The Sprague Dawley (SD) rat pups were purchased from SiPeiFu Biotechnology Co. and housed with their dams in plastic cages and provided with food and water ad libitum. Male neonatal rats of similar body weight in each litter were selected for the experiment. Each litter of neonatal rats was randomly divided into a control group (CON) and a maternal separation group (MS). All of the experiments were approved by the Institutional Animal Care and Use Committee of the Academy of Military Sciences (Permit No: IACUC-DWZX-2020-670).

Newborn rats were born with a birth diary of PND1, and for the next 20 days, dams were separated from their littermates for 3 h per day, and the rats were kept at a living environment temperature of 28 ± 2°C. The control group was not separated and was kept in the same environment as the separated group. All of the rats were fed until the weaning period of PND22, and only male rats were used in the present study to avoid variations due to hormonal cycles at PND30. After normal feeding until adult PND60, experiments were conducted and body weight and feed consumption were recorded regularly.

The semi purified diets used in this study were equivalent to diets produced by the American Institute of Nutrition (AIN-93G) (Reeves et al., 1993). All diets were isocaloric and compositionally the same in terms of carbohydrate and lipid component, and were obtained from SiPeiFu Biotechnology Co. The control diet (N) contained 18% protein, 63% carbohydrate, 7% fat, 5% fibers, and 2% L-leucine. The leucine-rich diet (H-LEU) and leucine-deficient diet (L-LEU) contained 5% and 0.22% L-leucine, respectively (Xiao et al., 2011; Viana and Gomes-Marcondes, 2013; Liu T. et al., 2018), and same amount carbohydrate, fat and fibers percentage as the control diet.

Rats were housed in open boxes of size 100 × 100 cm, with the inner walls and bottom painted black and the bottom divided into 25 20 × 20 cm squares. To eliminate the influence of the external environment on the rats, the experiments were conducted in a dedicated laboratory shielded from external sound, and the room temperature was maintained at 22 ± 2°C. To reduce the effect of rat odor, the open-air box was cleaned promptly after the previous rat finished the experiment, and then the next rat experiment was conducted. At the beginning of the experiment, the rat was placed in the center of the open field box, and the open field score, which was the sum of the number of squares crossed by the rat’s nose (crossing) and the number of hind limb stands (rearing), was recorded within 5 min. The rats’ autonomy and exploration behaviors in unfamiliar environment were evaluated comprehensively.

The Morris water maze test was performed in a pool with a diameter of 1.2 m and a water temperature of 25°C. Before the formal test, the subject rats were required to complete five training sessions of finding the platform for 3 min each, once every 12 h. During the training, the water level was higher than the platform to ensure that the rat’s body was mostly on the water surface after successfully finding the platform. During the training phase, the rat was randomly placed in one of the four quadrants of the water maze with its head facing the inner wall. The time taken by the rat to find the platform hidden under the water surface was recorded as the latency to find the platform (escape latency). If the 3-min internal platform search failed, the rat was led to the platform. During the test phase, the underwater platform was removed, and the time to swim to the original underwater platform location after entering the water and the time to traverse the platform area within 3 min (platform passes) were recorded. This experiment was used to evaluate the spatial learning and memory ability of the subject rats.

The object identification box was a 60 × 60 cm box with the interior and bottom painted black. The rats were acclimatized in the open box for 10 min the day before, and on the second day, two clean blocks of the same color and shape were placed 20 cm apart in the open box to record the cumulative objects explored by the rats in 10 min. One hour later, a clean block of completely different color and shape was replaced in the box, and the exploration time of the rat exposed to the new object (T_new) and the old object (T_old) within 10 min was recorded. The cognitive index = (T_new–T_old)/(T_new + T_old). This experiment reflected the ability of rats to recognize and remember new things. To avoid odor interference from previous rats, each rat was thoroughly cleaned and disinfected in an open box at the end of the experiment.

After completion of the behavioral test, anesthesia was administered with a combination of urea and chloral hydrate at a rate of 5 ml/kg according to body weight. In prone position, the posterior cervical muscles were blunted and separated to expose the foramen magnum of the occipital bone. The needle was inserted vertically, and a 200 μL microcentrifuge tube was inserted to allow cerebrospinal fluid to flow. The fluid was stored at 80°C. We placed 10 μL of cerebrospinal fluid sample in a microcentrifuge tube, added 40 μL of isotopic internal standard methanol solution, and shook it at 1200 rpm for 5 min. We then aspirated 10 μL of supernatant into a 1.5 mL EP tube and added 70 μL of borate buffer and 20 μL of LAQC derivatization reagent. The supernatant was shaken for 10 min at 55°C and 1200 rpm in a metal bath. The amino acids in the cerebrospinal fluid were detected by ultra-performance liquid chromatography–tandem mass spectrometry (Xevo TQ-S, Waters Corp., Milford, MA, USA).

Rats were dissected on ice, heads were cut off, dura mater was separated, skulls were stripped, and brains were isolated. Tissues were preserved in 4% paraformaldehyde, dehydrated, and sealed with wax, and tissues were embedded. The tissues were chilled on a 20° freezing table, and the solidified wax blocks were removed and trimmed; tissues were cut into sections 4 μm thick, affixed to glass slides, and baked in a 60°C oven. The sections were immersed in toluidine blue (Solarbio) for 5 min, then washed with distilled water, slightly fractionated with 1% glacial acetic acid, made transparent in xylene for 5 min, and sealed with neutral glue. Microscopic observation and photography were performed using ImageJ (version 1.45) image analysis software.

The rat brain was placed on an ice plate, and the left and right cerebral cortex were bluntly separated along the median sagittal line to expose the bilateral hippocampus. The maximum volume of tissue was 1 mm × 1 mm × 1 mm. The tissue was fixed with 0.1 m phosphate buffer PB (PH7. 4) 3 times for 15 min at 4°C, fixed at room temperature for 2 h, dehydrated, infiltrated with working solution (acetone:812 embedding agent = 1:1) for 2 h and another working solution (acetone:812 embedding agent = 2:1) for 5 h, transferred to an embedding plate for 5 h, and baked at 37°C for 12 h. The sections were baked and polymerized at 60°C for 48 h. The sections were double stained with uranium and lead and air dried at room temperature for 12 h. Images were acquired using a transmission electron microscopy.

A fully automated protein expression analysis system (WES) was used for the assay. Rat hippocampus tissues were taken, homogenized with an electric homogenizer, lysed by adding cell lysis solution, and centrifuged at 12,000 × g for 5 min at 4°C in a low-temperature, high-speed centrifuge; then, the supernatant was removed and set aside, and the samples were quantified with the BCA protein quantification kit. Based on the quantification results, the samples were adjusted to the recommended sample concentration of 2 μg/μL using the WES kit 0.1 × sample buffer. Next, 3 μL of prepared samples, 1 μL of Mastermix, 10 μL of primary antibodies Beclin-1, Lc3, Bcl-2, Bax, and β-actin (1:100, ABclonal), horseradish peroxide 10 μL (HRP), rabbit secondary antibody 10 μL, and luminescent solution 15 μL were loaded onto WES plates. After centrifugation (2500 g, 5 min), the WES plates (12–230 kDa, 25 capillaries) were loaded into a fully automated protein expression analysis system, and the results were analyzed using Compass for WES software (version 4.10). The grayscale values of the bands given by the software were used to calculate the relative protein expression, using β-actin as an internal reference.

HT-22 cells were inoculated into 24-well plates, ensuring a cell concentration of 1 × 10⁵/ml and 5% CO2, and cultured for 24 h at 37°C using custom leucine-deficient DMEM high-glucose medium. Infection with a fluorescent double-labeled lentivirus mRFP-GFP-LC3 system, MOI = 100, was observed under a fluorescent microscope.

We added 50 μL of cell suspension to a 96-well plate and incubated it overnight in 5% CO2 in a 37°C cell incubator to allow cells to grow against the wall. We added 10 μL of lysate to high-control wells and incubated them in 5% CO2 in a 37°C cell incubator for 30 min. After adding 100 mL of working solution per well, we incubated the samples at room temperature and protected them from light. After adding 50 μL of termination solution per well, we measured the absorbance at 490 nm with an enzyme marker within 5 min.

HT-22 cells were digested and inoculated into 6-well plates at a density of 2 × 10⁵ cells/mL, 3 mL per well. When cells reached 80–90%, they were given serum-free treatment and various factor interventions, such as leucine and B-A1. The medium in the 6-well plates was added to round-bottom flow tubes and labeled; cells in the wells were washed with PBS and digested with 500 μL of 0.25% EDTA-free trypsin at room temperature. We digested the cells for 3 min and added 500 μL medium to the flow tube to terminate the digestion, blew gently, pipetted into the flow tube, centrifuged at room temperature for 5 min using a medical centrifuge at 1000 r/min, discarded the supernatant, added 1 mL PBS for vortex shaking, centrifuged twice at room temperature for 5 min using a medical centrifuge at 1000 r/min, stained the samples according to the instructions of the Annex V and PI double-staining kit, and tested them in the machine after 30 min of protection from light.

Statistical software SPSS 26.0 was used for statistical analysis, and quantitative results were expressed as mean ± standard deviation (sd). Quantitative values between two groups were compared using the independent samples t-test at the level of α = 0.05. One-way ANOVA was used for multiple groups, and p < 0.05 was considered a statistically significant difference. Nissl staining was processed and analyzed with ImageJ software.

Behavioral tests were conducted to investigate the effects of MS stress on cognitive function. Compared with the CON group, the results showed significantly lower cognitive function in the MS group, including self-exploratory ability, novel object recognition, and spatial learning memory in the open field, novel object recognition test, and Morris water maze test (p < 0.05, p < 0.01; Figures 1A–G).

To confirm whether there was neuronal damage in the hippocampal region associated with MS-induced cognitive impairment in rats, we used Nissl staining to stain neuronal cells (mainly neurons) in the hippocampal region, and the results showed that neurons in the hippocampal CA1 and CA3 regions of MS rats were loosely arranged, lightly stained, and reduced in number compared with the CON group, with no differential changes in the DG region (p < 0.05, p < 0.01; Figures 2A,B). The leucine levels in CSF were significantly decreased in MS rats, and there was a positive correlation with the number of hippocampal neurons (p < 0.05, p < 0.01; Figure 2C).

To study the effect of different levels of leucine on cognitive function in MS animal models, after MS, the rats were fed a normal leucine diet (N, 2%), low leucine diet (L-LEU, 0.22%), or high leucine diet (H-LEU, 5%) for 40 days. A stress model of MS rats was established, and rats were grouped into CON + N (control normal diet), CON + L-LEU (control low-leucine diet), MS + N (stress group normal diet), MS + L-LEU (stress group low-leucine diet), and MS + H-LEU (stress high-leucine diet) groups. At PND 60, leucine in CSF was measured, and cognition-related behavioral tests were performed. The results showed that, compared to the control normal diet group, the leucine levels were lower in the low leucine diet, MS normal diet, and MS low-leucine diet rats, while the MS high-leucine diet group had higher leucine levels than the MS low-leucine diet group (p < 0.05, p < 0.01; Figure 3A). Rats in the low-leucine diet group had lower scores in both the open field test and the novel object recognition test compared to the control normal diet group, and the MS high-leucine diet group had higher scores than the MS low-leucine diet group (p < 0.01; Figures 3B,C). The MS normal diet group and MS low-leucine diet group had higher scores during the assessment phase (p < 0.05, p < 0.01; Figure 3D). The number of stage penetrations was reduced in both the MS normal diet group and the MS low-leucine diet group compared with the control normal diet group, and there was a tendency toward an increased number of stage penetrations in the MS high-leucine diet group. The low-leucine diet caused a prolonged latency time in all groups of rats compared with the control normal diet group, and there was a decreased latency time in the MS high-leucine diet group compared with the MS low-leucine diet group in male rats (p < 0.05, p < 0.01; Figures 3E,F).

To investigate the effect of MS stress on rat hippocampal autophagy, samples were taken and observed using electron microscopy, which showed that autophagosomes were present in the hippocampus of the low-leucine diet CON and MS groups, and no autophagic vesicles were found in the high-leucine diet MS group, as shown in Figure 4A. The hippocampus of rats was taken to detect Beclin-1 expression, which was increased in the normal diet and low-leucine diet MS group but decreased in the high-leucine diet MS group compared with the low-leucine diet MS group (p < 0.05, p < 0.01; Figure 4B). The Bcl-2/Bax ratio was decreased in the low-leucine diet CON group, normal diet MS group, and low-leucine diet MS group (p < 0.05; Figure 4C).

To further verify the role of neuronal autophagy upregulation by leucine deficiency in ELS-induced metal disorder rats, HT-22 stress cell models were cultured in a specially customized cell culture medium with different contents of leucine and autophagy inhibitor. The results showed increased expression of LC3II/I in leucine starvation neurons compared to the normal (routine culture of neural cells) group, and it was remarkably restored after adding different concentrations of leucine (p < 0.05; Figure 5A). Autophagy double-labeled lentivirus (mRFP-GFP-LC3) infection of neurons cultured with different concentrations of leucine medium showed enhanced bicolor fluorescence in the Leucine starvation group, which diminished with increasing leucine concentration, as shown in Figure 5B. Furthermore, leucine-deficient culture and autophagy inhibitor intervention in neurons were examined for LC3 protein expression, lactate dehydrogenase (LDH) release, and cell apoptosis. The results showed that the LC3II/I ratio, LDH activity, and cell apoptosis rate in the NOLEU group were markedly elevated compared to the CON group, but they were significantly decreased after treatment with the autophagy inhibitor B-A1 (p < 0.05, p < 0.01; Figures 5C–E).