The whole relative procedures were performed as per the flowchart (Fig. 1).

Fig. 1

Technical roadmap for in vivo and in vitro experiments

Ethical approval and experimental animals

This study received ethical approval from the Animal Ethics Committee of Guangxi Medical University (Approval No. 202306672). Animal handling adhered to the guidelines of the Chinese Experimental Animal System Ethics Review Committee. The required sample size was calculated using a resource equation method [32, 33]. We utilized 64 male, 8-week-old Sprague–Dawley rats (220–300 g) sourced from Guangxi Medical University Experimental Animal Center. The rats were randomly distributed into subgroups from the numbered cohort.

Rat model of cecal ligation and puncture (CLP)

Rats were fasted for 12 h with access to water prior to modeling. Sepsis was induced using the CLP method [34, 35]. Prior to surgery, rats were weighed and anesthetized with 3% sodium pentobarbital (50 mg/kg). The depth of anesthesia was monitored by applying pressure to the toe with forceps; adequate anesthesia was confirmed by the absence of limb withdrawal (e.g., no limb flexion). The abdomen was prepared by shaving and iodine disinfection. A 3 cm midline incision was made to access the cecum, which was ligated near the ileocecal valve and punctured with an 18G needle to release minimal intestinal content. The cecum was returned to the abdominal cavity, and the abdominal incision was closed in layers. Sham operations involved abdominal surgery without cecum ligation or puncture. Postoperatively, subcutaneous saline was administered to prevent shock.

Targeted temperature control

For rats in the hypothermic group, ice packs and an ALC-HTP animal temperature control system (Shanghai Alcott Biotech Co. Ltd., China) were used to rapidly lower body temperature and maintain rectal temperature between 32 °C and 34 °C. The ALC-HTP system’s animal-specific thermometer, which has a digital display, was utilized to measure rectal temperature. The target temperature was achieved within approximately 10 min. After 10 h of intervention, thermal blankets were used to gradually restore body temperature at a rate of 0.2–0.5 °C per hour, aiming for a temperature of 36–38 °C. The rectal temperature of the normothermic group was maintained between 36 °C and 38 °C. Anesthesia was sustained during temperature control through hourly intraperitoneal injections of 3% sodium pentobarbital (15 mg/kg).

Experimental design and grouping

Sixty-four male SD rats were used in total. To ensure that our study groups were allocated in a manner that was both fair and unbiased, we utilized a computer-generated random number table. Each animal was assigned a unique number, and a random number generator was used to determine the group allocation. Of these, 24 rats were randomly allocated into four groups: (1) control normothermia (CN), (2) control hypothermia (CH), (3) sepsis normothermia (SN), and (4) sepsis hypothermia (SH), with six rats per group. Twelve hours post-CLP, the rats were euthanized, and samples were collected for further analysis.

The remaining 40 rats were assigned to four groups of ten each for survival monitoring over a 5-day period. With hourly observations on the first day post-CLP, two-hourly assessments on the second and third days, and every 6 h on the following 2 days. The survival data were analyzed following the 5-day observation period.

Evaluation of sepsis models

Referring to the evaluation indexes of sepsis animal model published by Bradly Shruma et al [36], the success of sepsis modeling was comprehensively assessed using seven indices: the animal's appearance, level of consciousness, activity, response to stimulus, eye opening, respiration rate, and respiratory quality, In our experiments, CLP-operated septic rats exhibited erect hair, arched backs, reduced activity, depressed spirits, weakened or absent responses to stimuli, closed eyes with insensitivity to pain stimulation and crust-like corners, and increased respiratory rates. Upon opening the abdominal cavity, obvious intestinal dilatation, edema, congestion; ligated cecum with gray coloration or necrosis; mesenteric adhesion and pus moss formation; and the presence of large amounts of bloody, turbid ascites accompanied by a foul odor were observed. The lungs of sham-operated rats were elastic, smooth, and light red, whereas the lungs of septic rats were dark in color with obvious congestion, edema, and local hemorrhage, indicating successful induction of sepsis manifestations in rats.

Hematoxylin and eosin (H&E) staining

Lung tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated with ethanol, and embedded in paraffin. Thin sections were prepared and stained with HE for histological examination under a light microscope. Lung injury was assessed using scoring criteria from the American Thoracic Society's official report [35]. Detailed methodologies can be found in the supplementary materials.

Bronchoalveolar lavage fluid (BALF) collection and analysis

The rats were anesthetized, and the trachea was exposed via a cervical incision to occlude the right bronchus. A small cut was then made in the trachea to insert the lavage tube into the left bronchus. Subsequently, 1 ml of ice-cold saline was infused through the catheter into the left lung, and BALF was collected by suctioning three times. Samples were centrifuged at 3000 rpm for 10 min at 4 ℃ and the supernatant was collected and stored at -80℃ protein concentration was measured using a BCA assay kit (Beyotime, Shanghai, China).

Measurement of the wet/dry weight (W/D) ratio

To determine the severity of pulmonary edema, the W/D ratio was calculated. After successful modeling, the right upper lung was clipped and cleaned with PBS, and absorbent paper was used to remove surface moisture. The tissue was subsequently weighed to obtain wet weight (wet mass). After 48 h of oven drying, the tissue was weighed again to obtain the dry weight (dry mass). The W/D ratio was then computed.

Blood gas analysis

Rats were anesthetized, and their limbs were properly fixed. The midline was utilized to cut the abdominal cavity, and the intestines were moved aside using cotton. The abdominal aorta was fully exposed, blood samples were collected and analyzed using a Blood Gas Analyzer Model ABL 90 (Radiometer Medical Devices Ltd, Denmark).

Enzyme-linked immunosorbent assay (ELISA)

Interleukin-1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) levels were quantified using ELISA kits (FANKEW, Shanghai, China) according to the manufacturer's instructions. Absorbance at 450 nm was measured with a microplate reader (Huaan Magnech, Beijing, China).

MDA, SOD, Fe2+, ROS, and GSH measurements

Malondialdehyde (MDA), superoxide dismutase (SOD), ferrous iron (Fe2⁺), ROS, and GSH levels were quantified using specific assay kits: MDA (A003-1-2, Nanjing Jiancheng Bioengineering Institute, China), SOD (BC5165, Solarbio, China), iron (BC5415, Solarbio, China), ROS (BB-475015, BestBio, China), and GSH (A006-2-1, Nanjing Jiancheng Bioengineering Institute, China). Assays were conducted following the manufacturer's protocols.

Real-time quantitative PCR (RT-qPCR)

Total RNA was isolated using the RNAeasy™ Animal RNA Isolation Kit with Rotary Column (Beyotime, R0026), following the manufacturer's guidelines. RNA purity and concentration were assessed using a Nanodrop 2000 Ultra-Micro Spectrophotometer. Reverse transcription was carried out using the Takara RT kit (RR036A) according to the provided protocol, and the cDNA products were stored at − 20 °C. The TB Green PCR Kit (Takara, RR820A) was used for RT-qPCR, and relative mRNA expression was quantified using β-actin as an internal control.

Western blot analyses

Western blotting was utilized to evaluate the expression of P53, SLC7A11, and GPX4 proteins. Samples were stored at – 80 °C prior to protein extraction, which was carried out using RIPA lysis buffer (Beyotime, Shanghai, China). Extracted proteins were separated via SDS-PAGE and transferred onto a PVDF membrane (Millipore, USA). After blocking with 5% skim milk, the membrane was rinsed with TBST and incubated overnight at 4 °C with primary antibodies targeting P53 (1:5000, Proteintech), SLC7A11 (1:500, Zheneng Bio), GPX4 (1:500, Zheneng Bio), and β-actin (1:10,000, Zheneng Bio). The next day, after the membrane was removed from the primary antibody mixture, TBST was used to thoroughly rinse the membrane. The secondary antibody (1:10,000 in TBST, CST) was incubated for 1 h at room temperature, protected from light. Protein signals were visualized using the Li-Cor Odyssey Infrared Imaging System (Li-Cor, USA).

Cell culture

RLE-6TN cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Oricell, USA) and antibiotics. Cells were maintained at 37 °C in a 5% CO2 incubator. In vitro sepsis was induced by exposing cells to LPS for 24 h.

Cell viability assay

Cell viability was evaluated using the Cell Counting Kit-8(CCK-8) (Meilunbio, Dalian, China).Each well of a 96-well plate was seeded with 5,000 RLE-6TN cells and subsequently treated with LPS for 24 h. Groups were organized based on the experimental design: control normothermia (CN), control hypothermia (CH), sepsis normothermia (SN), and sepsis hypothermia (SH). The SN and SH groups were treated with varying concentrations of LPS for 24 h. Subsequently, the CH and SH groups were transferred to a separate incubator maintained at 33 °C. Cell viability was evaluated using a CCK-8 assay, where cells were incubated with a 1:10 dilution of CCK-8 solution for 1 h at 37 °C, and absorbance was recorded at 450 nm.

ROS measurement in cells

RLE-6TN cells were incubated overnight with 10 µg/mL LPS. Subsequently, the cells were treated with 500 µL of a 10 µM DCFH-DA fluorescent probe and incubated for 30 min in the absence of light. To remove any remaining unattached probe, the cells were rinsed with PBS. Fluorescence intensity was measured using ImageJ software (XLIH, USA) following imaging with a Nikon Eclipse Ci-L microscope (Nikon, Japan).

Statistical methods

Quantitative data are expressed as mean ± standard deviation. Data following a normal distribution were analyzed using one-way ANOVA, preceded by a homogeneity of variances test. For homogenous variances, pairwise comparisons were conducted using the least significant difference (LSD) test, while post hoc multiple comparisons utilized the Bonferroni correction. If variances were unequal, Welch's test was applied, followed by Dunnett's T3 test for multiple comparisons. For non-normally distributed data, the rank-sum test was performed. Survival time and survival rate of rats were assessed using Kaplan–Meier (K-M) analysis, and the log-rank test was employed for group comparisons. All statistical analyses were performed using the SPSS version 23.0 software (IBM Corp., Armonk, NY, USA). A P-value < 0.05 was considered statistically significant.