeasen Biotech Co. Ltd, Shanghai, China). Gene expression was normalized to housekeeping genes (glyceraldehyde 3-phosphate Dehydrogenase, Gapdh). The primer sequences were referred to previous study. Comparative CT method (2−ΔCT) was used to calculated the relative gene expression.
The total protein was obtained with the cell lysis buffer for western and IP containing a protease and phosphatase inhibitor (Beyotime Inc., Shanghai, China). We collected the supernatants after centrifugation and then measured the concentration via a bicinchoninic acid (BCA) assay (Boster Biological Technology co.ltd., Wuhan, China).
The prepared protein samples were separated in 10% Bis-Tris SDS-polyacrylamide gels and then transferred to a nylon-PVDF Immobilon-PSQ membranes at 200 mA. The membranes were blocked in blocking buffer for 1 h at room temperature, and then were incubated overnight with primary antibody at 4C. The primary antibodies were included Tau5 (1:5000, Abcam, Cat # ab80579), p-Ser199 (1:5000, Abcam, Cat # ab81268), p-Ser396(1:5000, Abcam, Cat # ab109390), p-Thr231(1:5000, Abcam, Cat # ab151559) and β-actin(1:10000, Proteintech, Cat # 66009-1-Ig). After washing for several times, the membrane was incubated with secondary antibodies (1:7500; Proteintech, Cat # SA00001-1 and SA00001-2) for 2 h. The immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection (Biosharp, Beijing, China), and were scanned using GelView 6000 Pro (antpedia, China). Band density was measured using Image J software.
The immunohistochemistry analysis was detected as previous study . The immunoreactivities of the CA1 hippocampal region were measured with specific primary antibodies against anti-beta Amyloid 1-42 antibody (1:200, Abcam, ab201061). We used the streptavidin–horseradish peroxidase method and the reaction was visualized with the diaminobenzidine (DAB) detection process. The Aβ 1–42 immunoreactivities were calculated by counting blindly the numbers of Aβ plaques in CA1 regions under 10x, 20x and 40x magnification. Data were collected from four random fields in every section (n=3-5) for statistical analysis.
All statistical analyses were assessed by Student’s t test, one‐way and two‐way ANOVAs using SPSS (version 24.0 for Windows). Figures were plotted with GraphPad Prism 8. We used post hoc with Bonferroni’s correction when appropriate. Results were regarded as significance at P < 0.05.