Protective role of local hypothermia in taurocholate-induced acute pancreatitis: an in vivo and in vitro study

Abstract

BACKGROUND:

Acute pancreatitis (AP) is an inflammatory disease characterized by pancreatic autodigestion and systemic inflammatory responses, which can lead to severe complications and high mortality. Hypothermia has shown potential protective effects against AP-related injury; however, the metabolic mechanisms underlying hypothermia-mediated protection against AP remain unclear. In this study, we aimed to investigate serum metabolic changes in a rat model of AP treated with hypothermia with intragastric cooling and to identify potential protective metabolites and explore their biological roles.

METHODS:

The entire study included both in vivo and in vitro experiments. In the in vivo experiments, 18 male Sprague Dawley rats were randomly assigned to three groups (n=6 per group): the sham operation (SO), AP, and AP treated with hypothermia (APH) groups. AP was induced by retrograde intraductal injection of 3.5% sodium taurocholate. In the APH group, hypothermia with intragastric cooling (25-27 °C) was achieved using a gastric cooling balloon, while the rectal temperature was maintained at physiological levels. Serum amylase levels, pancreatic histopathology, and inflammatory cytokine expression were assessed using an enzyme-linked immunosorbent assay, Schmidt scoring system, immunohistochemistry, and Western blotting. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics was performed to identify differentially expressed serum metabolites associated with hypothermia. Through in vitro experiments, the biological effects of key metabolites were further investigated in sodium taurocholate-treated AR42J pancreatic acinar cells.

RESULTS:

In vivo, serum amylase levels, pancreatic pathological injury, and the expression of inflammatory cytokines, including interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and nuclear factor-κB (NF-κB), were significantly lower in the APH group than in the AP group. GC-MS metabolomic profiling revealed 41 differentially expressed metabolites between the APH and AP groups. In vitro experiments demonstrated that LY294002, pinitol, tocopherol and their combination reduced reactive oxygen species production, decreased α-amylase activity, and modulated Nrf2 mRNA expression in AR42J cells.

CONCLUSION:

In this study, local hypothermia attenuated inflammatory cytokine release, serum amylase elevation, and pancreatic injury in a rat model of AP. These protective effects may be associated with the upregulation of serum pinitol and tocopherol levels. These metabolites may exert potential protective effects through pathways related to inflammation and oxidative stress.

INTRODUCTION

Acute pancreatitis (AP) is a complex inflammatory disorder characterized by pancreatic acinar cell injury, the premature activation of digestive enzymes, and systemic inflammatory responses.^[1] Severe forms of the disease may rapidly progress to multiple organ failure and are associated with considerable morbidity and mortality.^[2] Despite advances in supportive care, effective targeted therapies for AP remain limited because of the complex and multifactorial mechanisms underlying disease progression.

Therapeutic hypothermia has been explored as a protective strategy in several inflammatory and ischemic conditions. Experimental studies have demonstrated that hypothermia can attenuate inflammatory responses, oxidative stress, and cellular injury.^[3] In the context of pancreatitis, hypothermia has been reported to reduce pancreatic injury and inflammatory cytokine release. However, systemic hypothermia may cause adverse physiological effects. Recently, local pancreatic hypothermia using transgastric cooling has been proposed as a promising approach for minimizing systemic temperature reduction while preserving protective effects on pancreatic tissue. de Oliveira et al^[4] demonstrated that transgastric local pancreatic hypothermia (25 °C) via a gastric cooling balloon effectively minimized systemic hypothermia and decreased pancreatic necrosis, apoptosis, and inflammation.

Although the anti-inflammatory effects of hypothermia have been partially described, the underlying metabolic mechanisms remain unclear. Metabolomics provides a powerful tool for identifying metabolic alterations associated with disease processes and therapeutic interventions. In this study, we used gas chromatography-mass spectrometry (GC-MS)-based metabolomics to investigate serum metabolic changes in a rat model of AP treated with hypothermia with intragastric cooling. Furthermore, we aimed to identify potential protective metabolites and explore their biological roles.

METHODS

Ethical approval

All the experimental procedures were approved by the Institutional Animal Care and Use Committee of Hunan Provincial People’s Hospital (Approval No. 2022-182) and were conducted in accordance with the relevant guidelines and regulations.

Chemicals, reagents, and instruments

Methanol, acetonitrile, formic acid, pyridine, n-hexane, methoxyamine hydrochloride, L-2-chlorophenylalanine, and N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) containing 1% TMCS were purchased from Sigma-Aldrich (USA). Antibodies purchased from Abcam (UK) included anti-IL-6 (EPR23819-103) (ab290735), anti-NF-κB (E381) (ab220803), anti-TNF-α (52B83) (ab1793), and anti-IκB-α (E130), β-actin (66009-1-Ig), glycogen synthase kinase-3β (GSK-3β; 22104-1-AP), and phosphorylated protein kinase B (P-AKT) (66444-1-Ig) were purchased from Proteintech (Rosemont, USA). Amylase enzyme-linked immunosorbent assay (ELISA) kits, pinoxazone (441252-500MG), tocopherol (T3251-5G), and sodium taurocholate were purchased from Sigma-Aldrich (USA). A reactive oxygen species (ROS) detection kit was purchased from Beyotime Technology Co., Ltd. (China). AR42J cells (AW-CNR085) were purchased from Changsha Abiowell Biotechnology Co., Ltd. (China).

The YZA-09A temperature maintenance recorder, manufactured by Hefei Spac Instrument Technology Co., Ltd. (China), included an electronic temperature recorder, two temperature measuring electrodes, and a heating pad.

In vivo experiments

Animal model and experimental design

Male Sprague-Dawley (SD) rats (360-400 g) were obtained from the Experimental Animal Center of the Medical College of Hunan Normal University. The animals were housed under controlled environmental conditions (23±1 °C, 60% humidity, 12-h light/dark cycle) with free access to food and water.

The rats were randomly assigned to three groups (n=6 per group): the sham operation (SO), AP, and AP treated with hypothermia (APH) groups.

After fasting for 12 h (free access to water), the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL/kg). AP was induced in the AP and APH groups by retrograde injection of 3.5% sodium taurocholate (0.1 mL/100 g body weight) into the biliopancreatic duct. Rats in the SO group underwent identical surgical procedures without taurocholate infusion.

Gastric cooling balloon

A 20-mm flexible rubber balloon was made using two 8-Fr pediatric silicone catheters (Guangzhou Weilide Medical Co., Ltd., China) for transgastric and pancreatic cooling. The two catheters were bundled and securely connected to a medical rubber finger cot (Shandong Lanfan Health Technology Co., Ltd., China).

Hypothermia with intragastric cooling

In the APH group, a 0.5-cm gastrectomy was performed after AP. A 20-mm flexible rubber balloon (with two silicone catheters, one for inflow and one for outflow) was inserted into the stomach, and the stomach wall was sutured. A temperature electrode (with a lead wire fixed to the abdominal skin) was placed in the gastropancreatic interstice, and another temperature electrode (fixed with tape) was placed in the rectum to avoid systemic hypothermia. After abdominal suturing, local abdominal hypothermia treatment was initiated; the rats were kept on a heating pad. The pancreatic surface temperature was maintained at 25-27 °C, and the rectal temperature was maintained at 37-39 °C (continuously recorded throughout the experiment) by adjusting the ice water circulation rate and the heating pad power. No temperature monitoring/regulation was performed in the SO or AP groups.

Sample collection

All rats received lactated Ringer’s solution (4 doses of 5 mL with an interval of 1 h) postoperatively. Blood samples were collected from the orbital vein at 1 h and 3 h and from the abdominal aorta at 5 h. Serum was extracted by centrifugation (3,000 rpm, 10 min, 20 °C). Serum samples were stored at −80 °C for further analysis. The rats were euthanized by cervical dislocation; stomach and pancreatic tissues were preserved in 10% neutral-buffered formaldehyde, while serum and partial pancreatic tissues were stored at ˗80 °C until use.

Serum amylase analysis

Serum amylase levels were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions.

Histological analysis

Using a microtome (KD3368; Boyn Industrial, China), the pancreatic tissues were cut into 4-μm-thick slices. They were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned (4 μm). They were then stained with hematoxylin and eosin and evaluated by two blinded pathologists using the Schmidt scoring system^[5] for pancreatic injury.

Immunohistochemistry (IHC) of pancreatic tissues

Immunohistochemistry was performed to detect the expression of nuclear factor-κB (NF-κB), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and inhibitor kappa B-α (IκB-α) in pancreatic tissues. Paraffin-embedded pancreatic tissue sections were dewaxed, treated with hydrogen peroxide for 10 min, subjected to antigen retrieval with citrate buffer for 10 min, and blocked with goat serum for 30 min. After blocking, the sections were incubated with primary antibodies for 12 h (phosphate buffered saline [PBS] as a negative control), followed by hematoxylin counterstaining, xylene clearing, neutral resin mounting, and diaminobenzidine staining. Brownish-yellow intracellular granules indicated a positive result. The percentage of positive cells was quantitatively analyzed using Image-Pro plus 6.0 software (Media Cybernetics, USA).

Western blotting analysis

The protein concentration was determined using a bicinchoninic acid (BCA) assay. A 20-μg sample was loaded per well. After transfer, the membrane was blocked at 20 °C for 1 h with a 5% skim milk Tris-buffered saline solution containing 0.05% Tween 20, followed by overnight incubation at 4 °C with primary antibodies against IL-6, TNF-α, NF-κB, and IκB-α. After 1 h of incubation with secondary antibody, chromogenic reagent was added for development (β-actin was used as an internal control). The protein band intensity was analyzed using Quantity One software (Bio-Rad Laboratories, USA), and the relative protein expression level was calculated.

GC-MS-based metabolomic analysis

Serum samples were subjected to metabolomic analysis using GC-MS. Metabolites were extracted using methanol/acetonitrile and derivatized with methoxyamine hydrochloride and N,O-bis (trimethylsilyl) trifluoroacetamide.

Chromatographic separation was performed with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm; Agilent J&W Scientific, USA). Mass spectrometry data were collected in full-scan mode (m/z 50-500). The raw data were processed for peak detection, alignment, and normalization. Metabolite identification was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Human Metabolome Database.

Multivariate statistical analysis was performed using SIMCA-P14.1 software (Sartorius, Germany). Differentially abundant metabolites were screened based on the following criteria: variable importance in projection (VIP)>1; fold change (FC)>1.2 or <0.8; P<0.05; and a false discovery rate (FDR)<0.1. A heatmap cluster analysis was subsequently conducted. Analysis of differentially abundant metabolites was conducted utilizing MetaboAnalyst 4.0 software.

In vitro experiments

Cell culture

AR42J pancreatic acinar cells were cultured in F12K medium supplemented with 20% fetal bovine serum at 37 °C with 5% CO₂. The cells were divided into seven groups: the control (A), sodium taurocholate (B), LY294002 (C), pinitol (D), tocopherol (E), pinitol+tocopherol (F), and hypothermia (G) groups. Cells were pretreated with sodium taurocholate to simulate pancreatic injury and then subjected to different treatments for 24 h.

Measurement of ROS and α-amylase

Intracellular reactive oxygen species (ROS) levels were measured using dichlorofluorescein diacetate (DCFH-DA) staining followed by flow cytometry analysis. α-amylase levels in cell supernatants were determined using ELISA kits.

Western blotting analysis

Total cellular protein was extracted using radioimmunoprecipitation analysis lysis buffer supplemented with 1% benzyl sulfonyl fluoride. The protein concentration was determined using the BCA method, with 20 μg of total protein loaded. Electrophoresis was performed on a 10% sodium dodecyl sulfate-polyacrylamide gel, followed by membrane transfer and blocking. The membranes were incubated with primary antibody overnight at 4 °C. The next day, the membrane was incubated with the secondary antibody at room temperature for 1 h. Colorimetric and imaging analyses were performed using enhanced chemiluminescence. The primary antibody concentrations used were as follows: β-actin (1:200), IL-6 (1:1000), phosphorylated AKT (1:1000), GSK-3β (1:1000), and NF-κB (1:1000). The secondary antibody concentration was 1:5000.

Reverse transcription-polymerase chain reaction (RT-PCR)

TRIzol reagent (1 mL) was added to the cells in the culture dish and mixed thoroughly with a pipette. The lysate was transferred to a centrifuge tube and incubated at room temperature for 5 min to ensure complete cell lysis. Chloroform and precooled isopropanol were subsequently added, and the mixture was stored in a refrigerator at −20 °C overnight before centrifugation to obtain the supernatant. RNA was precipitated with 75% ethanol, and the reaction mixture was prepared following the instructions specified in the kit. Finally, the results were analyzed using a gel imaging system. An ABI PRISM 7500 machine (Applied Biosystems, USA) was used to conduct RT-PCR. The sequences of primers used were as follows: Tlr4 forward: 5′-GGCTTCTAACCTCAACGACCT-3′ and reverse: 5′-ATGATTCTTTGCCTGAGTTGCTT-3′; Pi3k forward: 5′-AGCCACAGATCCACTTAACCC-3′ and reverse: 5′-CTTGCTGTCCCCACTTTACTGA-3′; Nrf2 forward: 5′-ACGGCTAAAACTTCCTACTGTGA-3′ and reverse: 5′-ACACTTACACAGAAACTAGCCCAA-3′; Caspase-3 forward: 5′-ATCAGCCTAATTTTACAGACC-3′ and reverse: 5′-TCTCCTTTCCTTACGCTCT-3′.

Statistical analysis

The data are presented as the mean±standard deviation. Statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software Inc., USA). Comparisons between two groups were conducted using unpaired Student’s t-tests, while multiple-group comparisons were performed using one-way analysis of variance (ANOVA). A P-value < 0.05 was considered to indicate statistical significance.

RESULTS

In vivo experiments

Hypothermia reduced serum amylase levels

No significant differences were observed among the three groups at baseline or 1 h after surgery. However, at 3 h and 5 h, serum amylase levels were significantly elevated in both the AP and APH groups compared with those in the SO group (P<0.001), confirming successful induction of AP. Serum amylase levels were significantly lower in the APH group than in the AP group (P<0.001), indicating that local hypothermia attenuated pancreatic enzyme release.

Hypothermia alleviated pancreatic histological injury

Histological examination further confirmed the protective effects of hypothermia. Pancreatic tissue from the SO group showed normal morphology without edema or inflammatory infiltration. In contrast, the AP group exhibited marked pathological changes, including pancreatic edema, inflammatory cell infiltration, and extensive acinar cell necrosis (Figure 1 A-C). The pathological injury of the rats in the APH group was milder and characterized by reduced edema and focal necrosis. Quantitative analysis using the Schmidt scoring system revealed lower histological scores in the APH group than in the AP group (5.5±1.5 vs. 10.7±1.6, P<0.0001), indicating that hypothermia mitigated pancreatic tissue injury (Figure 1 G).

Figure 1. In vivo histological examination and IHC. A-C: pancreatic tissue of the SO group (A) showing no signs of edema or inflammatory cell infiltration, AP group (B) showing pancreatic tissue edema (blue arrow), inflammatory cell infiltration, and massive cell necrosis (red arrow), and APH group (C) showing edema (blue arrow) and patchy and focal necrosis (red arrow); D-F: gastric tissue of the SO group (D), AP group (E) and APH group (F) showing no cellular edema, cell necrosis and bleeding, inflammatory cell infiltration changes; G: Schmidt score of the pancreatic tissue; H: IHC of pancreatic tissue. ^***P<0.001; ^**P<0.01; ^*P<0.05. IHC: immunohistochemistry; SO: sham operation; AP: acute pancreatitis; APH: acute pancreatitis with hypothermia; NF-κB: nuclear factor-κB; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α; IκB-α: inhibitor kappa B-α.

Hypothermia suppressed inflammatory signaling in pancreatic tissue

IHC analysis revealed significantly increased expression of NF-κB, IL-6, and TNF-α in pancreatic tissues in the AP group compared with the SO group (P<0.01). In contrast, IκB-α expression was significantly reduced in the AP group. Compared with AP treatment, hypothermic treatment significantly decreased NF-κB, IL-6, and TNF-α expression but increased IκB-α expression (Figure 1 H) (P<0.05 or P<0.001).

Consistent with these findings, Western blotting analysis revealed that the protein levels associated with the TLR4/NF-κB signaling pathway (e.g., IL-6, TNF-α, IκB-α, and NF-κB) were significantly elevated in the AP and APH groups than in the SO group (P<0.001). In the APH group, the expression of IL-6, TNF-α, and NF-κB significantly decreased, whereas the expression of IκB-α increased compared with the AP group (P<0.01) (Figure 2).

Figure 2. Protein expression in the pancreatic tissue of rats. ^***P<0.001; ^**P<0.01. SO: sham operation; AP: acute pancreatitis; APH: acute pancreatitis with hypothermia; NF-κB: nuclear factor-κB; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α; IκB-α: inhibitor kappa B-α.

GC-MS analyses revealed distinct metabolic alterations following hypothermia

A total of 53 differentially abundant metabolites were identified between the APH group and the AP group. After FDR correction, 41 metabolites remained significantly altered (FDR<0.1), including 15 up-regulated and 26 down-regulated metabolites in the APH group compared with the AP group. The core metabolites being up-regulated included pinitol (FDR=0.032) and tocopherol (α-tocopherol: FDR=0.028; γ-tocopherol: FDR=0.041) (Figure 3).

Figure 3. Top 18 differentially expressed metabolites between the acute pancreatitis with hypothermia and acute pancreatitis groups.

In vitro experiments

Compared with the control group, the ROS levels in the sodium taurocholate, LY294002, pinitol, tocopherol, pinitol+tocopherol, and hypothermia groups were higher (P<0.0001). Furthermore, the ROS levels were lower in the LY294002, pinitol, tocopherol, pinitol+tocopherol, and hypothermia groups than in the sodium taurocholate group (P<0.0001), with the most obvious reduction observed in the LY294002 and pinitol+tocopherol groups.

Compared with the control group, the α-amylase levels in the sodium taurocholate, LY294002, pinitol, tocopherol, and hypothermia groups were higher (P<0.0001). The level of α-amylase in the pinoresinol + tocopherol group also increased, but the degree was lower than that in the other groups (P=0.0075). Compared with the sodium taurocholate group, the α-amylase levels decreased in the LY294002, pinitol, tocopherol, pinitol+tocopherol, and hypothermia groups.

Western blotting and RT-PCR analyses further revealed that sodium taurocholate increased the protein expression of NF-κB, IL-6, P-AKT, and GSK-3β, as well as TLR-4, PI3K, and Caspase-3 mRNA levels, while reducing Nrf2 expression. Treatment with pinitol, tocopherol, pinitol+tocopherol, LY294002, or hypothermia partially reversed these changes, suggesting that these metabolites modulate inflammatory and oxidative stress pathways involved in pancreatic injury (Figure 4).

Figure 4. Protein (A) and mRNA expression (B) in AR42J cells under various treatments. Group A: control; Group B: sodium taurocholate; Group C: LY294002; Group D: pinitol; Group E: sodium taurocholate+tocopherol; Group F: pinitol+tocopherol; Group G: hypothermia. ^***P<0.0005, ^**P<0.005, ^*P<0.05 compared with group A. P-AKT: phosphorylated protein kinase B; GSK-3β: glycogen synthase kinase-3β; PI3K: phosphatidylinositol 3-kinase; Nrf2: nuclear factor erythroid-2-related factor 2.

DISCUSSION

Severe AP may rapidly progress to systemic inflammatory response syndrome and multiple organ failure, resulting in high morbidity and mortality.^[1,6] In the present study, we demonstrated that local hypothermia with intragastric cooling significantly alleviated pancreatic injury in a sodium taurocholate-induced rat model of AP. Hypothermia reduced serum amylase levels, attenuated pancreatic histological injury, and suppressed inflammatory signaling pathways. Importantly, metabolomic analysis revealed that pinitol and tocopherol were markedly upregulated following hypothermic treatment, suggesting that these metabolites may contribute to the protective effects of hypothermia.

Therapeutic hypothermia has been investigated in conditions involving inflammatory and ischemic injury.^[7,8] Hypothermia can reduce inflammatory mediator release and alleviate tissue damage in pancreatitis models.^[9,10] Local cooling, in particular, has been proposed as a promising strategy because it minimizes systemic hypothermia while maintaining local protective effects. Our findings are consistent with those of previous reports demonstrating that local hypothermia reduces pancreatic necrosis and inflammatory responses.

Metabolomic analysis revealed that the levels of pinitol and tocopherol, as two key metabolites, increased after hypothermic treatment. Pinitol is a naturally occurring inositol derivative known for its anti-inflammatory and antioxidant properties.^[11] Pinitol can inhibit the activation of the NF-κB signaling pathway by reducing the phosphorylation of IκB-α, thereby suppressing inflammatory responses.^[12] Tocopherols, particularly α-tocopherol and γ-tocopherol, are major isoforms of vitamin E that play essential roles in antioxidant defense.^[13] These compounds can scavenge ROS and regulate inflammatory signaling pathways.^[14] In the present study, in vitro experiments demonstrated that both pinitol and tocopherol significantly reduced ROS production and inflammatory factor expression in sodium taurocholate-treated AR42J cells. Furthermore, these metabolites modulated signaling pathways involving PI3K/AKT, TLR4/NF-κB, and Nrf2, suggesting that the protective effects of hypothermia may involve coordinated regulation of oxidative stress and inflammatory responses.

Several limitations of this study should be acknowledged. First, local hypothermia was achieved using a surgically inserted gastric cooling balloon, which may differ from clinically applicable approaches. Future studies should explore less invasive cooling methods that could be translated into clinical practice. Second, the sample size was relatively small, and larger studies are needed to confirm the metabolomic findings. Third, although in vitro experiments suggested potential mechanisms involving pinitol and tocopherol, the in vivo regulatory pathways remain to be fully elucidated. Additional studies are needed to determine the sources of these metabolites and to clarify their roles in pancreatic protection.

CONCLUSION

Local hypothermia attenuated inflammatory cytokine release, serum amylase elevation, and pancreatic injury in a rat model of taurocholate-induced AP. These in vivo protective effects were linked to the upregulation of serum pinitol and tocopherol. These two metabolites exerted pancreatic protective effects through pathways related to inflammation and oxidative stress. Further research is needed to explore the clinical applicability of local hypothermia and the precise regulatory network of pinitol and tocopherol in vivo.

Funding: This study was supported by projects of the Scientific Research Program of the Health Commission of Hunan Province (D202310006192), the Natural Science Foundation of Hunan Province (2021JJ70018), and the Guangxi Clinical Research Centre for Critical Care Medicine.

Ethical approval: The study was approved by the Ethics Committee of Hunan Provincial People’s Hospital (2022-182).

Conflicts of interest: All authors declare no potential conflicts of interest.

Contributors: FY performed the experiments and dratfed the manuscript. ZHT and XTH designed the experimental scheme. XL collected the experimental data. LLL performed experiments and revised the manuscript. ZT and YC performed experiments.