Chemicals, reagents, and instruments
Methanol, acetonitrile, formic acid, pyridine, n-hexane, methoxylamine hydrochloride, L-2-chlorophenylalanine, and N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) were purchased from Sigma Aldrich (St. Louis, MO, USA). All chemicals used were of analytical grade. Anti-IL-6 [EPR23819-103] (ab290735), anti-NF-κB [E381] (ab220803), anti-TNF-α [52B83] (ab1793), and anti-IκBα [E130] (ab32518) antibodies were purchased from Abcam (Cambridge, UK). Amylase ELISA kits and sodium taurocholate were purchased from Sigma.
A temperature maintenance recorder (YZA-09A) was purchased from Hefei Sipeik Instrument Technology Co. Ltd. (Hefei, China) and assembled with one electronic temperature recorder, two temperature measuring electrodes, and one heating pad.
Intragastric cooling balloon design
Two 8-Fr silicone catheters for children (Wellead Medical Co. Ltd., Guangzhou, China) were bound and securely connected to a medical rubber finger cover (Bluesail Medical Co. Ltd., Shandong, China) as a 20-mm flexible rubber balloon and used for transgastric cooling of the pancreas.
Animals and treatment
All animal experiments were performed in accordance with the guidelines of Ethics Committee of Hunan Provincial People's Hospital (Approval No. 2022 − 182). Male Sprague–Dawley rats weighing 360–400 g were purchased from the Experimental Animal Center of Medical College of Hunan Normal University. All rats were housed in an environmentally controlled room (23 ± 1°C and 60% humidity) with a 12:12 h light–dark cycle and had free access to food and water. Only male SD rats were used in the process of the experiment to avoid the influence of gender differences on data and metabolomic results, and to reduce variables.
Eighteen rats were randomly divided into three groups: sham operation (SO, n = 6), acute pancreatitis (AP, n = 6), and acute pancreatitis hypothermia (APH, 25–27°C, n = 6) groups. All rats were fasted for over 12 h and offered free access to tap water before the operation. The weights of the rats in the three groups were measured, and the rats were then anesthetised via intraperitoneal injection of 3 mL/kg 10% chloral hydrate. The pancreas was turned several times in the SO group, followed by closure of the abdomen. AP was induced in the AP and APH groups via retrograde injection of 3.5% sodium taurocholate solution at a volume of 0.1 mL/100 g into the biliopancreatic duct. Five to ten minutes after injection of sodium taurocholate solution, localised or diffuse hyperaemia and oedema of the pancreas were regarded as successful modelling in the AP and APH groups, and then, the abdominal cavity was closed in the AP group. In the APH group, after the pancreatitis model was successfully established, gastrotomy was performed near the cardia with a 0.5-cm incision and a 20-mm flexible rubber balloon with two inflow and outflow silicone catheters, which were placed inside the stomach of the rat through the incision, and the gastric wall was sutured. A temperature-measuring electrode was placed in the gastropancreatic space, and the electrode lead was sutured and fixed with the skin outside the abdominal cavity. Finally, the local hypothermia intervention in the stomach was implemented immediately after the abdominal cavity was closed. Simultaneously, the rats in the APH group were placed on a heating pad to prevent systemic hypothermia, and another temperature-measuring electrode was inserted into the rectum of the rats, through the anus, and fixed with tape to prevent experimental errors caused by the removal of the temperature-measuring electrode. The inflow silicone catheter was connected to a peristaltic pump device. By controlling the speed of ice water circulation and adjusting the output power of the heating pad, the pancreatic surface temperature was controlled at 25–27°C. The rectal temperature was controlled at 36–39°C to achieve pancreatic surface temperature reduction while avoiding systemic hypothermia. The temperatures of the gastropancreatic space and rectum were continuously monitored during the whole experiment. Pancreatic and rectal temperatures were not monitored and controlled in the SO and AP groups. Lactated Ringer’s solution was subcutaneously administered to all animals as a volume replacement. Rats received 20 mL of Lactated Ringer’s solution divided into four doses of 5 mL each, which were administered every 1 h. After 1 and 3 h of operation, blood samples were collected from the orbital vein, and serum was obtained via centrifugation at 3,000 rpm for 10 min at room temperature (20°C). At 5 h post-surgery, blood samples were collected from the abdominal aorta, and serum was obtained via centrifugation at 3,000 rpm for 10 min at room temperature 20°C. Rats were euthanised via cervical dislocation, and a portion of the pancreatic and gastric tissue was fixed in 10% neutral-buffered formaldehyde solution. Serum and a part of the pancreatic tissues were rapidly stored in a -80°C refrigerator until use.
Analysis of serum amylase levels
All sera were isolated via centrifugation for 10 min at 3,000 rpm and stored in a -80°C refrigerator until use. Serum amylase was determined using an amylase ELISA kit (Sigma) according to the manufacturer’s instructions.
Histological examination
The pancreatic tissues were sliced into 4-µm thick sections using a microtome (KD3368, Shanghai, China), stained with haematoxylin and eosin, and analysed according to histologic scoring[8] for AP by two pathologists who were blinded to the experiment.
Immunohistochemistry (IHC) of the pancreatic tissues
IHC was performed to observe NF-κB, IL-6, TNF-α, and IκBα expression in pancreatic tissues. Paraffin-embedded pancreatic tissue sections were deparaffinised, incubated with hydrogen peroxide for 10 min, repaired with citrate buffer for 10 min, and blocked with goat serum for 30 min. After incubation with goat serum, the slides were incubated overnight (12 h) with primary antibodies against NF-κB, IL-6, TNF-α, and IκBα. The negative control was replaced with phosphate-buffered saline, followed by diaminobenzidine colour development, haematoxylin counterstaining, and xylene transparent and neutral resin sealing. The appearance of brown-yellow particles in the cells was considered a positive result. The percentage of NF-κB-, IL-6-, TNF-α-, and IκBα-positive cells was calculated using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
Western blot analysis
The pancreatic tissues were homogenised, and the total protein was extracted. The concentration and content of pancreatic tissue proteins were determined using bicinchoninic acid. A 20-µg sample was loaded into each well. After being transferred to blocking buffer [5% milk in Tris-buffered saline containing 0.05% Tween 20] at room temperature (20°C) for 1 h, membranes with diverse bands were probed with anti-IL-6, anti-TNF-α, anti-NF-κB, and anti-IκBα antibodies overnight at 4°C. The secondary antibody was added and incubated for 1 h, and a chromogenic solution was added for colour development, with β-actin as the internal reference. The brightness of each protein band was analysed using Quantity One Analysis software (Bio-Rad Laboratories, Hercules, CA, USA). The protein levels of IL-6, TNF-α, NF-κB, and IκBα in pancreatic tissues were expressed as the grey value of the protein bands of IL-6, TNF-α, NF-κB, and IκBα/the grey value of the β-actin band.
GC-MS procedures
The sera were thawed at room temperature (20°C), and 200 µL was added to a 1.5-mL EP tube, with 10 µL of 2-chloro-1-phenylalanine dissolved in methanol (0.3 mg/mL) as an internal standard. Then, 300 µL of a mixture of ice-cold methanol and acetonitrile (at a ratio of 2/1) was added, followed by vortexing for 1 min. The samples were then stored at -20°C for another 10 min and centrifuged at 15,000 rpm for 10 min at 4°C. Quality control samples were prepared by mixing all samples to produce a mixed sample. Aliquots (200 µL) of the supernatant were transferred to glass sampling vials for vacuum drying at room temperature (20°C). Then, 80 µL of methoxyamine hydrochloride solution containing 15 mg/mL pyridine was added. Next, 80 µL of BSTFA containing 1% TMCS and 20 µL of hexane were added, followed by vigorous vortexing for 2 min. This was followed by derivatisation at 70°C for 60 min, and finally, the mixture was left at ambient temperature for 30 min until GC-MS analysis.
Derived samples were analysed using an Agilent 7890 B gas chromatography system (Agilent Technologies Inc., Santa Clara, CA, USA). Chromatographic separation was performed using a DB-5MS fusion silica gel capillary column (30 m × 0.25 mm × 0.25 µm; Agilent J&W Scientific, Folsom, CA, USA). Helium (99.99%) was used as the carrier gas and passed through the column at a steady flow rate of 1 mL/min. The injector temperature was maintained at 260°C in advance. The oven temperature was initially set to 70°C. Subsequently, the temperature was gradually increased at a rate of 8°C/min to 125°C, 5°C/min to 210°C, 10°C/min to 270°C, 20°C/min to 305°C, and the temperature was finally maintained at 305°C for 5 min. The collision energy was set to 70 eV. MS data were collected in full scan mode (m/z 50–500).
LC-MS procedures
Sera were thawed at room temperature (20°C). Serum (200 µL) was added to a 1.5-mL EP tube containing 10 µL of 2-chloro-L-phenylalanine (0.3 mg/mL) dissolved in methanol and vortexed for 10 s. A mixture of methanol and acetonitrile at a ratio of 2/1 (300 µL) was vortexed for 1 min, sonicated for 10 min at room temperature (25–28°C), and stored at -20°C for 30 min. The extract was centrifuged at 15,000 rpm for 15 min at 4°C, and 1 mL of the supernatant was added to a brown glass vial and dried in a freeze-concentrated centrifuge dryer. Next, 300 µL of a mixture of methanol and water (1/4, v/v) was added to each sample. Samples were vortexed for 30 s and allowed to stand at 4°C for 2 min. Samples were then centrifuged at 15,000 rpm for 5 min at 4°C. The supernatant from each tube was collected with a crystal syringe, filtered through a 0.22-µm microfilter, and transferred to LC vials. The vials were stored at -80°C until LC-MS analysis.
Metabolic profiles in positive and negative ion modes were analysed using an ACQUITY UHPLC system (Waters Corporation, Milford, MA, USA) coupled to an AB SCIEX Triple TOF 5600 system (AB SCIEX, Framingham, MA, USA). The column was ACQUITY UPLC BEH C18 (1.7 µm, 2.1 mm × 100 mm). The binary gradient elution system consisted of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v). Gradient elution was as follows: 0 min, 5% B; 2 min, 20% B; 4 min, 60% B; 11 min, 100% grade B; 13 min, 100% B. The column temperature was set to 45°C, and the flow rate was 0.4 mL/min. All samples were stored at 4°C. Full scan mode (m/z 70–1000) combined with information-dependent mode was used for data acquisition. The parameters of the mass spectra were as follows: ion source temperature: 550°C (+) and 550°C (-); ion spray voltage: 5500 V (+) and 4500 V (-); curtain gas: 35 psi. The clustering potentials were 100 V (+) and − 100 V (-). The collision energies were 10 eV (+) and − 10 eV (-). Interface heater temperature: 550°C (+) and 600°C (-). Information-dependent acquisition (IDA) analysis was performed in the m/z range from 25 to 1,000 with a collision energy of 30 eV.
Identification and quantitative analysis of multiplatform metabolomics
Raw data acquired by the analytical instruments were processed for peak detection and alignment. Full scan mass spectra of these metabolites were further searched and analysed using biochemical databases, including the Human Metabolome Database and Kyoto Encyclopaedia of Genes and Genomes. The peak areas of metabolites were normalised against the internal standard to semi-quantitatively determine metabolite levels.
Statistical analyses
Serum amylase levels and protein levels of IL-6, TNF-α, NF-κB, and IκBα in pancreatic tissue were analysed using SPSS 22.0 software (IBM, Armonk, NY, USA). IL-6, TNF-α, NF-κB, and IκBα positive cell rates in pancreatic tissue were in accordance with the normal distribution. Analysis of variance was used for comparisons among the three groups, and P < 0.05 was considered statistically significant.
The metabolic information data detected by the instrument were pre-processed using MetaboAnalyst 4.0 software (https://www.metaboanalyst.ca) for t-test and fold change analysis to obtain P-values and fold-change (FC) values. The metabolic information data detected by the device were imported into SIMCA-P14.1 software (Sartorius, Gottingen, Germany) after the relevant pre-treatment, and partial least square-discriminant analysis was performed to obtain the variable important in projection (VIP) value. The differential metabolites between SO, AP, and APH groups were screened under the conditions of FC > 1.2 or FC < 0.8, VIP > 1, and P < 0.05, and heat map cluster analysis was performed. Differential metabolites were analysed using MetaboAnalyst 4.0 software.