Animals
Thirty wild‐type male 6‐week‐old C57BL/6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China, license [SCXK (Zhejiang) 2021-0006]), weighing 22–25g. All mice were kept in an SPF animal feeding room at the Shandong University of Traditional Chinese Medicine [SYXK (Lu) 20220009]. The mice were housed individually (20-22 °C), a relative humidity of 60%–70%, light time 7:00–19:00. Animal Ethics SDUTCM20230407002.
Model and groups
After fasting for 12 h, 15 mice were randomly selected to undergo abdominal anesthesia with tribromoethanol dissolved in tert-amyl alcohol (0.1 mL/10 g) to relieve pain. The brain stereotaxator was used for positioning: the dural membrane was exposed at 1.5 mm and 1.0 mm behind the anterior fontanel and 1.5 mm away from the sagittal sutures. Liquid was injected vertically into both lateral ventricles, the needle was inserted 2mm, the injection was completed 5min, and the incision was sutured. This modeling method is consistent with our previous research [16]. The control group was injected with 5μl artificial cerebrospinal fluid on day 1 and day 3. Another 15 mice were injected with 5μl streptozotocin (STZ) solution (prepared with artificial cerebrospinal fluid, concentration 6 mg/mL, injection volume 3 mg/kg) on day 1 and 3. Antibiotics were given for 3 days to prevent infection.
Reagents
Tribromoethanol (Batch No. A103417, Sigma-Aldrich, Darmstadt, Germany), Antibeta Amyloid 1-42 (1:1000, ab201060, Abcam, Shanghai, China), Animal non-immune serum (sheep) (SP KIT-B2, Fuzhou Maixin Biotech. Co., Ltd., Fuzhou, China), Fast enzyme-labeled sheep anti-rabbit IgG polymer (KIT-5004, Fuzhou Maixin Biotech. Co., Ltd.), Enhanced DAB Plus Kit (DAB-2031, Fuzhou Maixin Biotech. Co., Ltd.), Silver glycine staining kit (G1052-500T, Wuhan Servicbio. Co., Ltd.,Wuhan, China).
Anhydrous ethanol (Chengdu Chron Chemicals Co., LTD, Chengdu, China), TRIzol (Ambion,Austin, Texas, USA), primer (Tsingke, Beijing, China), chloroform (Chengdu Guerda Rubber Industry Co., LTD, Chengdu, China), no RNase water (Servare Biotech Inc.,Wuhan, China), and isopropyl alcohol (Chengdu Guerda Rubber Industry Co., LTD) were used.
Equipment of this study included: S1000™ Thermal Cycler common PCR instrument (BIO-RAD, California, USA), CFX Connect real-time quantitative fluorescent PCR instrument (BIO-RAD), 96-well quantitative PCR plate (Servare Biotech Inc), PCR plate sealing film (LABSELECT, Beijing, China), Chemiluminescence imaging system ChemiScope6100 (Shanghai Qinxiang Scientific Instrument Co., LTD, Shanghai, China), H1 16KR table refrigerated high-speed centrifuge (Hunan Ke Cheng instrument Co., LTD, Hunan, China), SC-3610 low speed centrifuge (Anhui USTC ZONKIA scientific instruments Co., LTD, Anhui, China), Handheld instantaneous centrifuge (SCLLOGEX, Shanghai, China), Vortex oscillator SCL-VS (SCLLOGEX), Electrophoresis apparatus (164-5050) (BIO-RAD), 10 μL, 20 μL, 200 µL, and 1000 µL gun head (Beijing Labgic Co., LTD, Beijing, China), Microsample feeder (Thermo scientific, Massachusetts, USA), Handheld instantaneous centrifuge CF2800M (LABGIC, Beijing, China), Grinding machine (Servare Biotech Inc), Sealing instrument (Servare Biotech Inc).
Bioinformation analysis
The GSE122063 dataset (100 samples) included 44 normal samples and 56 AD samples, and the GSE5281 dataset (161 samples) included 74 normal samples and 87 AD samples were obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Agilent-039494 SurePrint G3 Human GE v2 8×60K Microarray 039381 (Feature Number version) and [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array. Akt and Wnt pathway genes were obtained from the official GSEA website (https://www.gsea-msigdb.org/gsea/msigdb/). R language limma package, pheatmap package, clusterProfiler, R package pROC, R package clusterProfilerj, org.Hs.eg.db package and R package “ggcorrplot” were used. The GeneMINIA, MiRDB, miRWalk, Starbase, and Cytoscape databases were used. The process is shown in Fig.1.
Step-down test
The test was performed after one month of feeding [17]. The experiment included one- day training period and one- day test period, and the training period was not recorded. The time that the mice required to jumped off the platform (step down delay (SDL)) and the number of electric shocks (error time (ET)) were recorded during the test period. If the mouse stays on the platform and does not jump off, ET is 0 and the observation time (300 s) is SDL.
Fresh tissue specimens
After the mice were killed, hippocampal tissue was dissected on the ice. The hippocampal tissue placed in the EP tube was transferred to a liquid nitrogen tank at -180℃ for preservation. Each group had 10 samples.
Paraffin section
Euthanized the mice after behavioral tests. All brains were dehydrated, embedded in paraffin, and sectioned. The brains were sliced sequentially (4 μm). Four mice were in each group.
Silver plating staining
The slices were placed in xylene I for 10 min, xylene II for 10 min, xylene Ⅲ for 10 min, anhydrous ethanol Ⅰ for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 85% alcohol for 5 min, and then washed in distilled water 3–5 times. Silver glycine dye solution C was added to the chemical ring, dyed for 5 min and wash with distilled water three times; Silver glycine dye solution B (preheated at 37 ℃) was added into the histochemical ring and treated for 3–5min. The section was removed, the residual silver glycine dye solution B on the tissue was quickly shaken off, and placed into silver glycine dye solution A (preheated 15 min 45 °C), while observing the reduction effect. Then the section was removed after a few seconds and quickly shaken and placed into fresh silver glycine dye solution A (preheated 15 min at 45 °C). After a few seconds, the samples were removed and washed with distilled water. If the dyed background was too deep, it was treated with silver glycine dye solution D and washed three times with distilled water. The slices were dehydrated using anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, dimethyl I for 5 min, and xylene II for 5 min, and sealed with neutral gum. SlideViewer image analysis software was used to observe neurofibrillary tangles (NFTs) in each group (20×).
Immunohistochemical
The slices were placed in xylene I for 10 min, xylene II for 10 min, xylene Ⅲ for 10 min, anhydrous ethanol Ⅰ for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 85% alcohol for 5 min, and then washed in distilled water.
The naturally cooled slides were placed in PBS (pH 7.4), shaken for 3 times for 5 minutes each time, blocked with endogenous peroxidase, placed in 3% hydrogen peroxide solution, incubated at 24℃ away from light for 15 minutes, placed in PBS (pH 7.4), shaken for 3 times for 5 minutes each time.
The tissue on the slide was evenly covered with animal non-immune serum (sheep) and closed at room temperature for 30min. Shake off the sealing solution, add monoantibody drops, and overnight in a wet box at 4℃. Add the secondary antibody and return to room temperature. Place the slide in a pH 7.4 PBS and shake three times for 5 minutes each time. Tissue was covered with secondary antibody (HRP label) and incubated at room temperature for 30 min.
he tissue was placed on PBS (pH 7.4) and shaken three times for 5 minutes each time. Add DAB color developing solution. The color development time is controlled under the microscope, and the color development is terminated by washing. The positive colors are brown and yellow.
Hematoxylin was dyed for about 3 minutes and washed with water. Hematoxylin differentiation solution was added for 6 seconds and then washed with water.
After dewatering, 4 samples were taken from each group, and the number of positive neurons in temporal lobe was observed under light microscope. SlideViewer image analysis software was used to record the number of positive cells in each group (30× magnification).
Quantitative reverse transcription (RT-q) PCR
Total RNA extraction
RNA was extracted from 20 frozen brain tissue samples, including 10 AD and 10 control samples. Take 50mg of tissue for each sample and add 1ml TRIzol reagent. The sample was fully homogenized and placed on ice for 10 minutes to fully lysate the cells. 300ul chloroform was added and violently shaken for 30 seconds. The liquid was stratified at 24℃for 10 minutes and centrifuged at 12000g and 4℃for 15 minutes. Carefully absorb the upper water phase and put it into another EP tube, add the same volume of ice isopropyl alcohol, mix it upside down, let it stand for 10 minutes, and centrifuge at 12000g and 4℃ for 10 minutes. White RNA precipitates can be seen at the bottom of the tube. Gently tilt the tube mouth to discard the supernatant, add 1ml of 75% ethanol to the precipitate, reverse several times to make the precipitate float, let it stand for 2 minutes, centrifuge at 7500g and 4℃ for 5 minutes, and then make the precipitate adhere to the bottom of the tube again. Repeat this step twice. Discard the supernatant, put it into a super clean table and dry it, make the ethanol and water volatilize as much as possible, and the RNA precipitation becomes transparent. 20-50ul of RNase-free water was added to the dried RNA precipitation and left for 15 minutes to completely dissolve the RNA. 1ul was taken for concentration detection by Nano drop, and the RNA purity/concentration was recorded to calculate the loading amount for subsequent reverse transcription steps. The remaining RNA was immediately reverse-transcribed or frozen in the refrigerator at -80℃.
RNA detection
RNA (1 µL) was used for detection using a NanoPhotometer N50. Reverse transcription of the mRNA was performed using the SureScript First Strand cDNA Synthesis Kit (Servare Biotech Inc.). The components of the reverse transcription kit were melted at 24℃ and centrifuged 1000g×1min, placed on ice, and the following reagents and solutions were added in the following order on ice: 4 µL of 5× reaction buffer, 1 µL of miRNA RT enzyme mix, 0.1 ng to 5 µg total RNA, and nuclease-free water up to 20 μL. After centrifugation 1000g×1min, the reverse transcription reaction was conducted in an ordinary PCR apparatus using the following thermal cycle: 25℃ for 5 min, 50℃ for 15 min, 85℃ for 5 s, and held at 4℃. The cDNA was diluted 5–20 times in double-distilled H2O (RNase/DNase-free). The qPCR reaction was then performed according to the following reaction system: 3 μL of cDNA, 5 μL of 2× Universal Blue SYBR Green qPCR master mix, 1 μL of Forward primer (10 µM), and 1 μL of Reverse primer (10µM). After centrifugation 1000g×1min, 40 cycles of reaction were performed with CFX96 real-time quantitative fluorescence PCR according to the following conditions: predenaturation 95℃ for 1 min, denaturation 95℃ 20 s, annealing 55℃ 20 s, and extension 72℃ 30 s. The amplification and dissolution curves were prepared, and the CT values were determined. The primer sequences are listed in Table 1. The relative expression was eventually calculated using the 2–∆∆Ct method. Finally, the 2-∆∆Ct value was calculated, and the p-value was calculated using GraphPad Prism 9.
Statistical analysis
SPSS software (version 19.0) and GraphPad Prism (version 9.4.1) were used to perform statistical analyses and data mapping. A one-way analysis of variance was used for intragroup data analysis. An independent sample T-test was used for intergroup data analysis. p < 0.05 determined that statistical significance was established at p < 0.01 used to flag a higher significance level.