Experimental animals
This experimental protocol was conducted on 60 adult male SD rats, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., with production license number SCXK (Lu) 20190003. After purchase, the rats were first fed adaptively in the animal room of the Human Anatomy Department of our hospital for one week, all under the same conditions. During this period, they can freely access food and water. During the experiment, the laboratory was ensured to have sufficient lighting, clean and well ventilated, and the room temperature was controlled at around 24 ℃. All relevant operations during the experimental process comply with the requirements of international health research institutions and the Ethics Committee of our institution regarding animal experiments.
Experiments Design
Experiment 1: Evaluate whether a rat model of hyperhomocysteinemia was successfully established. Randomly divide rats weighing 230-250g into a normal group (12 rats) and a model group (12 rats)(Fig. 1).
Experiment 2: On the basis of Experiment 1, explore the improvement effect and mechanism of Quercetin on the learning and memory ability of AD model rats based on the AMPK/mTOR pathway. 24 rats injected with Hcy via tail vein were randomly divided into a model group (12 rats), a Quercetin group (12 rats), and an inhibitor group (12 rats) (Fig. 1).
Model preparation and Drug Treatment
Model preparation
The rats were placed in a rat fixator, and their tails pierced through the small hole below the fixator pad. They were wiped with an alcohol swab and then soaked in 37 ℃ warm water for 2 minutes to achieve cleaning, disinfection, and dilation of blood vessels. At the junction of the tip of the rat's tail to the center 1/3 of the base of the tail, the needle is inserted diagonally upwards. Each rat were injected with Hcy (400ug/kg/d, Sigma, #H4628) through the tail vein.
Drug Treatment
Control group: On the day of modeling, rats in the Control group were weighed and injected with the actual dose of normal saline (NS) through the tail vein. Continuous injection for 14 days.The success of establishing a rat model of hyperhomocysteinemia was evaluated using a water maze and Western blot experiments.
Hcy group: After confirming successful modeling, only 0.9% sodium chloride solution (2ml/kg) was administered by gavage without any other treatment.
QT group: After confirming successful modeling, QT (15mg/kg/day, Bioss, #D10747) was administered by gavage for 4 consecutive weeks.
CC group: After confirming successful modeling, QT was administered by gavage for 4 consecutive weeks, and Compound C(AMPK inhibitor) (10mg/kg/day, MCE, #HY-13418A ) was administered by gavage for 2 consecutive weeks.
Morris water maze test
Morris water maze (Beijing Zhongshi Dichuang Science༆Technology Development Co., Ltd.) test was used to detect the spatial learning and memory ability of rats. The rats were placed in a water maze (black, 2cm below the water surface, about 10cm in diameter) to find a hidden platform, and the time when the rats found the platform was recorded, that is, the escape latency. The positioning navigation experiment was to train for 6 consecutive days, randomly select one of the four quadrants as the start, put the rat into the pool facing the pool wall by hand, if the rat did not find the hidden platform within 90 seconds, guide it to the platform, let the rat stay on the platform for 30 seconds and take it out, and record the escape latency of the rat as 90 seconds. At an interval of one day after the positioning navigation experiment, the platform was removed for the space exploration experiment, and the rats entered from the adjacent quadrant of the original platform quadrant in a pseudo-random manner. The number of times of crossing the original platform and the residence time in each quadrant were recorded. The longer the rats stayed in the quadrant where the previous platform was, the better their spatial memory ability was. A video tracking system was used to record the motion trajectory of each rat and the time to find the platform in each experiment. During the experiment, keep the quiet environment of the experiment.
After the behavioral study, the materials were obtained under anesthesia. Six rats were used for western blot and six rats were used for morphological experiment.
Western blot
The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the brains were sacrificed. The brain tissues were stored in a -80 ℃ freezer for subsequent protein extraction. A small amount of tissue block was added to the lysate for grinding, and was fully lysed. After centrifugation and standing, the supernatant was collected, and 5 × The loading buffer was boiled, and then the Western blot experiment was performed. Prepare separation gel first, then concentrated gel, insert the comb for loading, SDS-PAGE gel electrophoresis concentrated gel voltage 80V, separation gel voltage 120V, until bromophenol blue electrophoresis to the lower end of the gel to stop electrophoresis; After electrophoresis, the gel was cut and the membrane was transferred according to the molecular weight of marker molecule and target protein; After the membrane transfer, the skimmed milk powder was blocked, slowly shaken on a shaker for 2 hours, and the primary antibody was added. The primary antibody was diluted and mixed with 5ml of universal antibody diluent according to the appropriate ratio in the antibody instructions, including GAPDH Antibody (1:10000,Affinity, #AF7021), AMPK antibody (1:1000, Affinity, #AF6423), p-AMPK antibody(1:1000, Affinity, #AF3423), mTOR antibody (1:800,boster,#BM4182), p-mTOR antibody(1:1000, Affinity, #AF3308), p-Tau antibody (1:1000, Signalway Antibody, #11110) and incubated overnight at 4℃; After TBST washing, add the secondary antibody. The secondary antibody is diluted and mixed with 5ml of universal antibody dilution solution according to the appropriate proportion of antibody instructions (HRP-labeled Goat Anti-Rabbit IgG(H + L) (1:1000, Beyotime, #A0208)) and incubated for 1hour at room temperature on a shaking table; TBST was exposed after washing, and the gray values of AMPK, p-AMPK, mTOR, p-mTOR and p-Tau were observed to calculate their relative expression.
Nissl staining
After paraffin sections were dewaxed, brain slices were stained with Nissl staining solution(G1430, Solarbio)at 56 ° C for 1h, and then washed with deionized water. The slices were placed in Nissl differentiation solution༈G1430, Solarbio༉for differentiation for several seconds to 2 minutes, and the background was observed under the microscope until it was nearly colorless. After dehydration with gradient alcohol, the film is sealed with neutral gum and dried naturally; Microscopic imaging analysis system was used to observe the cells in CA1 and CA3 regions of the hippocampus and take photos for analysis.
Immunohistochemistry
After the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, the hearts were perfused. During perfusion, 100ml of 0.9% NaCl was used first, followed by 300ml of 4% paraformaldehyde. The brain was removed and placed in 4% paraformaldehyde for 24 hours, i.e. post fixation. The brain was removed from the fixative solution, the liquid on the surface was blotted with filter paper, and the brain segment where the hippocampus was located was separated according to the anatomical atlas of rats and embedded in paraffin. Paraffin sections were cut into 5 µm. After drying, the film was dewaxed by xyleneⅠ, xyleneⅡ, 100% alcohol Ⅰ, 100% alcohol Ⅱ, 95% alcohol, 75% alcohol gradient alcohol for 5min each; Rinse with PBS once, add 3% hydrogen peroxide solution to inactivate endogenous enzyme for 10min; Wash with PBS, add citric acid buffer (ph = 6.0), and cool naturally after antigen retrieval in microwave oven; After washing with PBS, add 5% BSA 37 ℃ for 40 min, shake it dry, and add primary antibody (Rabbit Anti-phospho-AMPK alpha 2 (Ser173) antibody(1:200,bioss, #bs-5575R); Phospho-mTOR (Ser2448) Antibody (1:200 ,Affinity biosciences, #AF3308);p-Tau(Phospho-Thr231) Antibody(1:100, Signalway Antibody, #11110) )at 4 ℃ overnight; The next day, after being taken out at 4 ℃, it was re incubated at room temperature, then rinsed with PBS and added with HRP-labeled Goat Anti-Rabbit IgG(H + L) (1:50,Beyotime, #A0208), and incubated at 37 ℃ for 30min; Rinse with PBS, drop the prepared DAB chromogenic solution (HRP:liquid A:liquid B = 1000: 50:50), and rinse with double distilled water after the cytoplasm is fully colored under the microscope; After dehydration with gradient alcohol, the film is sealed with neutral gum and dried naturally; Microscopic imaging analysis system was used to observe the cells in hippocampal CA1 and CA3 regions and take photos for analysis.
Data analysis
SPSS 26.0 statistical software was used for statistical analysis, and one-way analysis of variance was used for comparison between groups. The results were expressed as mean ± standard deviation (\(\stackrel{-}{\text{x}}\pm \text{s}\)), and p<0.05 indicated statistical significance.