Experimental animals and groups
Specific pathogen-free (SPF) male rats (n = 100; 3 months; 200 ± 20 g) were obtained from the Anhui Center of Laboratory Animals [certificate number: SCXK (Su) 2017-0003]. All rats were kept in a sterile room at 22 ± 3 °C and humidity of 55 ± 10%. They were provided with food and water ad libitum for 1 week. Subsequently, rats were randomized into two groups: control (n = 8) and model (n = 92). Model rats were fed on a high-fat, high-sugar diet for 30 days and intraperitoneally injected with STZ (25 mg/kg) [19]. After 72 h, tail vein blood was collected to determine random blood glucose levels (≥ 16.7 nmol/L represented diabetes in rats) [20]. Seventy-two rats were successfully modeled. Cognitive disorders were examined using the Morris water maze. The mean escape latency, and difference ratio between the model and control groups were calculated. A value >20% indicated successful modeling of cognitive impairment [21]. These rats were randomized into four groups: model (n = 8), Electroacupuncture application (EA) (n = 8), autophagy (rapamycin; n = 8), and EA+3-methyladenine (3-MA; n = 8). All experiments were carried out according to the “Guiding Opinions on Treating Laboratory animals” (Ministry of Science and Technology). All rats were anesthetized with intraperitoneal 0.3% sodium pentobarbital (30 mg/kg).
In the EA group, rats were first fixed and kept awake. The target sites were Yishu (EX-B3), Zusanli (ST36), BaiHui (GV 20), and Dazhui (DU14). Subcutaneous needles (size: 0.3525 mm; needle penetration depth: 4 mm) were used to probe Yishu and Zusanli. All puncture points were connected to Han's Electroacupuncture instrument. After reviewing the literature [22, 23], EA was performed using a stimulation current of 1 mA, frequency of 15 Hz, duration of 30 min, and once a day [14].
The rapamycin group received rapamycin (2 mg/kg) intraperitoneally once daily, and the rest of the treatment was identical. Rapamycin has been shown to induce autophagy [24].
The EA+3-MA group received 3-MA (1.5 mg/kg) intraperitoneally once daily (30 min after EA), and the rest of the treatment was identical. Different from rapamycin, 3-MA can inhibit autophagy [25].
Rats were treated for 4 weeks (1 day of rest after 6 days of intervention). The model and control groups were not treated.
Experimental design
The experimental design is presented below (Fig. 1).
Morris water maze
The Morris water maze test determined the learning and memory function after treatments. One day before the formal training, rats were placed in the pool and allowed to swim freely for 2 min to adapt to the new environment. The experiment included two learning tests: the place navigation test and the spatial probe test. (1) The navigation test was conducted to evaluate spatial learning. Rats were trained for two trials a day for four consecutive days. During the test, rats were positioned in the pool, facing the pool wall at the four starting locations. Each rat was allowed 2 min to find the hidden platform. Escape latency time was calculated as the time to find the hidden platform. If rats failed to reach the hidden platform within the 120 s, they were guided to the platform. After staying on the platform for 20 s, the escape latency time was recorded as 120 s. The crossing route to the platform was recorded with a video camera. (2) Memory was determined with a spatial test. Once the place navigation test was complete, the hidden platform was eliminated, and well-trained rats were permitted to swim for 2 min. The frequency of entering the hidden platform and percentage of distance traveled, defined as the distance traveled in the target quadrant divided by the distance traveled across all quadrants, were recorded within 120 s.
Hematoxylin–eosin (H&E) staining
Paraformaldehyde (4%) was used to fix hippocampal tissues before paraffin-embedded. They were sliced into 5 µm sections and fixed on glass slides. Tissue morphology was examined after H&E staining. Images were captured with a microscope (Olympus BX53).
Rats were intraperitoneally anesthetized with 0.3% sodium pentobarbital (30 mg/kg), then fixed on the operating table (supine position). After disinfection, the thorax of the rats was cut open, and perfusion was performed from the heart. Once the rats became stiff, the heads were cut off and harvested on an ice plate. Rats' cerebral cortex and hippocampus were removed for alcohol dehydration and transparent with xylene. The paraffin wax (60 °C) was dipped three times and embedded into paraffin blocks. The wax blocks were sectioned with a microtome, the tissue pieces were placed in a water bath (40 °C) for layering, and slides were inserted at an angle to collect the sections. Sections were attached to slides in the oven (60 °C, 3 h). A series of xylene [xylene (i) 20 min, (ii) 20 min, and (iii) 20 min], ethanol [anhydrous ethanol (i) 5 min and (ii) 5 min], and alcohol (95% 5 min, 90% 5 min, 80% 5 min, and 70% 5 min) was applied. Next, they were stained with Mayer's hematoxylin solution and eosin. Slices were dehydrated with ethanol and sealed with neutral gum. Finally, the morphological changes of hippocampal cells were observed under a microscope.
Enzyme-linked immunosorbent assay (ELISA)
First, rats were anesthetized with intraperitoneally-injected 0.3% sodium pentobarbital (30 mg/kg) and decapitated. The collection of hippocampal tissues was performed on ice, homogenized with a homogenizer, and centrifuged for 10 min at 10,000 rpm at 4 °C. Next, 1% of lysates were subjected to ELISA following the manufacturer’s instructions. The expression levels of Aβ1-42 were evaluated using a microplate reader at 450 nm.
Sample preparation for electron microscopy
Briefly, 1 mm × 1 mm × 1 mm pieces of the hippocampal CA1 region were fixed in 2.5% glutaraldehyde phosphate buffer (4 °C, pH 7.4) for 4 h, then dehydrated with ethanol and acetone. They were embedded and cut into 60 nm sections for staining with lead citrate for 5–6 min and uranyl acetate for 20 min. Imaging was conducted by TEM (JEM1230).
Immunofluorescence and immunohistochemistry staining
Rats from all groups were anesthetized by intraperitoneal injection of isoflurane. The chest cavity was quickly opened to expose the heart. Then, 0.9% NaCl solution was administered with a needle into the apex of the myocardium to flush blood from the vasculature. A small incision was cut on the right atrial appendage to observe the outcomes. After standard cardiac perfusion, the prefrontal hippocampus was fixed with 4% paraformaldehyde. Tissues were sliced into 3 µm sections, immersed thrice in xylene and a series of ethanol solutions, and rinsed with running water. Sections were incubated with goat serum for 0.5 h after antigen retrieval, then incubated for 1 h at 37 °C in the presence of primary antibodies (COXIV: 1:100; Lamp2: 1:100), and washed three times using 1× PBS buffer. Next, samples were incubated with fluorophore-conjugated secondary antibodies for 30 min at 37 °C in the dark. Slices were mounted using anti-fading reagents. Images were captured by fluorescence microscopy to determine the mean fluorescence of target proteins.
Western blotting
Proteins were extracted from hippocampal tissues using RIPA lysis buffer. The concentration of extracted proteins was determined with a BCA protein assay kit. Equal protein amounts were separated on an SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes for blocking for 2 h with 5% non-fat milk, and incubated with the following primary antibodies overnight at 4 °C: GAPDH: 1:1000 LC3: 1:1000; BCL2: 1:1000; P62: 1:1000; Beclin1: 1:2000; DISC1: 1:1000. After washing with 1× TBST buffer, membranes were incubated with HRP conjugated secondary antibodies (1:5000) for 120 min at room temperature (RT). The enhanced chemiluminescence reagent was used to develop protein blots, which were analyzed with Image J software.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNAs from 100 mg hippocampus tissues were extracted using Trizol reagent and reverse-transcribed into cDNA with the HiScript III 1st Strand cDNA Synthesis Kit. The thermocycling conditions were: 25 °C/5 min, 50 °C/15 min, 85 °C/5 min, and 4 °C/10 min. DISC1 expression was examined using PowerUp™ SYBR® Green. The thermocycling conditions were: 95 °C/10 min; 40 cycles of 95 °C/15 s and 60 °C/60 s; 95 °C/15 s, 60 °C/60 s, and 95 °C/15 s. The internal control in this assay was β-actin. Relative expressions of DISC1 mRNA were determined using the 2−ΔΔCt method. The primers used in this assay are presented in Table 1.
Statistical analysis
Data are shown as means ± standard deviations (SDs) and were analyzed using SPSS 27.0. Multiple groups were compared by one-way analysis of variance (ANOVA). The mean values of two groups were compared with Fisher's least significant difference (LSD) method. A p ≤ 0.05 was considered statistically significant.