Chemicals, reagents and antibodies

Amresco (USA) supplied ACR with a purity of 99.9%. DMEM medium was purchased from Procell (China). Fetal bovine serum (FBS) was purchased from Wenren. 4% paraformaldehyde was purchased from Biosharp (China). Glutaraldehyde fixative solution (2.5%, for electron microscopy) and 50×TAE were purchased from LEAGENE (China). DAPI dye, BCA protein assay kit, and cell counting kit-8 (CCK-8) were obtained from Beyotime (China). eEF2K-siRNA was sourced from RIBOBIO (China). The PAGE Gel Fast Preparation kit and RT-qPCR assay kit were purchased from Epizyme (China) and Toyobo (Japan), respectively. The gene identification kit was purchased from TIANGEN (China).

The primary antibodies included eukaryotic elongation factor 2 (eEF2) (Cat. ABP58457, Abbkine), Phospho-eEF2 (Thr56) antibody (Cat. ABP58456, Abbkine), eEF2K antibody (Cat. GTX111496, GeneTex), Synapsin 1 (SYN1) (Cat. ABP0125, Abbkine), tropomyosin receptor kinase B (TrkB) (Cat. ABP55433, Abbkine), brain-derived neurotrophic factor (BDNF) (Cat. GTX132621, GeneTex), lysophosphatidylcholine acyltransferase 1 (Lpcat1) (Cat. DF12033, Affinity), β-actin (Cat. TA811000, ORIGENE). The secondary antibodies used were FITC-conjugated goat anti-mouse IgG (Cat. TA130013, ORIGENE) and Cy3-conjugated goat anti-rabbit IgG (Cat. A23920, Abbkine). HRP-conjugated antibodies included goat anti-mouse IgG (Cat. A21010, Abbkine) and goat anti-rabbit IgG (Cat. A21020, Abbkine).

Study subjects

We defined the workers in the ACR monomer production workshop of a factory in northern China as the ACR exposure group for this study, and the administrative workers in the same factory, who were strictly not exposed to ACR, as the ACR non-exposed group. We investigated the basic situation of the factory, hygiene and protective measures, as well as personal protective equipment. A standardized questionnaire was used to record the general information, occupational history, and personal protective measures of the survey subjects. We also investigated the occurrence of subjective symptoms related to learning and memory in the ACR-exposed group and the non-exposed group. The questionnaire survey was conducted face-to-face to ensure independent and careful completion by each respondent, minimizing survey result bias. Exclusion criteria for the study encompassed long-term use of antipsychotic drugs, systemic diseases impacting the central nervous system’s function, metabolic disorders, other conditions affecting blood metabolites, and diabetes resulting from prolonged medication use. Approval for the study was granted by the Ethical Committee of Harbin Medical University, and all subjects provided informed consent [26,27,28,29].

A total of 100 people were surveyed in the study, including the ACR-exposed group (n = 50; mean age, 42.16 ± 4.39 years; mean working years, 16.62 ± 5.24 years) and the non-exposed group (n = 50; mean age, 41.64 ± 3.47 years; mean working years, 16.20 ± 2.46 years). We randomly selected 10 individuals with subjective symptoms related to learning and memory from the ACR-exposed group (n = 50), and 10 individuals without self-reported symptoms related to learning and memory from the non-exposed group (n = 50). The selected ACR-exposed group (n = 10; age, 42.10 ± 3.98 years; working years, 17.02 ± 4.37 years) and non-exposed group (n = 10; age, 43.80 ± 3.85 years; working years, 18.00 ± 2.11 years) were subjected to serum proteomic analysis.

Serum collection

Both groups adhered to identical dietary restrictions and refrained from alcohol consumption for the three days preceding the collection of blood samples. Morning fasting cubital venous blood (4 mL) was collected from all subjects using blood collection tubes with sodium heparin. The blood underwent rapid centrifugation at 3000×g for 10 min at room temperature, leading to the separation of serum, which was then stored at -80 °C until further analysis.

Population serum proteomics

The serum samples were taken out from − 80 °C and centrifuged at 4 °C, 12,000×g for 10 min. 600 µg of the supernatant was taken and the Pierce™ Top 12 Abundant Protein Depletion Spin Columns Kit from Thermo was used according to the instructions to remove high-abundance proteins. The protein concentration was determined using the BCA assay kit. The proteins were then enzymatically digested into peptides using trypsin, followed by desalting with Strata X C18 (Phenomenex) and vacuum freeze-drying. The peptides were dissolved in 0.5 M TEAB and labeled according to the instructions of the TMT assay kit. The labeled peptides were analyzed using liquid chromatography mass spectrometry, and the results were finally organized and analyzed (Supplementary Method 1).

Cell culture and transfection

Well-differentiated rat pheochromoma cells (PC12) were obtained from Procell Life Science & Technology Co., Ltd., Wuhan, China. The cells were cultured in cell culture medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 89% DMEM. The cells were maintained at 37℃ in a 5% CO2 incubator and passaged using Trypsin digestion solution for sub-culturing.

PC12 cells were mainly used for two parts of research. The first part of the research is to expose PC12 cells to different concentrations of ACR solutions to observe the expression of eEF2K in PC12 cells. PC12 cells were divided into control group, 1.25 mM ACR group and 2.5 mM ACR group, and the exposure time was 24 h. The second part of the research is to use eEF2K-siRNA to inhibit the expression of eEF2K in PC12 cells and explore whether inhibiting the expression of eEF2K in PC12 cells can improve the neurotoxicity caused by ACR. The PC12 cells were divided into four groups: the control group, the eEF2K-siRNA group (final concentration 10 µM), the ACR group (2.5 mM ACR), and the combined eEF2K-siRNA (final concentration 10 µM) and ACR (2.5 mM ACR) group. The exposure duration for each group was 24 h. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was employed for cell transfection. siRNA used in the transfection were synthesized by Thermo Fisher Scientific (Massachusetts, USA).

Animal feeding and grouping

Eighteen 8-week-old SPF male SD rats were purchased from Vital River Laboratories (Beijing, China). The license number is SCXK (JING) 2021-0011. eEF2K heterozygous C57BL/6J mice (eEF2K+/−) were purchased from Gempharmatech Co., Ltd (Jiangsu, China), and the strain number is T037867. We obtained wild-type (WT) and KO (eEF2K−/−) pups by crossing heterozygous mice (eEF2K+/−). Twenty-four 8-week-old male WT mice and Twenty-four 8-week-old male KO mice were used for experimental studies. All rats and mice were subjected to 1 week of acclimatization feeding and were housed under a 12:12 h light/dark cycle, at a temperature of 22 ± 1 °C and a relative humidity of 55 ± 10%. They had ad libitum access to food and water. All procedures adhered to the regulations set by the Ethical Committee for Research on Laboratory Animals, as reviewed and approved by the Medical Ethics Committee of Harbin Medical University (Harbin, China). The approval process aligns with guidelines established by the National Institutes of Health (USA) [27].

Eighteen male SD rats were randomly assigned to three groups: control, low-dose ACR, and high-dose ACR (n = 6 per group). The low-dose ACR group received a dose of 6 mg/kg body weight, while the high-dose ACR group received a dose of 18 mg/kg body weight. The control group was administered distilled water. Twenty-four male WT mice and 24 male KO (eEF2K−/−) mice were randomly divided into four groups, including WT group, KO group, WT + ACR group, and KO + ACR group (n = 12 each group). The WT group and KO group were given distilled water, while the WT + ACR group and KO + ACR group were given a dose of 18 mg/kg body weight of ACR. Both rats and mice were orally gavaged with a dose equivalent to 1% of their body weight, and the duration of treatment was 28 days. Following the last oral gavage of ACR, both rats and mice were euthanized, and the entire hippocampal tissue was isolated based on specific experimental needs. The dosage of ACR administered to rats and mice in this study was determined based on our previous research [27].

Cell viability assay

PC12 cells were evenly distributed in a 96-well plate, with each well containing 1 × 104 cells/mL. The experimental group and eEF2K transfection group were treated separately. Each concentration group was set up with 6 duplicate wells and cultured at 37 °C and 5% CO2 for 24 h. 10 µL of CCK-8 solution was added to each well 1 h before the end, and the culture was continued in the incubator for 2 h. The absorbance was measured using a microplate reader at 490 nm wavelength. The cell survival rate is presented as the relative survival rate, calculated as the ratio of the absorbance value in the experimental group and eEF2K transfection group to the absorbance value in their corresponding control group.

Behavior tests

Rats and mice were subjected to behavioral tests continuously within 3 days after completing the exposure. Open field test. The open field test equipment included an open field box and a top camera. The equipment was purchased from Shenzhen Ward Life Science Co., Ltd., China. The activity of each rat was individually monitored for 10 min in the open field (L × W × H: 100 × 100 × 40 cm). The activity of each mouse was individually monitored for 5 min in an open field (L × W × H: 50 × 50 × 40 cm). All data were recorded using video-tracking software (SMART, Panlab Harvard Apparatus Bioscience Company, USA). Parameters evaluated included Movement time in Periphery and Resting Time in Zone (s) - Center. Shuttle Box Test. The experiment used a shuttle box with dimensions of 696 × 348 × 445 mm (Chengdu Taimeng Technology Co., Ltd., China). Mice were given 2 min to adapt in the shuttle box, including walking freely in the shuttle box to become familiar with the learning environment. At the experiment’s outset, position the mouse in the left compartment of the shuttle box, near and facing the end wall. If the mouse relocates to the opposite compartment during the conditioned stimulus (CS), the conditioned response is automatically logged. The CS lasted for a maximum of 10 s, followed by an unconditioned stimulus (US) for a maximum of 10 s. Mice were studied for three consecutive days and tested on the fourth day. STT-100 shuttle experiment video analysis software was used to analyze and make statistics on the experimental data. The parameters evaluated include the percentage of conditioned responses and the percentage of unconditioned responses.

Histopathological observation of hippocampus

Rats and mice were anesthetized and subjected to transcardial perfusion with 200 mL of cold normal saline (4 °C) followed by 300 mL of ice-cold 4% paraformaldehyde (PFA) (4 °C). Rats and mice hippocampal tissues were fixed in 4% PFA, dehydrated, and embedded in paraffin. Hippocampal tissues were sectioned into 4 μm-thick serial sections and stained with hematoxylin and eosin (HE). Each slide received 50 µL of neutral gum, and the sections were examined under a light microscope (BH-2; Olympus Corporation, Tokyo, Japan). A pathologist, blinded to group assignment, conducted the histological examination.

Immunohistochemical analysis

The fixation methods of rat and mouse hippocampal tissues were the same as those for histopathological observation. Following the fixation of hippocampal tissues, the tissues were immersed in 30% sucrose in 0.01 M PBS until they sank to the bottom of the container. Brains were then mounted on poly-L-lysine-coated slides and sliced into 4 µM sections using a cryostat. These sections underwent a 30-minute incubation with 2% normal goat serum to prevent nonspecific binding. Then the diluted primary antibody was added dropwise, and rat hippocampal tissues were incubated with eEF2K (1:500) and mouse hippocampal tissues were incubated with eEF2K (1:300) overnight (15 h) at 4 °C. After washing three times with PBS, HRP-labeled goat anti-rabbit/mouse secondary antibody was added dropwise and incubated at 37 °C for 30 min. The sections were detected using a DAB color development kit (Servicebio, China). 50 µL of neutral gum was added to each slide, and the slides were observed under a microscope (Fi3, Nikon, Japan). Subsequently, image analysis was performed.

Immunofluorescence analysis

The fixation and sectioning procedures for rat hippocampal tissues mirrored those employed for histopathological observation and immunohistochemical analysis. Following completion, the sections underwent antigen retrieval (4.75 mL antigen retrieval agent and 500 mL double-distilled water), were boiled for 10 min, and then washed three times with PBS for 5 min each. Subsequently, 100 µL of blocking solution was added, and the sections were incubated at room temperature for 30 min. We added rabbit anti-rat primary antibody eEF2K (1:500) to the samples and incubated them overnight at 4 °C. After three PBS washes, slices were treated with a dark incubation of fluorescent (Cy3)-labeled goat anti-rabbit IgG secondary antibody for 60 min. Subsequently, three additional PBS washes, each lasting 5 min, were performed. DAPI was introduced and allowed to incubate for 5 min in the dark, followed by three PBS washes, each lasting 5 min. Images were observed and captured using a fluorescence microscope (Fi3, Nikon, Japan).

PC12 single cell suspension was prepared, and the cell concentration was adjusted to 1 × 10³ cells/mL before being inoculated in a 6-well plate. After the cells were adherent and grown, they were divided into experimental groups and treated with poison for 24 h. 4% PFA was added for fixation for 30 min and then 0.1% Triton-100 was added for permeabilization for 30 min. After a 30 min blocking step with 2% BSA, rabbit anti-rat eEF2K (1:1000) was applied and incubated overnight at 4 °C. The secondary antibody (goat anti-rat IgG-FITC) was introduced, followed by a 1 h incubation at 37 °C, and DAPI was added for 10 min. Each step involved three washes with PBS. Subsequently, images were observed and captured for analysis using a fluorescence microscope (Fi3, Nikon, Japan).

Observation of morphology in hippocampal tissue and PC12 cells using transmission Electron Microscopy

Rat and mouse hippocampal tissues, along with PC12 cells, were transformed into sample blocks measuring 1 mm³ and promptly immersed in 2% glutaraldehyde fixative for 2 h. The tissues underwent three washes with 0.1 M phosphate buffer (pH = 7.4), each lasting 15 min. Following a 1 h treatment with 1% osmic acid, the samples were rinsed three times with 0.1 M phosphoric acid solution, with each rinse lasting 15 min. Ethanol and acetone were employed for a 15 min gradient dehydration process. The tissue was embedded in a porous rubber embedding template (embedding agent: anhydrous ethanol = 1:1) and subsequently dried in an oven to create an embedding block. Sections, approximately 70 nm in thickness, were then produced using an ultramicrotome (Leica Ultracet R, Germany). Ultimately, double staining with 3% uranyl acetate and lead citrate was performed, and transmission electron microscopy (JEM1230, Japan) was utilized for image observation and collection for subsequent analysis.

Western blot analysis

The hippocampal tissues from rats and mice, along with PC12 cells, were lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1% PMSF based on the experimental groups. The sample suspension underwent centrifugation at 4 °C, 12,000×g for 10 min, and the resulting supernatant was collected. Total protein concentration was determined using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Equal aliquots of samples containing 50 g of protein were subjected to 15% (w/v) SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline and Tween 20 (TBST) for 2 h. Following this, blots were incubated overnight at 4 °C with rabbit polyclonal primary antibodies against eEF2K (1:1000), eEF2 (1:1000), p-eEF2 (1:1000), BDNF (1:1000), SYN1 (1:1000), TrkB (1:1000), Lpcat1 (1:500), and β-actin (1:1000). The membranes underwent five washes with TBST and were then incubated at room temperature for 2 h with a secondary antibody (goat anti-mouse/rabbit) diluted at 1:10000. Target proteins were detected using an enhanced chemiluminescence detection kit, and the relative intensity bands were analyzed using a gel imaging analysis system. β-actin served as an internal control.

RNA extraction and real-time qPCR analysis

RNA extraction from hippocampal tissue (rats and mice) and cell lysates utilized the E.Z.N.A.® Total RNA Kit I (Omega, China), following the manufacturer’s instructions. Subsequent to extraction, RNA underwent reverse transcription using the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan). The resulting cDNA served as a template for amplification with THUNDERBIRDTM Next SYBR® qPCR Mix (TOYOBO, Japan). Gene expression levels were quantified by normalizing to β-actin. Detailed primer sets for genes are provided in Supplementary Table 1 and Supplementary Table 2.

Mouse hippocampal tissue proteomics

The appropriate amount of mouse hippocampal tissue was taken out from a -80 °C freezer and was ground. After tissue grinding, the lysate was subjected to centrifugation at 12,000×g for 10 min at 4 °C, and the resulting supernatant was collected for protein concentration determination. Peptide fragments obtained from trypsin digestion were desalted using Strata X C18 (Phenomenex) and subsequently vacuum freeze-dried. The peptides were dissolved in 0.5 M TEAB and labeled following the TMT reagent kit instructions. After dissolving the peptide fragments in mobile phase A, separation was achieved using an EASY-nLC 1200 ultra-high-performance liquid chromatography system. Ionization of the separated peptides was performed in the NSI ion source, and analysis was carried out using an Orbitrap Exploris™ 480 mass spectrometer (ThermoFisher Scientific). Finally, the results were organized and analyzed (Supplementary Method 1).

Molecular docking and bioinformatics analysis

The HDOCK online platform (http://hdock.phys.hust.edu.cn/) was utilized as the molecular docking program in this study (Supplementary Method 2). We utilized Gene Ontology (GO) terms to analyze differentially expressed proteins identified through proteomic analysis, including Cellular Component (CC), Molecular Function (MF), and Biological Process (BP). For the functional enrichment analysis of GO and pathways, a two-tailed Fisher’s exact test was applied to evaluate the enrichment of differentially expressed proteins among all annotated proteins. Pathway analysis utilized the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/). The outcomes of GO enrichment and KEGG analysis were presented using R Studio software. To identify protein-protein interaction networks, the differentially expressed proteins’ database identifiers or protein sequences from the comparative group were cross-referenced with the STRING (v.11.5) database. The protein-protein interaction relationships for differentially expressed proteins were extracted based on a confidence score > 0.4 (middle confidence), and the network was visualized using the Cytoscape tool.

Statistical analysis

Epidata 3.0 software (http://www.epidata.dk) was employed for data entry and analysis. Results are presented as means ± SD. GraphPad Prism9 software was utilized for the analysis of diverse experimental data, with all experiments independently conducted at least three times. Continuous variable data underwent analysis through independent samples t-test or one-way analysis of variance, while categorical variables were assessed using the chi-square test. A P-value of ≤ 0.05 was deemed statistically significant.