Mouse model of SAE and grouping

A total of 81 mice (C57BL/6 J male, 6–8 weeks old, 20–25 g) were obtained from the Animal Experiment Center of Xuzhou Medical University. The ethical approval code for the animal experiments is 2021–12-W023. The mice (with unrestricted access to food and water) were kept in a setting with the temperature ranging from 22 °C to 26 °C and a humidity between 40 and 70%. They were randomly divided into three groups: sham, SAE, and DFO. Based on the duration of the experiment, each group was divided into three subgroups: 1 day, 3 days, and 7 days. The SAE and DFO groups were established using the CLP method [11]. Specifically, the mice underwent a 12-h fast (food and water) prior to the operation, then they were anesthetized via an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), followed by a 1-cm incision along the midline of the abdomen to expose the cecum. A silk thread was used to bind the midpoint between the ileocecal valve and the end of the cecum, after which a 21G needle was used to puncture the distal cecum and squeeze out a little amount of excrement. The cecum was restored into the abdominal cavity, and the peritoneum and skin were sutured. In the sham group, the cecum was exposed after the laparotomy, without ligation or puncture. After surgery, all mice were exposed to an intraperitoneal injection of preheated normal saline (37 °C, 50 mL/kg). The DFO group received three intraperitoneal injections of DFO (50 mg/kg) at 12 h before, immediately after, and 12 h after the operation, while the other groups received intraperitoneal injections with normal saline (same volume). The animals were observed every 12 h after the administration and operation. The method used for euthanizing and collecting specimens from the experimental animals was the same as that used before modeling with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).

Clinical sample collection

Ten children admitted to the Pediatric Intensive Care Unit of Xuzhou Children’s Hospital from September 2022 to August 2023 with SAE were randomly selected as the study group (the SAE group), and 10 sepsis patients without associated brain dysfunction were selected as the control group (the non-SAE group). This study was approved by the Medical Ethics Committee of Xuzhou Children’s Hospital (Approval No. 2022–05-07-H07). The criteria for inclusion in the SAE group were as follows: (1) diagnosis of sepsis and organ dysfunction based on the 2020 version of the Surviving Sepsis Campaign International Guidelines for the Management of Septic Shock and Sepsis-Associated Organ Dysfunction in Children [14]; (2) age of > 28 days and ≤ 14 years; (3) presence of an altered mental status upon admission, such as seizures, delirium, or coma, with a Glasgow Coma Scale score ≤ 9; and (4) abnormalities observed in the electroencephalogram and cranial imaging. The exclusion criteria were as follows: (1) underlying neurological disorders affecting the nervous system; (2) diagnosis of intracranial infection, intracranial hemorrhage, or other intracranial organic lesions during hospitalization; and (3) brain disorders caused by dysfunction of other organ systems, such as hepatogenic encephalopathy, renal encephalopathy, etc. The baseline data, Glasgow Coma Scale scores, and white blood cell count, C-reactive protein, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, serum creatinine, and blood culture results from the two groups of hospitalized pediatric patients were collected. Blood samples from patients were obtained at 24, 48, and 72 h following admission, and the serum and peripheral blood mononuclear cells (PBMCs) were separated. The levels of S100 calcium-binding protein beta (S-100β), glial fibrillary acidic protein (GFAP), superoxide dismutase (SOD), and malondialdehyde (MDA) at each time point were detected in the serum by using enzyme-linked immunosorbent assays (ELISAs). The ELISA kits were purchased from Shanghai Lanpai Biological Technology Co., Ltd. The extracted PBMCs were preserved in 1 mL of TRIzol reagent (Vazyme Biotech, Nanjing, China) and stored in a -80 °C freezer.

Total RNA extraction and real-time polymerase chain reaction (qPCR)

PBMCs were extracted using the procedure for lymphocyte separation. The procedure consisted of collecting the patient’s blood specimen (usually 2 mL) and centrifuging it at 2000 rpm for 5 min to separate the blood cells. Next, 4 mL of PBS buffer was added to the separated blood cells, and the mixture was mixed well. Subsequently, 2 mL of Ficoll reagent (New England Biolabs Inc., Beijing, China) was slowly added, and the mixture was allowed to form two layers, with the lower layer being a liquid. The two layers underwent centrifugation at 1800 rpm for 30 min. The solution separated into three layers, with the PBMC layer in the middle appearing as a cloudy layer. The PBMC layer in the middle was carefully removed, PBS buffer was added to give a total volume of 10 mL, and it was centrifuged at 2000 rpm for 5 min. The PBMCs adhered to the bottom of the centrifuge tube, so they were washed twice with PBS, 1 mL of TRIzol was added, and the tube was sealed and stored at -80 °C for subsequent qPCR experiments. The total RNA isolated from the PBMCs of the patients was reverse transcribed to complementary DNA by a PrimeScript™ RT reagent kit (Vazyme Biotech, Nanjing, China) and stored at –20℃. The qPCR was carried out to detect the mRNA levels. Using β-actin as the internal reference, the cDNA was amplified using SYBR Green Mix (Biosharp, Hefei, China) on a LightCycler 96 fluorescence qPCR system. The PCR conditions were as follows: 1 min of incubation at 94 °C, then 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. The relative expression level of the target gene was calculated using the 2−ΔΔCt method. Table 1 lists the primer sequences used to amplify the target genes.

Table 1 Primer sequences for qPCRNeuroethological score

The neurobehavioral changes in the different groups at 1-day postoperation were evaluated by the neuroethological scores [15], according to the following procedures and criteria: (1) The corneal reflex was tested by gently touching the mouse’s cornea with a cotton swab to elicit blinking or shaking the head. (2) The auricular reflex was considered normal if the mouse’s auricle was touched and caused a strong head rotation. (3) The righting reflex was normal if the mouse was placed in the supine position and quickly returned to the prone position, flattening the front and rear feet. (4) The Tai-flick reflex was normal if the mouse turned around and escaped injury after the tail was briefly stimulated. (5) The escape reflex was normal if the mouse received a temporary stimulus and escaped. The scoring criteria were as follows: normal reflex (within 1 s), 2 points; reflex dullness (1–10 s), 1 point; and areflexia, 0 points. The total score was calculated by summing each item, with a scale of 1 to 10.

Hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM) detection

One day after the operation, three mice from each group were sacrificed, and their brains were removed. The hippocampi of the left brains were isolated and immediately immersed in a 4% paraformaldehyde universal tissue fixative (Biosharp, Hefei, China). The tissue embedding, tissue sectioning, and H&E staining were performed before the section scanning. The right brains of the mice were also removed, immediately immersed in a TEM fixative (Servicebio, Wuhan, China), and stored at 4 °C. After 30 min, the samples were taken out, and the hippocampi were immediately placed in an ice bath. Tissue samples (2 mm × 2 mm) were prepared and immersed in the TEM fixative, followed by storage at 4 °C. Finally, the tissues were analyzed by TEM.

Protein extraction and western blot

First, the tissue sample was rapidly frozen in liquid nitrogen to maintain its integrity and stability. Next, the frozen tissue sample was placed into a prechilled homogenization tube. Then, a homogenizer was used to break down the sample. Radioimmunoprecipitation assay lysate (Beyotime Biotechnology, Shanghai, China) and phenylmethylsulfonyl fluoride (Beyotime Biotechnology, Shanghai, China) were mixed in a ratio of 50 to 1 to extract the total protein in the hippocampal tissues (n = 3 in each group). The protein concentration of the extract was determined by a bicinchoninic acid kit (Beyotime Biotechnology, Shanghai, China), and then all samples were diluted to the same protein concentration. A sodium dodecyl sulfate–polyacrylamide gel electrophoresis rapid preparation kit (Beyotime Biotechnology, Shanghai, China) was used to prepare a 10–12% gelatinous plate, and the same amount of tissue protein (20 µg) in each group was added to each well. After gel electrophoresis and transfer, the polyvinylidene fluoride (PVDF) membranes were washed and blocked with 5% skim milk powder for 1 h before incubation with primary antibodies against GPX4 (Abcam, Cambridge, UK, ab125066, 1:1000), PEBP-1 (Abcam, Cambridge, UK, ab76582, 1:1000), 15-LOX (Proteintech, Chicago, IL, USA, 13073–1-AP, 1:500), and GAPDH (Affinity, Changzhou, China, AF7021, 1:10,000) at 4 °C overnight. After washing, the PVDF membranes were treated with horseradish peroxidase-coupled antibody (Bioworld, Shanghai, China, BS13278, 1:10,000) at room temperature for 1 h. Pierce ECL GAPDH served as the internal control for total protein when detecting the protein bands using Chemiluminescent Substrate (Biosharp, Hefei, China).

ELISA

The levels of S-100β, neuron-specific enolase (NSE), and 4-hydroxynonenal (4-HNE) in the serum as well as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and MDA in the hippocampal tissues of the mice were measured using commercial ELISA kits (Lanpaibio, Shanghai, China) (n = 4 for each group). The blood was obtained by removing the mouse eyeballs, followed by centrifugation to separate the serum. The hippocampus was obtained and homogenized in normal saline. After centrifugation, the protein concentrations were measured, and the samples were subsequently diluted to the same protein concentration. The levels of S-100β, MDA, total SOD, and GFAP in the serum of patients were measured using commercial ELISA kits (Lanpaibio, Shanghai, China).

Fifty microliters of a standard working solution at various concentrations was applied to each well in a 96-well plate. Next, the samples were added to the tested wells, with 10 μL in each well, followed by 40 μL of sample diluent in each well. Next horseradish peroxidase-labeled antibody (100 μL) was added to each well and incubated for 60 min at 37 °C. After discarding the liquid, the plate was patted dry on absorbent paper, followed by each well being filled with detergent and incubation for 1 min. The plate was once more patted dry and then cleaned five times after disposing of the detergent. Each well was filled with 50 μL of substrate working solutions A and B, which were then incubated for 15 min at 37 °C in the dark. Following the addition of 50 μL of the stop-working solution to each well, the optical density (OD) value of each well at 450 nm was determined in less than 15 min using a microplate reader.

Detection of ferrous ions by a colorimetric method

The concentration of ferrous ions was determined in hippocampal tissues (n = 4 per group) using a Ferrous Ion Colorimetric Kit (Elab Science, Wuhan, China). Colorimetry was used in this study to detect ferrous ions. The principle is as follows: Ferrous ions in the sample bind to a probe, resulting in the formation of a substance that exhibits a strong absorption peak at a wavelength of 593 nm. Within a certain range, the OD of this substance is linearly correlated with the concentration of ferrous ions, reflecting the total content of ferrous ions. The hippocampal samples and iron standard working solution were prepared. Next, 150 μL of the chromogenic solution was added to 300 μL of the standard working solution and the sample to be tested, and the mixture was thoroughly mixed. The mixture was incubated at 37℃ for 10 min and subsequently centrifuged at 12,000 × g for 10 min. Then, it was added to the enzyme-labeled plate, and the OD value was measured at 593 nm. The concentration of ferrous ions in the hippocampal tissues was calculated according to the provided instructions.

Detection of the mitochondrial membrane potential (MMP) with JC-1

The mitochondria were extracted from the hippocampal tissues of mice (n = 4 per group) using a mitochondrial extraction kit (Solarbio, Beijing, China). The homogenization of hippocampal tissue was performed in an ice-cold bath at 0 °C, followed by centrifugation at 4 °C to extract the mitochondria, which were then rapidly transferred and stored at -70 °C. The proteins were measured by the bicinchoninic acid (BCA) method and then diluted to the same concentration. JC-1 staining working solution was prepared following the instructions provided in the MMP detection kit (Beyotime Biotechnology, Shanghai, China) and was subsequently mixed with the extracted hippocampal mitochondria. The preparation and addition of JC-1 working solution were also performed in a sterile ice-cold bath at 0 °C, followed by centrifugation at 4 °C. The fluorescence intensities of the JC-1 polymer and monomer were measured at 525/590 nm and 490/530 nm, respectively.

Detection of the mitochondrial ATP content in the hippocampus

The ATP content was determined utilizing the luciferin–luciferase luminescence technique. Hippocampal mitochondria were isolated from mice (n = 4 per group), and the protein content was measured by a BCA protein assay reagent. The sample (50 μL) and the luciferin–luciferase reaction kit reagent (50 μL) (Beyotime Biotechnology, Shanghai, China) were reacted to quantify the ATP content. According to the instructions, ATP in the samples is unstable at room temperature but can remain stable for up to 6 h on ice (0 °C). Therefore, sample lysis, working solution preparation, and other operations were conducted under sterile conditions in an ice bath at 0 °C, and centrifugation was performed at 4 °C. Luminescence was measured using a luminometer, and the ATP content was quantified using a standard curve.

Statistical analysis

The analysis of the data was done with SPSS Statistics 23.0. The chart was created using GraphPad Prism 8.0 software. The data were obtained from at least three independent tests and presented as the mean ± standard deviation. The t-test (two-independent-sample) was used to compare the normally distributed data of the two groups, and one-way analysis of variance was utilized. P < 0.05 was used as the significance criterion to assess statistical significance.