A total of 192 male specific pathogen-free Sprague-Dawley rats (weight, 300±20 g) were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China) and housed under standard laboratory conditions (12/12 h light-dark cycle, 21±2˚C, humidity 50±10%) with food and water provided ad libitum during the study. All animal procedures were conducted in accordance with the Guidelines for Animal Care of Guangzhou University of Chinese Medicine and were approved (approval no. ZYD-2020-028) by the Animal Care and Use Committee of Guangzhou University of Chinese Medicine (Guangzhou, China). All efforts were made to minimize animal suffering.

Male Sprague-Dawley rats were divided into the following four groups using the random weight method: i) Sham, ii) normal saline (NS), iii) 3.5% NaT, and iv) 5% NaT. Sham group rats underwent Sham operation and were treated similarly without infusion (n=16), NS group rats were infused with NS solution and underwent Sham operation (n=16), whereas SAP was induced in 3.5% NaT group rats (n=80) (3, 6, 12, 24, 48 and 72 h) and 5% NaT group rats (n=80) (3, 6, 12, 24, 48 and 72 h) according to a previously reported method (13,15,16). Rats were fasted for 12 h before the operation. Anesthesia was induced using isoflurane (RWD Life Science) and an R550 Multi-output Anesthesia Machine (RWD Life Science) before the onset of surgery. Briefly, a midline laparotomy was performed after sterilization, and the duodenum was identified and gently rotated to expose the posterior surface and pancreas. The proximal biliopancreatic duct was then temporarily occluded with a microclip. The cannula was passed through the duodenal wall to the papilla and into the pancreatic duct. Next, 3.5 or 5% NaT (Sigma-Aldrich; Merck KGaA) at 1 ml/kg was infused into the pancreatic duct at a constant rate (6 ml/h) and pressure, using a syringe pump (Leienly Company). After the infusion, the cannula and microclips were removed from the biliopancreatic duct. Finally, the peritoneum and skin were sutured using a 4-0 prolene suture. After surgery, all the rats were administered NS (1 ml/kg) and placed on a heated pad (37˚C) until they recovered. Animals in the Sham and NS groups were euthanized at 72 h, whereas NaT-induced SAP animals were euthanized at 3, 6, 12, 24, 48 and 72 h after overdosage of isoflurane anesthesia (≥5% for >1 min after cessation of breathing stops). Due to the difficulty of surgery and the high mortality rate in this model, particularly in the 5% NaT group, no rats survived in the 5% NaT-72 h group. The rest of the sample distribution was as follows: Sham group (n=16), NS group (n=16), 3.5% NaT-3 h (n=15), 3.5% NaT-6 h (n=15), 3.5% NaT-12 h (n=13), 3.5% NaT-24 h (n=14), 3.5% NaT-48 h (n=5), 3.5% NaT-72 h (n=4), 5% NaT-3 h (n=15), 5% NaT-6 h (n=13), 5% NaT-12 h (n=11), 5% NaT-24 h (n=7), 5% NaT-48 h (n=3), and 5% NaT-72 h (n=none).

Blood was collected from the abdominal aorta, and ascitic fluid was collected under isoflurane anesthesia before euthanasia. All blood and ascitic fluid samples were centrifuged at 1,000 x g, 4˚C for 15 min, and maintained at -80˚C until use. Tissue samples from the pancreas, lung, ileum, liver and kidneys were harvested and fixed in 4% paraformaldehyde (Dalian Meilun Biology Technology Co., Ltd.) at room temperature for over 24 h, embedded in paraffin blocks, or stored at -80˚C until use.

Tissue sections were stained with hematoxylin and eosin (H&E) for histopathological evaluation. After dewaxed and dehydration, tissue sections were stained with hematoxylin at room temperature for 5 min, followed by differentiation and bluing, then stained with eosin at room temperature for 1 min. For each group, the pancreatic histopathological scores of three independent tissue sections were blindly assessed by two pathologists according to the scoring criteria developed by Schmidt et al (23) and simplified by Liu et al (15). Briefly, the pancreatic histopathological scoring criteria included four aspects: i) interstitial edema: 0 points=none, 1 point=interlobular, 2 points=lobule involvement, and 3 points=isolated island-like acinar cells; ii) leukocyte infiltration: 0 points=none, 1 point=<20%, 2 points=20-50%, and 3 points=>50%; iii) acinar cell necrosis: 0 points=none, 1 point=<5%, 2 points=5-20%, and 3 points=>20%; (iv) hemorrhage: 0 points=none, 1 point=1-2, 2 points=3-5, and 3 points=>20%. Pulmonary histopathological changes were assessed by areas of alveolar septal thickening, leukocyte infiltration, alveolar capillaries dilation and congestion and hemorrhage.

The serum and ascitic fluid amylase levels were determined using starch-iodine colorimetry and α-amylase assay kits (Nanjing Jiancheng Bioengineering Institute). Phosphate-buffered saline (PBS) was used to dilute the pancreatitis rat serum and ascites fluid samples. First, 0.1 ml of serum and ascitic fluid samples were added into 0.5 ml 4 g/ml substrate buffer and placed in a water bath with a water temperature of 37˚C for 7.5 min. Then, 0.5 ml 0.1 mol/l iodine solution and 3 ml distilled water were added. The absorbance was immediately detected at 660 nm on a UV spectrophotometer (Shanghai Metash Instrument Co., Ltd.).

The serum and ascitic fluid lipase levels were determined after dilution (1:1 and 1:10) with PBS using a routine colorimetric method and a lipase assay kit (Nanjing Jiancheng Bioengineering Institute). The substrate buffer was preheated at 37˚C and added to 50 µl fresh serum or ascitic fluid, and the absorbance was detected at 420 nm in the first 30 sec as A1. After 10 min of reaction, the absorbance (A2) was read using a UV spectrophotometer. The lipase activity was measured according to the manufacturer's protocol.

The ileum tissues were lysed in normal saline containing a protease inhibitor. Tissues were ground using an automatic tissue lyser (Jingxin Industrial Development Co., Ltd.). The lysate was collected and centrifuged at 1,000 x g and 4˚C for 15 min. The protein concentration was measured using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). All samples were stored at -80˚C until use. Enzymatic immunoassays for IL-1β, IL-6, and TNF-α were performed using ELISA kits (cat. nos. CRE0006, CRE0005 and CRE0003; Beijing 4A Biotech Co., Ltd.) according to the manufacturer's protocol. The standard, serum, and ascitic fluid samples were added into a 96-well plate (100 µl/well), which was precoated with IL-1β, IL-6, and TNF-α, respectively. Then, they were incubated at 37˚C for 2 h and washed five times with washing buffer. The corresponding biotinylated antibodies (IL-1β 1:100, IL-6 1:100 and TNF-α 1:100), enzyme-binding antibodies, and tetramethylbenzidine solution were added. Finally, 100 µl stop solution was added to stop the reaction. The absorbance was detected at 450 nm using MULTISKAN GO (Thermo Fisher Scientific, Inc.), the concentrations of each sample were calculated according to the standard curve, and protein concentrations were normalized to tissue cytokine levels.

Ileum tissue was embedded in an Optimum Cutting Temperature embedding agent, sliced into 10-µm thick sections, and fixed with 4% paraformaldehyde at 4˚C for 30 min. Next, the tissue sections were incubated with 100 µl of 5% donkey serum blocker solution at room temperature for 2 h to block non-specific antigens. A diluted antibody-containing 5% donkey serum PBST (0.05% Tween-20) was prepared of ZO-1 (1:50; cat. no. sc-33725, Santa Cruz Biotechnology, Inc.) or occludin (1:50; cat. no. sc-133256, Santa Cruz Biotechnology, Inc.). Next, 50 µl of the diluted antibody solution was added to each sample and incubated overnight at 4˚C. After washing with PBST for 10 min thrice, the samples were incubated with a PBST-diluted AF488-conjugated secondary antibody (1:200; cat. no. 1010-30, Southern Biotech) for 2 h at room temperature. Then, they were washed with PBST thrice for 10 min each, and stained with Hoechst 33342 (1:1,000; cat. no. 561908, BD Pharmingen) at room temperature for 30 min. An LSM 8000 laser confocal microscope (Carl Zeiss AG) equipped with a camera was used to observe the stained tissue samples and to obtain images.

Serum alanine transaminase (ALT), aspartate transaminase (AST), creatinine (CRE), and urea levels were measured using a Hitachi 7080 automatic biochemical analyzer (Hitachi, Ltd.). Lung myeloperoxidase (MPO) levels were determined using MPO ELISA kits (cat. no. CSB-E13689Rb, Cusabio Technology LLC) and malondialdehyde (MDA) activity was determined using the Lipid Peroxidation MDA Assay kit (cat. no. S0131S, Beyotime Institute of Biotechnology) according to the manufacturer's protocol.

All data were analyzed using the SPSS software (version 25.0; IBM Corp.) and are presented as the mean ± standard error. After the homogeneity of variance test, one-way ANOVA followed by the least significant difference (LSD) or Games-Howell test was performed for multiple group comparisons. Data were displayed using GraphPad Prism (version 7.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

SAP rat serum and ascitic fluid were collected at different time points (3, 6, 12, 24, 48 and 72 h) and the amylase and lipase levels were detected. As revealed in Fig. 1A, the mortality rate in the 3.5% NaT-72 h group was ~50%, and most animals succumbed within 24 h. The mortality rate in the 5% NaT-72 h group was ~100%; therefore, a sample from this group was unobtainable.

The ascites volume increased 3 h after surgery. Significant increases in the ascites volume were observed in the 3.5% NaT (3, 6 and 12 h) and 5% NaT (3, 12 and 48 h) groups compared with those in the NS group (Fig. 1B). Serum amylase levels significantly increased after pancreatic duct perfusion with 3.5% NaT (3, 6, 12 and 24 h) and 5% NaT (3, 6, 12 and 24 h) compared with those in the NS group (Fig. 1C). The amylase levels in ascitic fluid also significantly increased after perfusion with 3.5% NaT (6 and 12 h) and 5% NaT (3, 6, 12 and 24 h), as demonstrated in Fig. 1D. In the 5% NaT group, the serum and ascitic fluid amylase levels exhibited a sustained increase and were maintained at a high level without reduction. No statistically significant difference in amylase levels was observed in the 5% NaT-48 h group. Lipase activity in the 3.5% NaT and 5% NaT groups significantly increased in the serum and ascitic fluid at 3, 6 and 12 h after the infusion and recovered within 24 h (Fig. 1E and F).

Histopathological scores were assessed (Table I) according to the levels of pancreatic interstitial edema, leukocyte infiltration, acinar cell necrosis and hemorrhage using H&E staining. Representative images (magnification, x200) of the H&E-stained pancreatic sections are revealed in Fig. 2. No remarkable difference was observed in the histopathological images of the pancreas between the Sham and NS groups. In the 3.5 and 5% NaT groups, the pancreas was critically damaged in a time- and dose-dependent manner, compared with the NS group. Furthermore, the pancreas in the 5% NaT group exhibited earlier severe acinar cell necrosis, interstitial edema, and hemorrhage compared with that in the 3.5% NaT group (Fig. 2 and Table I). However, leukocyte infiltration was mild in both groups (mostly lymphocytes, plasmacytes, and eosinophils rather than neutrophils).

The protein levels of IL-1β, IL-6, and TNF-α were measured using ELISA to evaluate the inflammatory response in the serum and ascitic fluid of rats with NaT-induced pancreatitis. The levels of IL-1β in ascitic fluid were significantly increased in the 5% NaT-12 h group compared with those in the NS group (P<0.05; Fig. 3D). The levels of IL-6 in ascitic fluid significantly increased in the 5% NaT group at 3, 6 and 12 h, and were alleviated within 48 h (P<0.05 compared with the 12 h group; Fig. 3E). The levels of IL-6 significantly increased in serum at 5% NaT-48 h, Whereas the level of IL-1β and TNF-α in the serum scarcely increased after 3.5 or 5% NaT infusion (Fig. 3A-C). Again, the levels of TNF-α in the serum and ascitic fluid were not elevated (Fig. 3C and F).

Compared to the NS group, the AST levels were significantly elevated after infusion in the 3.5% (6 and 12 h) and 5% NaT groups (3, 6 and 12 h; P<0.05; Fig. 3G). The level of ALT in 5% NaT groups (12 h) was significantly increased (P<0.01; Fig. 3H). In 3.5% NaT groups, ALT and AST levels declined back to almost normal levels at 72 h, whereas in the 5% NaT groups a nonsignificant downward trend was observed after 24 h (Fig. 3G and H). Furthermore, the CRE levels increased only in the 5% NaT-3 h group (P<0.01), compared with those in the NS group (Fig. 3I), whereas no statistically significant increase in the serum urea levels was observed (Fig. 3J).

According to the liver histopathology results (Fig. S1), only slight hepatocellular edema and hepatic sinusoid dilation were observed at 12 h (1/3) and 24 h (1/3) in the 5% NaT group. In the kidneys, ~1/3 of the 5% NaT rats at each time point exhibited slight renal tubule edema or renal vascular congestion, and only one rat among all animals developed periglomerular interstitial hemorrhage and ischemic tubular necrosis at 24 h. The most severe pathological findings are shown in Fig. S1. However, changes in the liver and kidneys were considered insufficient for the diagnosis of pathological damage. In addition, no apparent pathological changes in the kidneys and livers were observed in the 3.5% NaT group, even after 72 h (data not shown).

Lung sections were stained with H&E and images were obtained (Fig. 4). Histopathological results showed that the 3.5 and 5% NaT groups showed alveolar septum thickening with partial alveolar collapse, alveolar capillary dilation with congestion, or local hemorrhage. Alveolar septum thickening was widely observed in all NaT groups (Table II). Slight neutrophil infiltration was observed in certain cases (Table II). MPO and MDA levels in lung tissue were not significantly increased (Fig. S2).

Intestinal mucosal injury was observed in the ileum in the 5% NaT groups using H&E staining (magnification, x200; Fig. 5). In the 5% NaT group, the intestinal mucosal epithelium exhibited abscission, necrosis and hemorrhage at 3 and 6 h after infusion, suggesting that 5% NaT induced mucosal injury in the early stage of SAP. No significant pathological changes in the ileum were observed in the 3.5% NaT group compared with those in the NS group until 48 h (1/3) and 72 h (1/3). Furthermore, immunofluorescence was used to detect alterations in ileal tight junction morphology in the 5% NaT group (24 and 48 h). As expected, disruption of ZO-1 and occludin tight junction constituents was detected in the 5% NaT-48 h group (Fig. 6).

The levels of IL-1β, IL-6 and TNF-α in ileum tissue were detected using ELISA (Fig. S3). The results showed that the IL-1β and TNF-α levels in the 5% NaT group were significantly increased in the ileum at 12 h compared with those in the NS group (P<0.01). No statistical difference in the IL-6 levels was observed in either the 3.5 NaT or 5% NaT groups.