Animals
30 Male Sprague Dawley rats (250–280 g) were used in this study. The rats were maintained at an ambient temperature of 22–24°C and 50–60% humidity, under a 12 h light: 12 h dark cycle with food and water available ad libitum.The animals were randomly divided into three groups: (1) normal control group, (2) STZ injection group, and (3) STZ injection plus treadmill exercise group. The study was approved by the institutional ethics committee and complies with the Declaration of Helsinki.
Reagents and instruments
RAGE antibody (Cell SignalingTechnolo-gy); HMGB1,NF-κB-p65 antibody (American Santa Cruz Biotechnology Co., Ltd.) HMGB1, NF-κB-p65 antibody, IκBα, GAPDH antibody, immunohistochemical kit, glycosylated hemoglobin kit, PBS phosphate buffer, 4% paraformaldehyde (Nanjing Jiancheng Biology Co., Ltd.), streptozotocin (Sigma Co., Ltd.); Before streptozotocin was used, the solution was prepared with 0.1mol/L citric acid buffer (0℃ ~ 4℃, pH=4.2) at the ratio of 1 to 100. Morris water maze (Shanghai Jiliang Software Technology Co., Ltd.); Eon full-wavelength enzyme labeling instrument of Shanghai Jisun Software Technology Co., Ltd. (BioTek); Sanolian Blood glucose Meter (SanNuo Biosensor Co., Ltd.); BI-2000 medical image analysis system (Chengdu Tai Meng Software Co., Ltd.).
Induction of diabetes
To induce diabetes, Four weeks after high fat diet (Basic feed 59%, sucrose 20%, lard 18%, egg yolk 3%), a single intraperitioneal injection of STZ (35 mg/kg, dissolved in 0.01-M citrate buffer at pH 4.5; Sigma Chemical Co.) was given to each animal, as the previously described method.15One week later, rats fasted for 10 hours and then blood glucose levels were measured by a glucometer (Roche, Germany). The rats whose blood glucose exceeded 12 mmol/L had diabetes and were utilized for the following study. A total of 10 rats were enrolled in the group after exclusion of rats with substandard blood glucose. Morris water maze was utilized to detect whether the rat model had cognitive impairment16 (compared with the blank control group, whether the platform could be found in the shortest time).
Exercise protocol
Before beginning the formal 60 day exercise protocol, animals were familiarized to treadmill running (5-20 min/day) for 5 consecutive days. After this period of habituation, the exercised animals performed 5 days of consecutive treadmill exercise (60 min/day) with 22 m/min speeds17. At the beginning of 60 min exercises, to warm up the rats, treadmill speed had been set at 5 m/min and progressively increased to 22 m/min. At the final of 60 minute exercises, the speed progressively decreased to 5 m/min to cool down. Mild electrical shock was accustomed the negligible amount to motivate animals to run. Control animals did not carry out treadmill exercise but were put on a nonmoving treadmill for 60 min/day for 5 days a week. Exercised animals were studied 24 h after their last exercise session.
Morris Water Maze Task
The Morris water maze task was used to evaluate memory function according to a previously described method.16 Four marked points are identified in the middle of the four quadrants of the maze, and a black platform is set in the water. The morris water maze test was performed 1 hour after the last exercise. Each marked point was trained once in the morning and once in the afternoon, each training interval was more than 30 min for four consecutive days. On the 5th day, the platform was removed, and the rat was thrown into the water from the marked point of the quadrant I toward the pool wall. The target rat's swimming time in the target quadrant and the number of time crossed the virtual platform were recorded within 90 seconds.
Sampling and specimen handling
After the Morris water maze task, all rats were fasted for 12 hours, and their body weight and fasting blood glucose were recorded. The blood was obtained from the orbital venous plexus of 8 rats in each group, and the blood was centrifuged with 3500r/min for 10 minutes. The serum was taken to determine glycosylated hemoglobin according to the instructions of the kit. After blood samples were collected, 4 rats were randomly selected from each group for anesthesia (10% chloral hydrate 3 ml / kg), fully fixed with PBS intracardiac perfusion, and craniotomy was performed to take 3 mm to 4 mm tissue blocks in the coronary coronal position. Hippocampus DG area)18,19, placed in 4% paraformaldehyde, fixed at 4℃ for 7 days, routine paraffin embedding and coronal sectioning, RAGE and NF-κB immunohistochemical staining, the remaining brain tissues were frozen with liquid nitrogen for using.
HE staining of hippocampus DG region of brain tissue
The isolated hippocampus tissue was placed in 4% paraformaldehyde and fixed at 4 ℃ for 24 days. before dehydration, fixed tissue was washed overnight with tap water before dehydration. The hippocampus tissue was dehydrated in different gradients of ethanol, transparent with xylene, twice immersed in paraffin, and embedded in conventional paraffin. 5 μm of tissue sections were stained with HE, transparent with xylene, and sealed with gum for electron microscope observation.
Immunohistochemical detection
The isolated hippocampus tissue was placed in 4% paraformaldehyde and fixed at 4 ℃ for 7 days, and then the coronal sections were prepared after routine paraffin embedding. According to the instructions of the immunohistochemical kit, the sections were routinely dewaxed and eliminated the activity of endogenous peroxidase. After washing, antigen repair was carried out, and the sections were closed and incubated overnight with corresponding primary antibodies (target protein primary antibodies: RAGE, p-NF-кBp65) at 4℃. The blot was washed and incubated with the secondary antibody combined with the primary antibody at room temperature for 2 h. The sections were stained with hematoxylin, dehydrated with ethanol, transparent with xylene and coated with neutral glue.
Western blot analysis
The frozen brain tissue was washed with PBS and incubated in lysis buffer, and then phosphatase inhibitor was added, the tissue was ground with a grinding rod until completely crushed, centrifuged twice at 4°C, and the supernatant was taken, the brain protein concentration was quantified using the BCA protein assay kit, then mixed with the loading buffer and heated with 2-mercaptoethanol at 100 ℃ for 5 min. The extracted protein was separated by SDS-PAGE and transferred to PVDF membrane. In TBST, the nonspecific binding sites were blocked by 5% skimmed milk powder, and then incubated overnight at 4℃ with corresponding primary antibodies (target protein primary antibodies: HMGB1, RAGE, p-NF-кBp65, p-IкBα, internal reference protein first antibody: NF-кBp65, IкBα, GAPDH). The blot was washed and incubated with the secondary antibody combined with the primary antibody at room temperature for 2 h. The ultra-sensitive ECL chemiluminescence kit and Clinx chemiluminescence imaging system display specific bands. The grayscale analysis of WB bands was carried out by ImageJv1.8.0, and the results were introduced into Excel. GAPDH (if the target protein is phosphorylated, total protein is used) as a reference, the gray ratio of bands is used as the expression result, and SPSS is used to analyze the results of each group.
Statistical analysis
Data are presented as the mean ± standard error of the mean. SPSS software version 23.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. One way ANOVA was used when the normal distribution was uniform and the variance was homogeneous. Nonparametric test was used when the normal distribution was not uniform or the variance was not homogeneous. One way ANOVA was employed to the test results.